Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells

Apoptosis is involved in the pathogenesis of Sjögren’s syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H₄R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H₄R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H₄R agonist HST-10. Expression and internalization of H₄R were studied by immunofluorescence staining ± clathrin inhibitor methyl-β-cyclodextrin (MβCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H₄R internalization was inhibited by MβCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H₄R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H₄R-stimulated samples. H₄R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H₄R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H₄R activation on apoptosis of human salivary gland cells. Diminished H₄R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.     

Discovery and design of benzimidazolone based inhibitors of p38 MAP kinase

A new class of benzimidazolone p38 MAP kinase inhibitors was discovered through high-throughput screening. X-ray crystallographic data of the lead molecule with p38 were used to design analogues with improved binding affinity and potency in a cell assay of LPS-induced TNF production. Herein, we report the SAR of this new class of p38 inhibitors.     

Seed germination ecology and salt stress response in eight Mediterranean populations of Sarcopoterium spinosum (L.) Spach

This study aims to deepen the analysis of seed germination ecology and salinity tolerance of Sarcopoterium spinosum (Rosaceae). Germination tests were conducted to evaluate the effect of the fruit’s spongy tissue and the intraspecific variability in seed germination among eight populations of the species on responses to light and total darkness, constant and alternating temperatures, salt stress and germination recovery. The effect of the presence of the spongy tissue varied among populations, with significant results for seed germination. For all populations, optimum germination temperatures were observed in the range of 10–20°C, indicating that S. spinosum and its germination in the field might occur preferably in the period between autumn and early spring. The high water availability due to rainfall during this period could be a considerable advantage for the seed germination of this species. Seeds of S. spinosum showed the ability to germinate in up to 250 mM NaCl in the substrate, and their ability to recover after salt exposure may be interpreted as adaptation to the coastal habitats in which they generally grow. These results give this species a halo-tolerant character. Great inter-population variability is detected in this study in several aspects, which indicated that the Mediterranean populations of S. spinosum differ considerably and are adapted to their local conditions. This study provides new information about S. spinosum seed ecology, which could help to preserve and apply effective conservation measures for this species, which in several areas of its distribution range is endangered.     

Phylogenetic diversity and genome sizes of Astragalus (Fabaceae) in the Lebanon biogeographical crossroad

The Lebanese mountain range is an important zone of plant species richness and endemism where the genus Astragalus constitutes a principal component of plant biodiversity. Most of endemic Astragalus taxa, living in mountains and arid zones of Mounts Lebanon and anti-Lebanon, are characterized by a cushion, spiny vegetative form, named “tragacanthic”, which is a remarkable example of vegetative convergence evolution. Because of determination difficulties, taxonomic uncertainties, and discrepancy in the number of taxa listed according to authors, new data are hardly needed to improve systematics of Astragalus and to investigate the role of the Lebanese mountain range as refugia of biodiversity. Before this study only two values on the genome size of Astragalus were reported in the literature and no previous molecular studies had been carried out on Astragalus genus in Lebanon. We examined the utility of rDNA ITS molecular markers to distinguish Astragalus species of Lebanese mountain range and the variation range of their genome size. The main results revealed a striking diversity in Lebanese Astragalus species with the emphasis of a huge variation of genome sizes, an important inter-specific chromosome polymorphism and the existence of a high phylogenetic diversity. The strict endemic species of the Lebanese mountains are positioned throughout the phylogeny. These results confirm that the Lebanon and anti-Lebanon mounts constitute a third diversity center for Astragalus and that high altitude areas are important refugia of plant biodiversity despite centuries of exploitation by humans.     

Method for generation of human hyperdiversified antibody fragment library

The selection of antibody fragments from libraries using in vitro screening technologies has proven to be a very good alternative to the classical hybridoma technology, and has overcome the laborious process of antibody humanization. However, the complexity of the library is critical in the probability of being able to directly isolate a high affinity antibody specific to a target. We report a method to make hyperdiversified antibody fragment libraries, based on human immunoglobulin variable genes mimicking the somatic hypermutation process. This mutagenesis technology, MutaGen, was used for the first time on the entire variable domain (frameworks and CDRs) of large repertoires of human variable antibody domains. Our MutaGen process uses low-fidelity human polymerases, known as mutases, suggested to be involved in the somatic hypermutation process of immunoglobulin genes. Depending on the mutases used, we generated complementary mutation patterns with randomly distributed mutations. The libraries were generated with an average of 1.8 mutations per 100 amino acids. The hyperdiversified antibody fragment libraries constructed with our process should enable the selection of antibody fragments specific to virtually any target.     

Cross-talk between the paired domain and the homeodomain of Pax3: DNA binding by each domain causes a structural change in the other domain, supporting interdependence for DNA Binding

The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.     

Combinatorial engineering to enhance thermostability of amylosucrase

Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C.