Virally and physically transgenized equine adipose-derived stromal cells as a cargo for paracrine secreted factors

Background  

Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated.     

Population dynamic of the extinct European aurochs: genetic evidence of a north-south differentiation pattern and no evidence of post-glacial expansion

Background

Various evolutionary models have been proposed to interpret the fate of paralogous duplicates, which provides substrates on which evolution selection could act. In particular, domestication, as a special selection, has played important role in crop cultivation with divergence of many genes controlling important agronomic traits. Recent studies have indicated that a pair of duplicate genes was often sub-functionalized from their ancestral functions held by the parental genes. We previously demonstrated that the rice cell-wall invertase (CWI) gene GIF1 that plays an important role in the grain-filling process was most likely subjected to domestication selection in the promoter region. Here, we report that GIF1 and another CWI gene OsCIN1 constitute a pair of duplicate genes with differentiated expression and function through independent selection.

Results

Through synteny analysis, we show that GIF1 and another cell-wall invertase gene OsCIN1 were paralogues derived from a segmental duplication originated during genome duplication of grasses. Results based on analyses of population genetics and gene phylogenetic tree of 25 cultivars and 25 wild rice sequences demonstrated that OsCIN1 was also artificially selected during rice domestication with a fixed mutation in the coding region, in contrast to GIF1 that was selected in the promoter region. GIF1 and OsCIN1 have evolved into different expression patterns and probable different kinetics parameters of enzymatic activity with the latter displaying less enzymatic activity. Overexpression of GIF1 and OsCIN1 also resulted in different phenotypes, suggesting that OsCIN1 might regulate other unrecognized biological process.

Conclusion

How gene duplication and divergence contribute to genetic novelty and morphological adaptation has been an interesting issue to geneticists and biologists. Our discovery that the duplicated pair of GIF1 and OsCIN1 has experienced sub-functionalization implies that selection could act independently on each duplicate towards different functional specificity, which provides a vivid example for evolution of genetic novelties in a model crop. Our results also further support the established hypothesis that gene duplication with sub-functionalization could be one solution for genetic adaptive conflict.     

Porous silicon-based optical microsensor for the detection of L-glutamine

The molecular binding between the glutamine-binding protein (GlnBP) from Escherichia coli and L-glutamine (Gln) is optically transduced by means of a biosensor based on porous silicon nano-technology. The sensor operates by the measurement of the interferometric fringes in the reflectivity spectrum of a porous silicon Fabry-Perot layer. The binding event is revealed as a shift in wavelength of the fringes. Due to the hydrophobic interaction with the Si-H terminated surface of the porous silicon, the GlnBP protein, which acts as a molecular probe for Gln, penetrates and links into the pores of the porous silicon matrix. We can thus avoid any preliminary functionalization process of the porous layer surface, which is also prevented from oxidation, at least for few cycles of wet measurements. The binding of Gln to GlnBP has also been investigated at different concentration of GlnBP.     

The application of a 3D-QSAR (autoMEP/PLS) approach as an efficient pharmacodynamic-driven filtering method for small-sized virtual library: application to a lead optimization of a human A3 adenosine receptor antagonist

We have recently reported that the combination of molecular electrostatic potential (MEP) surface properties (autocorrelation vectors) with the conventional partial least squares (PLS) analysis can be used to produce a robust ligand-based 3D structure-activity relationship (autoMEP/PLS) for the prediction of the human A3 receptor antagonist activities. Here, we present the application of the 3D-QSAR (autoMEP/PLS) approach as an efficient and alternative pharmacodynamic filtering method for small-sized virtual library. For this purpose, a small-sized combinatorial library (841 compounds) was derived from the scaffold of the known human A3 antagonist pyrazolo-triazolo-pyrimidines. The most interesting analogues were further prioritized for synthesis and pharmacological characterization. Remarkably, we have found that all the newly synthetized compounds are correctly predicted as potent human A3 antagonists. In particular, two of them are correctly predicted as sub-nanomolar inhibitors of the human A3 receptor.     

Demonstration of long-range interactions in a PDZ domain by NMR, kinetics, and protein engineering

Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.     

Role of CD45 Signaling Pathway in Galactoxylomannan-Induced T Cell Damage

Previously, we reported that Galactoxylomannan (GalXM) activates the extrinsic and intrinsic apoptotic pathways through an interaction with the glycoreceptors on T cells. In this study we establish the role of the glycoreceptor CD45 in GalXM-induced T cell apoptosis, using CD45^+/+ and CD45^−/− cell lines, derived from BW5147 murine T cell lymphoma. Our results show that whereas CD45 expression is not required for GalXM association by the cells, it is essential for apoptosis induction. In CD45^+/+ cells, CD45 triggering by GalXM reduces the activation of Lck, ZAP70 and Erk1/2. Conversely, in CD45^−/− cells, Lck was hyperphosphorylated and did not show any modulation after GalXM stimulation. On the whole, our findings provide evidence that the negative regulation of Lck activation occurs via CD45 engagement. This appears to be related to the capacity of GalXM to antagonize T cell activation and induce T cell death. Overall this mechanism may be responsible for the immune paralysis that follows GalXM administration and could explain the powerful immunosuppression that accompanies cryptococcosis.     

Mutational Profiling of Kinases in Human Tumours of Pancreatic Origin Identifies Candidate Cancer Genes in Ductal and Ampulla of Vater Carcinomas

Background

Protein kinases are key regulators of cellular processes (such as proliferation, apoptosis and invasion) that are often deregulated in human cancers. Accordingly, kinase genes have been the first to be systematically analyzed in human tumors leading to the discovery that many oncogenes correspond to mutated kinases. In most cases the genetic alterations translate in constitutively active kinase proteins, which are amenable of therapeutic targeting. Tumours of the pancreas are aggressive neoplasms for which no effective therapeutic strategy is currently available.

Methodology/Principal Findings

We conducted a DNA-sequence analysis of a selected set of 35 kinase genes in a panel of 52 pancreatic exocrine neoplasms, including 36 pancreatic ductal adenocarcinoma, and 16 ampulla of Vater cancer. Among other changes we found somatic mutations in ATM, EGFR, EPHA3, EPHB2, and KIT, none of which was previously described in cancers.

Conclusions/Significance

Although the alterations identified require further experimental evaluation, the localization within defined protein domains indicates functional relevance for most of them. Some of the mutated genes, including the tyrosine kinases EPHA3 and EPHB2, are clearly amenable to pharmacological intervention and could represent novel therapeutic targets for these incurable cancers.