Thermal desorption effects on fragment ion production from multi-photon ionized uridine and selected analogues.

Experiments on neutral gas-phase nucleosides are often complicated by thermal lability. Previous mass spectrometry studies of nucleosides have identified enhanced relative production of nucleobase ions (e.g. uracil+ from uridine) as a function of desorption temperature to be the critical indicator of thermal decomposition. On this basis, the present multi-photon ionization (MPI) experiments demonstrate that laser-based thermal desorption is effective for producing uridine, 5-methyluridine, and 2'-deoxyuridine targets without thermal decomposition. Our experiments also revealed one notable thermal dependence: the relative production of the sugar ion C5H9O4 + from intact uridine increased substantially with the desorption laser power and this only occurred at MPI wavelengths below 250 nm (full range studied 222-265 nm). We argue that this effect can only be rationalized plausibly in terms of changing populations of different isomers, tautomers, or conformers in the target as a function of the thermal desorption conditions. Furthermore, the wavelength threshold behavior of this thermally-sensitive MPI channel indicates a critical dependence on neutral excited state dynamics between the absorption of the first and second photons. The experimental results are complemented by density functional theory (DFT) optimizations of the lowest-energy structure of uridine and two further conformers distinguished by different orientations of the hydroxymethyl group on the sugar part of the molecule. The energies of the transitions states between these three conformers are low compared with the energy required for decomposition.

Potential use of surface-assisted LIBS for determination of strontium in wines.

Laser-induced breakdown spectroscopy (LIBS) is a well-established technique for elemental analysis and has been widely used for qualitative and quantitative analysis of different solid samples. LIBS is also well-known for not requiring sample preparation, but the analysis of liquids is actually a great challenge. In the present work, a novel approach of elemental analysis of liquids with an organic matrix has been performed, to the best of our knowledge, making a liquid-to-solid matrix conversion by drying wine samples on aluminum and silicon wafers, which have demonstrated an increase in the analytical performance of LIBS. A red wine from Slovakia (not blended with any other variety or wine from other regions or adulterants) was prepared according to the procedure consisting of drying 2 ml of wine dropped on a solid wafer having a flat surface area of about 25 cm2. Surface-assisted LIBS in combination with the conversion of liquid into solid avoids the difficulties and limitations of working with liquid samples by LIBS, improving the limit of detection (LOD), repeatability, and sensitivity. The quantification of strontium (Sr) has been realized by addition of the known concentration of SrCl2·6H2O in wine before the drying process. Sr is an important element among those usually used as markers for identification of the soils on which the vines grow. Two ionic (407.771 nm and 421.552 nm) and two neutral (460.733 nm and 481.188 nm) Sr lines were used to plot the calibration curves in order to study the LODs and the matrix effects for the analysis of Sr in the tested wines and for different wafer materials. This direct surface-assisted LIBS measuring method has been successfully applied for the determination of Sr in a red wine sample from Slovakia, and the obtained results with two kinds of substrates (Al and Si) were compared. Finally, a validation sample has been employed to test the accuracy of the established calibration curves.

The 60-kDa heat shock protein modulates allograft rejection.

Allograft rejection is a process of immune reactivity triggered by foreign transplantation antigens. We now demonstrate that the 60-kDa heat shock protein (hsp60), a molecule that is identical in the donor and the recipient, can regulate allograft immunity. In wild-type mice, hsp60 expression was greatly enhanced in allografts being rejected. By using MHC class II (Ealpha) promoter hsp60 transgenic mice either as donors of skin with enhanced expression of hsp60, or as allograft recipients with decreased hsp60 autoimmunity, we found that augmented expression of mouse hsp60 in the allograft accelerated its rejection, whereas reduced autoimmunity to mouse hsp60 in graft recipients delayed the process. Moreover, in nontransgenic mice, therapeutic administration of hsp60 or hsp60 peptides, known to modulate naturally occurring hsp60 autoimmunity, led to delayed allograft rejection. Thus, we demonstrate that hsp60 expression and hsp60 autoimmunity can influence and modify the immune response to foreign antigens. Hence, autoimmunity to self-hsp60 epitopes is not necessarily an aberration, but may serve physiologically and therapeutically to modulate foreign immunity.

Treatment of NOD diabetes with a novel peptide of the hsp60 molecule induces Th2-type antibodies.

A peptide from the sequence of hsp60 molecule, designated p277, has been shown to be functionally involved in modulating the development of auto-immune diabetes in the NOD mouse: administration of p277 to NOD mice can arrest the diabetogenic autoimmune process, even when far advanced. Is p277 the only hsp60 peptide able to modulate the disease? We mapped T cell responses to peptides spanning the mouse hsp60 molecule and identified an immunogenic peptide, designated p12, that is also functional in arresting NOD diabetes. Although no spontaneous T cell reactivity to p12 could be detected in NOD mice, subcutaneous administration of 100 microg of p12 in mineral oil to 10-week-old female NOD mice, similar to treatment with p277, significantly prevented progression of the disease. Administration of other immunogenic peptides was not effective. A peptide from the glutamic acid decarboxylase (GAD65) sequence, GADp35, and a peptide from the myco-bacterial hsp60 molecule did not influence the development of diabetes. The effectiveness of hsp60 peptides p12 and p277 was associated with the induction of antibodies to the peptides of the IgG1 and IgG2b isotypes, antibodies which appear to be regulated by anti-inflammatory cytokines. There was a negative correlation between the amounts of antibodies induced by the hsp60 peptides and the level of blood glucose. Thus, more than one peptide of the hsp60 molecule can be used to inhibit the development of NOD diabetes, and the effect of peptide therapy appears to be associated with the induction of specific antibody isotypes.

Molecular characterization of the diabetes-associated mouse MHC class II protein, I-Ag7.

The MHC class II molecule of the non-obese diabetic (NOD) mice, I-Ag7, is associated with susceptibility to autoimmune diabetes. To try to understand the molecular basis of this association, we analyzed the peptide binding properties and intracellular behavior of I-Ag7 in comparison with other I-A haplotypes. We found that I-Ag7 molecules manifested normal intracellular trafficking and lifespan, and a small but clearly detectable fraction of I-Ag7 in the cells formed SDS-resistant compact dimers. The binding of an antigenic reference peptide to I-Ag7 was stable and was accompanied by compact dimer formation. Our analysis of the binding specificity of I-Ag7 revealed a peptide binding motif of nine amino acids with a degenerate position at P1 and three conserved anchor positions: P4, P6 and P9. An allele-specific preference for negatively charged residues was found at P9, apparently due to the presence of the rare Ser residue at position 57 of the I-Ag7 beta chain. These findings could have implications for the mechanisms of MHC-mediated susceptibility to autoimmune diabetes in the NOD mice.