Linkage isomerism in nitrite reduction by cytochrome cd1 nitrite reductase

Nitrite reduction by cytochrome cd(1) nitrite reductase (cd(1)NIR) is currently accepted to involve coordination of the nitrite nitrogen atom to the ferrous d(1) heme. Here, density functional theory results are reported on the previously unexplored O-binding of nitrite to ferrous and ferric cd(1)NIR. Although the N-isomer (nitro) is energetically favored over the O-nitrite (nitrito), even one single strong hydrogen bond may provide the energy required to put the two isomers on level terms. When hydrogen bonding existent at the cd(1)NIR active site is accounted for in the computational model, the O-nitrite isomer is found to spontaneously protonate and thus yield a ferric-hydroxo species, liberating nitric oxide. An O-nitrite ferrous cd(1)NIR complex appears to be an energetically feasible intermediate for nitrite reduction. O-Coordination would offer an advantage since the end product of nitrite reduction would be a ferric-hydroxo/water complex, rather than the more kinetically inert iron-nitrosyl complex implied by the N-nitrite mechanism.     

The Effect of Calcium-Silicate Cements on Reparative Dentinogenesis Following Direct Pulp Capping on Animal Models

Dental pulp vitality is a desideratum for preserving the health and functionality of the tooth. In certain clinical situations that lead to pulp exposure, bioactive agents are used in direct pulp-capping procedures to stimulate the dentin-pulp complex and activate reparative dentinogenesis. Hydraulic calcium-silicate cements, derived from Portland cement, can induce the formation of a new dentin bridge at the interface between the biomaterial and the dental pulp. Odontoblasts are molecularly activated, and, if necessary, undifferentiated stem cells in the dental pulp can differentiate into odontoblasts. An extensive review of literature was conducted on MedLine/PubMed database to evaluate the histological outcomes of direct pulp capping with hydraulic calcium-silicate cements performed on animal models. Overall, irrespective of their physico-chemical properties and the molecular mechanisms involved in pulp healing, the effects of cements on tertiary dentin formation and pulp vitality preservation were positive. Histological examinations showed different degrees of dental pulp inflammatory response and complete/incomplete dentin bridge formation during the pulp healing process at different follow-up periods. Calcium silicate materials have the ability to induce reparative dentinogenesis when applied over exposed pulps, with different behaviors, as related to the animal model used, pulpal inflammatory responses, and quality of dentin bridges.     

Assessment of role of rosmarinic acid in preventing oxidative process of low density lipoproteins

This study aimed to assess the antioxidant compound effects on oxidisable substrates, using an effective bio-mimetic system based on human low density lipoproteins (LDL). Thermally generated radicals induce LDL oxidative changes to be identified and quantified. The bio-mimetic system thus developed responded linearly to radicals?? concentration over a range of 10^?6-10^?5 mol L^?1. Cu²⁺ accentuates lipoperoxidation but, when rosmarinic acid was present, Cu²⁺ produced an unexpected effect, i.e. increased antioxidant efficiency against lipoperoxidation. Rosmarinic acid inhibits production of lipoperoxides by up to 30 % in the absence of Cu²⁺ and up to 70 % in its presence when the rosmarinic acid-to-Cu molar ratio is 1: 1.     

Anisotropy resolved multidimensional emission spectroscopy (ARMES): A new tool for protein analysis

Structural analysis of proteins using the emission of intrinsic fluorophores is complicated by spectral overlap. Anisotropy resolved multidimensional emission spectroscopy (ARMES) overcame the overlap problem by the use of anisotropy, with chemometric analysis, to better resolve emission from different fluorophores. Total synchronous fluorescence scan (TSFS) provided information about all the fluorophores that contributed to emission while anisotropy provided information about the environment of each fluorophore. Here the utility of ARMES was demonstrated via study of the chemical and thermal denaturation of human serum albumin (HSA).     

Structural and dynamical characterization of poly-gamma-glutamic acid-based cross-linked nanoparticles

This work describes the formation of water-soluble hydrophilic nanoparticles from biosynthetic poly-γ-glutamic acid (PGA). Nanoparticles were formed by cross-linking using 2,2′-(ethylenedioxy) diethylamine in the presence of water-soluble carbodiimide. The structure was determined by nuclear magnetic resonance spectroscopy and the particle size by transmission electron microscopy (TEM), size exclusion chromatography (SEC), and dynamic light-scattering (DLS) measurements. The results from TEM, SEC, and DLS reveal that the particle size depends on the ratio of cross-linking. Particle size values measured by TEM were between 20 and 90 nm. Formation of cross-linked nanoparticles results in a dramatic viscosity drop compared to the viscosity of the corresponding solution of the parent PGA. The viscosity and DLS experiments disclose an intriguing interplay between intrachain and interchain cross-linking of the polymer chains, depending on the cross-linker density and polymer concentration. The SEC measurements show that the retention time of the major portion of particles increase because of the higher cross-linking ratio. At moderate cross-linker concentration, intramolecular cross-linking is the dominant process, whereas at higher cross-linker densities, the interpolymer cross-linking plays an important role. As a result, large clusters are also formed.     

Hydrolysis-condensation of tetraethoxysilane in nonparental solvents studied by GC-MS

Gas-chromatography coupled with mass spectrometry was used to investigate the hydrolysis and condensation of tetra-ethoxysilane (TEOS) in nonparental solvents (MeOH and nPrOH). In the presence of nonparental solvents, the reaction that advanced at highest rate is alkoxy group scrambling, that changes the precursor at the molecular level. Subsequent stages of hydrolysis-polycondensation process are essentially determined by the reactivity of the new molecular species formed.     

Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for Cdc7 kinase

Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [(33)P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [(33)P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.