Descripción: The effect of pore size to H2O2 detection by macroporous and mesoporous Prussian blue type electrocatalysts is reported in the present paper. The macroporous electrocatalysts were prepared employing spherical colloidal particles of different sizes (300, 460, 600 and 800 nm) as sacrificial templates to synthesize a copper hexacyanoferrate/polypyrrole (CuHCNFe/Ppy) hybrid material. Surprisingly, macroporous and non-porous CuHCNFe/Ppy displayed very similar results, which led to a discussion that application of macroporous platforms in sensors must consider the material wettability and the influence of electrochemical kinetics on analyte detection. In order to evaluate the effect of smaller pores, the performance of the macroporous H2O2 sensors was also compared to electrocatalysts synthesised through the immobilization of Prussian blue and CuHCNFe layers inside the cavities of mesoporous TiO2 films with diameters of 13, 20 and 40 nm. In this scale, the results were superior than those achieved with the non-porous sensors, demonstrating the possibility of controlling the performance of H2O2 sensors according to the pore diameter and the amount of immobilized material. Among the tested porous materials, the H2O2 sensor with better performance was achieved using the 20-nm diameter TiO2 platform functionalized with Prussian blue, which presented a sensitivity of (930 ± 50) μA cm-2 mmol-1 L, detection limit of (0.49 ± 0.08) μmol L-1, response time of (6 ± 2) seconds and linear range up to (1.3 ± 0.1) mmol L-1. This performance was extremely satisfactory considering sensors operating by chronoamperometry. © 2013 Elsevier Ltd. All rights reserved.

Descripción: The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol-1), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol-1 from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: By taking advantage of the extreme stability of a protein-DNA complex, we have obtained two highly specific monoclonal antibodies against a predetermined palindromic DNA sequence corresponding to the binding site of the E2 transcriptional regulator of the human papillomavirus (HPV-16). The purified univalent antibody fragments bind to a double-stranded DNA oligonucleotide corresponding to the E2 binding site in solution with dissociation constants in the low and subnanomolar range. This affinity matches that of the natural DNA binding domain and is severalfold higher than the affinity of a homologous bovine E2 C-terminal domain (BPV-1) for the same DNA. These antibodies discriminate effectively among a number of double- and single-stranded synthetic DNAs with factors ranging from 125-to 20,000-fold the dissociation constant of the specific DNA sequence used in the immunogenic protein-DNA complex. Moreover, they are capable of fine specificity tuning, since they both bind less tightly to another HPV-16 E2 binding site, differing in only 1 base pair in a noncontact flexible region. Beyond the relevance of obtaining a specific anti-DNA response, these results provide a first glance at how DNA as an antigen is recognized specifically by an antibody. The accuracy of the spectroscopic method used for the binding analysis suggests that a detailed mechanistic analysis is attainable.

Descripción: Although transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-α (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3′,5-triiodo-L-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O2 uptake. In this context, NO utilization increased active O2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor Nω-nitro-L-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.