Characterization and inhibition of aminopeptidase A by α-mercapto-β-amino acyl dipeptides

Summary In order to study the physiological role of aminopeptidase A(APA), several α-mercapto-β-amino acyl dipeptides were synthesized to obtain compounds having a high affinity for APA and a high selectivity versus aminopeptidase N (APN). Sulfornamide and carboxylate moieties which have been shown to be recognized by the S₁ subsite of the enzyme were introduced on the side chain of the α-mercapto-β-amino acyl sub-unit, the latter being coupled to dipeptides optimized to interact with the S'₁ and S'₂ subsites by means of combinatorial chemistry. Good affinities (16nM) were obtained, the selectivity factors being up to 160-fold versus APN.     

Interleukin-22 is produced by invariant natural killer T lymphocytes during influenza A virus infection: potential role in protection against lung epithelial damages

Invariant natural killer T (iNKT) cells are non-conventional lipid-reactive αβ T lymphocytes that play a key role in host responses during viral infections, in particular through the swift production of cytokines. Their beneficial role during experimental influenza A virus (IAV) infection has recently been proposed, although the mechanisms involved remain elusive. Here we show that during in vivo IAV infection, mouse pulmonary iNKT cells produce IFN-γ and IL-22, a Th17-related cytokine critical in mucosal immunity. Although permissive to viral replication, IL-22 production by iNKT cells is not due to IAV infection per se of these cells but is indirectly mediated by IAV-infected dendritic cells (DCs). We show that activation of the viral RNA sensors TLR7 and RIG-I in DCs is important for triggering IL-22 secretion by iNKT cells, whereas the NOD-like receptors NOD2 and NLRP3 are dispensable. Invariant NKT cells respond to IL-1β and IL-23 provided by infected DCs independently of the CD1d molecule to release IL-22. In vitro, IL-22 protects IAV-infected airway epithelial cells against mortality but has no role on viral replication. Finally, during early IAV infection, IL-22 plays a positive role in the control of lung epithelial damages. Overall, IAV infection of DCs activates iNKT cells, providing a rapid source of IL-22 that might be beneficial to preserve the lung epithelium integrity.     

Did the Quaternary climatic fluctuations really influence the tempo and mode of diversification in European rodents?

The objective of this study was to establish whether the Quaternary climatic fluctuations influenced the tempo and mode of diversification in European rodents. Our case study is the subgenus Microtus (Terricola) distributed from western Europe to the Caucasus. Mitochondrial cytochrome b gene sequences from several representatives of all the species were used to generate maximum‐likelihood and Bayesian phylogenetic trees, to estimate divergence times, to identify biogeographic ancestral areas and to study the rate of diversification. Results showed that phylogenetic tree topologies were similar to previous published studies but with a better resolution at some nodes. The origin of Microtus (Terricola) is dated back to approximately 4.05 Myr in the Early Pliocene, and molecular dating for most Terricola species corresponds to several glacial periods of the Pleistocene. Results of the biogeographic ancestral area reconstruction suggest that Microtus (Terricola) diversified from the Caucasus/Turkey/Iran area through western Europe. Several periods of diversity variation were highlighted as follows: two period of diversity increase, between 3 and 2 Myr, and after 1 Myr; two periods of diversity decrease, before 3 Myr, and between 2 and 1 Myr. The diversification rate of Microtus (Terricola) was 0.353 ± 0.004 event/Myr, a rate similar to that of the Muridae family. To conclude, although the Pleistocene glacial conditions had an impact on the speciation events, the Quaternary does not appear however as a period with an exceptional rate of diversification for European rodents.     

Reversibility of germinative and endocrine testicular function after long-term contraception with a GnRH-agonist implant in the tom—a follow-up study

A significantly reduced gonadotropin and testosterone secretion is a well-described result of long-term administration of GnRH agonists in the male dog and cat. To date, no data are available about the duration of efficacy and the reversibility of treatment-induced effects after long-term treatment with a 4.7 mg deslorelin implant. Seven healthy male European Shorthair cats (3.2 ± 0.5 kg, 1–6 years) were treated with a 4.7 mg deslorelin implant. Blood samples (testosterone, T), testicular volume, penile spines, and mating behavior were recorded once weekly. Considering T > 0.5 ng/mL as the biological endpoint, mean duration of efficacy was 78.8 ± 12.9 weeks (range: 61.7–100.7 weeks) with T concentrations increasing rapidly after the last T less than 0.1 ng/mL (basal) (P < 0.0001), and pretreatment T concentrations being reached after 3 weeks. Testicular volume rapidly increased after the first increase of T (P < 0.001) with pretreatment testicular volume being reached after 6.9 ± 3.4 weeks (5–11 weeks). “Normal” libido reoccurred 88.7 ± 12.4 weeks after treatment, and “normal” mating behavior was observed even later. Fertile matings occurred 7 to 42 weeks after the last T less than 0.1 ng/mL with a mean of 4.0 ± 0.0 kittens, and 13.6 to 47.6 weeks afterwards testicular histology revealed normal spermatogenesis. The present data confirm that the use of slow-release GnRH-agonist implants containing deslorelin in tomcats represents an effective and safe reversible alternative for long-term contraception; however, as number of animals is low, further fertility trials are recommended.     

Targeting DNA with novel diphenylcarbazoles

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.     

Association of specific expansins with growth in maize leaves is maintained under environmental, genetic, and developmental sources of variation

We aimed to evaluate whether changes in maize (Zea mays) leaf expansion rate in response to environmental stimuli or developmental gradients are mediated by common or specific expansins, a class of proteins known to enhance cell wall extensibility. Among the 33 maize expansin or putative expansin genes analyzed, 19 were preferentially expressed at some point of the leaf elongation zone and these expansins could be organized into three clusters related to cell division, maximal leaf expansion, and cell wall differentiation. Further analysis of the spatial distribution of expression was carried out for three expansins in leaves displaying a large range of expansion rates due to water deficit, genotype, and leaf developmental stage. With most sources of variation, the three genes showed similar changes in expression and consistent association with changes in leaf expansion. Moreover, our analysis also suggested preferential association of each expansin with elongation, widening, or both of these processes. Finally, using in situ hybridization, expression of two of these genes was increased in load-bearing tissues such as the epidermis and differentiating xylem. Together, these results suggest that some expansins may be preferentially related to elongation and widening after integrating several spatial, environmental, genetic, and developmental cues.     

Proteome analysis of grape skins during ripening

The characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and beta-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process.