The folding of ␤ 2 -microglobulin (␤ 2 -m), the protein forming amyloid deposits in dialysis-related amyloidosis, involves formation of a partially folded conformation named I 2 , which slowly converts into the native fold, N. Here we show that the partially folded species I 2 can be separated from N by capillary electrophoresis. Data obtained with this technique and analysis of kinetic data obtained with intrinsic fluorescence indicate that the I 2 conformation is populated to ϳ14 ؎ 8% at equilibrium under conditions of pH and temperature close to physiological. In the presence of fibrils extracted from patients, the I 2 conformer has a 5-fold higher propensity to aggregate than N, as indicated by the thioflavine T test and light scattering measurements. A mechanism of aggregation of ␤ 2 -m in vivo involving the association of the preformed fibrils with the fraction of I 2 existing at equilibrium is proposed from these results. The possibility of isolating and quantifying a partially folded conformer of ␤ 2 -m involved in the amyloidogenesis process provides new opportunities to monitor hemodialytic procedures aimed at the reduction of such species from the pool of circulating ␤ 2 -m but also to design new pharmaceutical approaches that consider such species as a putative molecular target.Dialysis-related amyloidosis represents an inevitable and severe complication of long term hemodialysis (1-4). Under this pathological condition, protein aggregates known as amyloid fibrils, accumulate in essential tissues, such as the skeletal muscle, interfering with their normal functions. A major constituent of the amyloid fibrils related to this pathological condition is ␤ 2 -microglobulin (␤ 2 -m).1 In its native form, this 99-residue protein is constituted by two ␤-sheets packed against each other to form a fold typical of the immunoglobulin superfamily (5). The two ␤-sheets, constituted by three and four strands, respectively, interact by means of hydrophobic interactions and a disulfide bridge that stabilizes further the ␤-sandwich structure.␤ 2 -m constitutes the light chain of the major histocompatibility complex class I (MHCI). A significant pool of ␤ 2 -m is also normally present in the plasma as a consequence of the constant release from the MHCI to allow the process of catabolic degradation in the kidney (1, 2). In chronic dialysis patients, the artificial membrane induces an inflammatory reaction, which causes the production and release of ␤ 2 -m to increase significantly (6). In addition, ␤ 2 -m cannot be filtered efficiently through the artificial membrane, resulting in an increase of the levels of soluble ␤ 2 -m from 0.3 to 30 g/ml, the range of concentrations observed within healthy individuals, to ϳ40 g/ml (7). The increase of free circulating ␤ 2 -m, the preferential substrate for amyloid deposition by this protein, is responsible, at least in part, for this form of amyloidosis in these patients (1, 2). Big efforts have been expended to improve the biocompatibility and performance of dialysis approaches. ...

Hyperphosphorylation at tyrosine is commonly observed in tumor proteomes and, hence, specific phosphoproteins or phosphopeptides could serve as markers useful for cancer diagnostics and therapeutics. The analysis of such targets is, however, a challenging task, because of their commonly low abundance and the lack of robust and effective preconcentration techniques. As a robust alternative to the commonly used immunoaffinity techniques that rely on phosphotyrosine(pTyr)-specific antibodies, we have developed an epitope-imprinting strategy that leads to a synthetic pTyr-selective imprinted polymer receptor. The binding site incorporates two monourea ligands placed by preorganization around a pTyr dianion template. The tight binding site displayed good binding affinities for the pTyr template, in the range of that observed for corresponding antibodies, and a clear preference for pTyr over phosphoserine (pSer). In further analogy to the antibodies, the imprinted polymer was capable of capturing short tyrosine phosphorylated peptides in the presence of an excess of their non-phosphorylated counterparts or peptides phosphorylated at serine.

The possibility to monitor, in solution, the steps of beta-amyloid (Abeta) nucleation and therefore to describe this dynamic process by using capillary electrophoresis and under optimized experimental conditions is described. Striking differences in the electrophoretic patterns of Abeta 1-42 and Abeta 1-40 over time are here shown, and different aggregation states are elucidated, which reflect the very diverse oligomerization behavior of two very similar peptides. The isolation of one aggregated species of high molecular weight by ultracentrifugation allowed us to assess its role as toxic oligomer. The perturbation of the existing equilibrium among the identified species by the addition of small molecules can in principle interfere with the aggregation process of the peptides and ultimately prevent the plaque formation in vitro.

A series of urea-based vinyl monomers was synthesized and investigated for their ability to function as polymerizable hosts for the molecular imprinting of N-Z-D- or L-glutamic acid in polar media (DMSO or DMF). The monomers were synthesized in one step from a polymerizable isocyanate and a nonpolymerizable amine or vice versa, with yields typically over 70%. Prior to polymerization their solution binding properties vis-a-vis tetrabutylammonium benzoate in DMSO were investigated by 1H NMR, UV-vis and fluorescence monitored titrations. The affinities of the urea monomers for benzoate depended upon the substitution pattern of the urea, with all diaryl ureas exhibiting high affinity. EDMA-based imprinted polymers prepared in DMF or DMSO against Z-D-(or L)-glutamic acid using 2 equiv of the urea monomer and 2 equiv of base were able to recognize the imprinted dianion as well as larger molecules containing the glutamic acid substructure. The affinity, reflected in liquid chromatography retention data, correlated with the solution binding properties of the corresponding monomers.

Two approaches to synthesize molecularly imprinted polymers with affinity for folic acid and other substituted pteridines have been compared. In the first approach, the folic acid analogue methotrexate was used as template and functional monomers capable of generating selective binding sites were searched in a miniaturized screening system based on binding assessment in the batch mode. Highest selectivity was seen using 2-vinylpyridine as functional monomer, which was confirmed in the chromatographic mode for a batch synthesized on a gram scale. However, the retentivity and selectivity of this phase were insufficient for anticipated applications. In a second approach, using methacrylic acid as the functional monomer, organic soluble inhibitors for the enzyme dihydrofolate reductase were used to develop sites complementary toward the pteridine substructure. This resulted in materials showing enhanced selectivity for substituted pteridines when evaluated by HPLC. Thus, methotrexate and leucovorine were selectively retained in mobile phases of either low or high aqueous content, thus showing the typical bimodal retention behavior of previously reported MIPs. In organic mobile-phase systems, the inhibitor used as template had an influence on the retentivity and selectivity of the MIP. The polymer imprinted with trimethoprim retained all folic acid analogues strongly and showed the highest selectivity among the MIPs in an organic mobile-phase system. This was supported by Scatchard analysis resulting in biphasic plots and a quantitative yield of high-energy binding sites. All templates were shown to associate strongly with MAA in CDCl(3), the strength of association correlating roughly with the template basicity and the selectivity observed in chromatography. Nonparallel complexation-induced shifts indicated formation of 1:2 template monomer complexes at concentrations corresponding to those of the prepolymerization solutions.

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