Buffer catalyzed cleavage of uridylyl-3',5'-uridine in aqueous DMSO: comparison to its activated analog, 2-hydroxypropyl 4-nitrophenyl phosphate.

Buffer catalysis of the cleavage and isomerization of uridylyl-3',5'-uridine (UpU) has been studied over a wide pH range in 80% aq. DMSO. The diminished hydroxide ion concentration in this solvent system made catalysis by amine buffers (morpholine, 4-hydroxypiperidine and piperidine) visible even at relatively low buffer concentrations (10-200 mmol L(-1)). The observed catalysis was, however, much weaker than what has been previously reported for the activated RNA model 2-hydroxypropyl 4-nitrophenyl phosphate (HPNP) in the same solvent system. In the case of morpholine, contribution of both the acidic and the basic buffer constituent was significant, whereas with 4-hydroxypiperidine and piperidine participation of the acidic constituent could not be established unambiguously. The results underline the importance of using realistic model compounds, along with activated ones, in the study of the general acid/base catalysis of RNA cleavage.

Holistic systems biology approaches to molecular mechanisms of human helper T cell differentiation to functionally distinct subsets.

Current knowledge of helper T cell differentiation largely relies on data generated from mouse studies. To develop therapeutical strategies combating human diseases, understanding the molecular mechanisms how human naïve T cells differentiate to functionally distinct T helper (Th) subsets as well as studies on human differentiated Th cell subsets is particularly valuable. Systems biology approaches provide a holistic view of the processes of T helper differentiation, enable discovery of new factors and pathways involved and generation of new hypotheses to be tested to improve our understanding of human Th cell differentiation and immune-mediated diseases. Here, we summarize studies where high-throughput systems biology approaches have been exploited to human primary T cells. These studies reveal new factors and signalling pathways influencing T cell differentiation towards distinct subsets, important for immune regulation. Such information provides new insights into T cell biology and into targeting immune system for therapeutic interventions.

A human ImmunoChip cDNA microarray provides a comprehensive tool to study immune responses.

DNA microarray technology has developed rapidly in recent years and has become an essential tool, providing novel approaches to biomedical research. In this paper, we describe a self-designed ImmunoChip cDNA array for immunological research. With a comprehensive selection of genes of interest, we can focus on key signalling pathways and molecular mechanisms at relatively low cost compared to commercial platforms which are usually targeted at global screening of gene expression. To validate the efficiency of the ImmunoChip, we studied T helper cell polarization to functionally distinct subsets (Th1 and Th2). We also developed a tool for quality control of cDNA microarrays that assesses the technical quality of an ImmunoChip. The information produced with the quality control tool is shown to be valuable for extracting correct information from cDNA microarrays. Gene expression measurements with ImmunoChip are in agreement with the results obtained using oligonucleotide microarrays and with published quantitative RT-PCR data. The ImmunoChip provides reliable measurements and gives new insights into various aspects of human immune responses.

Three-dimensional models of alpha(2A)-adrenergic receptor complexes provide a structural explanation for ligand binding.

We have compared bacteriorhodopsin-based (alpha(2A)-AR(BR)) and rhodopsin-based (alpha(2A)-AR(R)) models of the human alpha(2A)-adrenengic receptor (alpha(2A)-AR) using both docking simulations and experimental receptor alkylation studies with chloroethylclonidine and 2-aminoethyl methanethiosulfonate hydrobromide. The results indicate that the alpha(2A)-AR(R) model provides a better explanation for ligand binding than does our alpha(2A)-AR(BR) model. Thus, we have made an extensive analysis of ligand binding to alpha(2A)-AR(R) and engineered mutant receptors using clonidine, para-aminoclonidine, oxymetazoline, 5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304), and norepinephrine as ligands. The representative docked ligand conformation was chosen using extensive docking simulations coupled with the identification of favorable interaction sites for chemical groups in the receptor. These ligand-protein complex studies provide a rational explanation at the atomic level for the experimentally observed binding affinities of each of these ligands to the alpha(2A)-adrenergic receptor.