Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of ...2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10(-5)), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.
This short review highlights recent results on the use of nanoclusters of gold protected by a monolayer of functional ligands in both molecular recognition and catalysis. Particular emphasis is given ...to the multivalent properties of these systems and, in the case of catalysis, to the cooperativity between functional groups present in the ligands of the monolayer. The outstanding catalytic efficiency of some of the functional nanoparticles synthesized led us to call them nanozymes by analogy with the activity of catalytic polymers (synzymes).
New donor-acceptor hybrids of Zn(II)-metallated 5,15-diaryl porphyrins have been designed and synthesised via the porphyrin interactions with an electron acceptor molecule, di-n-hexyl N-substituted ...1,2,4,8-naphthalenetetracarboxylic diimide (NDI). Binding interactions within these supramolecular complexes were investigated in the solid state by synchrotron X-ray diffraction and probed in solution by (1)H NMR spectroscopy. The systematic modulation of the porphyrin π-density was achieved, for the first time as multiple methoxy and fluorine groups were introduced as substituents to the 5,15-diaryls of the porphyrin. For these, the variation of the porphyrin-NDI binding strengths determined by (1)H NMR titrations was shown, using the Swain's type dual parameter approach, to be closely linked with the peripheral substitution pattern of the diaryl porphyrins validated by crystallography. The new 1:1 donor-acceptor complexes formed display characteristic features of the aromatic-stacked systems, i.e. the parallel arrangement and short interplanar separation between the substituted porphyrin and NDI. Synthetic modification of electron-density on the porphyrin surface by introducing substituents at peripheral sites of functionalised porphyrins represent a general solution towards electronically tunable aromatic surfaces: an understanding of their solution and solid state behaviour will significantly improve the rational design of new functional donor-acceptor supramolecular materials with potential applications ranging from new energy materials to dye-sensitised solar cells, photovoltaics and future drug delivery devices.
Context: Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors.
Objective: Validate the use of recombinant neuraminidase expressed in baculovirus ...located on the viral surface capsule to develop a neuraminidase inhibitor screening assay.
Materials and methods: Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2′-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor.
Results: The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments.
Discussion and conclusions: The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.
Circulating immune complexes formed by tumor antigens and immunoglobulin M (IgM) represent a novel class of biomarkers with diagnostic value for early cancer detection. The quantitative analysis of ...these immune complexes is achieved by enzyme-linked immunosorbent assay (ELISA) methods using a purified calibrator from samples of patients with cancer. These complexes obtained from samples of human origin are not suitable for cost-effective production processes with high safety standards. Given the ill-defined biomarker/IgM ratio in these complexes, semisynthesis with retention of functional properties is difficult to achieve and may vary widely according to the batch-to-batch heterogeneity of starting biological preparations. Here the authors describe the development of a combinatorial method for defining the optimal reaction conditions for the reproducible semisynthesis of biomarker-IgM complexes by exploiting the biotin-avidin technology. The method relies on screening by ELISA the 3D composition space defined by the combinatorial variation of biotinylated-biomarker, biotinylated-IgM, and avidin concentrations aiming to select those conditions leading to biomarker-IgM complexes with the highest immunoreactivity. The method allows the reproducible synthesis of species with immunoreactivity comparable to that of natural immune complexes and endowed with sufficient stability to be used as calibrators in ELISA.
Hydrogen-Bonded Helical Organic Nanotubes Pantoş, G. Dan; Pengo, Paolo; Sanders, Jeremy K. M.
Angewandte Chemie (International ed.),
03/2007, Volume:
46, Issue:
13
Journal Article
In a recent series of papers, Miller and co-workers were able to show that His(pi-Me)-based, terminally protected peptides are potent catalysts of the asymmetric acyl transfer reaction, useful for ...the kinetic resolution of alcohols. In a structure-supporting solvent, one of the most active compounds, an Aib-containing tetrapeptide, is folded in a doubly intramolecularly H-bonded beta-hairpin motif incorporating a type-II' beta-turn conformation. In this work, we have expanded the study of the Miller tetrapeptide by examining a set of analogues and shorter sequences (dipeptide amides), characterized by chiral C(alpha)-tetrasubstituted alpha-amino acids of diverging bulkiness and optical configuration. Peptide synthesis in solution, conformational analysis by FT-IR absorption and (1)H NMR techniques, and screening of catalytic activity as well have been performed. Our results confirm the close relationship between the beta-hairpin 3D-structure and the catalytic activity of the peptides. A tetrapeptide analogue slightly more selective than the Miller compound has been found. However, the terminally protected, industrially more appealing, dipeptide amides are poorly effective.
Squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) is a useful biomarker for the risk of development of hepatocellular carcinoma (HCC) in patients with cirrhosis due to its progressive ...increase associated to HCC evolution. In patients with cirrhosis, other assays have been affected by interfering reactivities of IgM. In this study, the analytical specificity of the SCCA-IgM assay was assessed by evaluating SCCA-IgM measurement dependence on different capture phases, and by measuring the recovery of SCCA-IgM reactivity following serum fractionation.
Serum samples from 82 patients with cirrhosis were analyzed. SCCA-IgM was measured using the reference test (Hepa-IC, Xeptagen, Italy) that is based on rabbit oligoclonal anti-squamous cell carcinoma antigen (SCCA) and a dedicated ELISA with a mouse monoclonal anti-SCCA as the capture antibody.
SCCA-IgM concentrations measured with the reference assay (median value=87 AU/mL) were higher than those measured with the mouse monoclonal test (median value=78 AU/mL). However, the differences in the SCCA-IgM distribution were not statistically significant (p>0.05). When SCCA-IgM concentrations measured with both tests were compared, a linear correlation was found (r=0.77, p<0.05). Fractionation of the most reactive sera by gel-filtration chromatography showed that total recovery of SCCA-IgM reactivity was seen only in the fractions corresponding to components with a molecular weight higher than IgM and SCCA (>2000 kDa) with both tests.
The equivalence of both SCCA-IgM assays and the absence of reactivity not related to immune complexes support the analytical specificity of SCCA-IgM measurements. The results validate the assessment of SCCA-IgM for prognostic purposes in patients with cirrhosis.
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