Natural compounds with P2X7 receptor-modulating properties

The adenosine 5′-triphosphate (ATP)-gated P2X7 receptor is a membrane-bound, non-selective cation channel, expressed in a variety of cell types. The P2X7 senses high extracellular ATP concentrations and seems to be implicated in a wide range of cellular functions as well as pathophysiological processes, including immune responses and inflammation, release of gliotransmitters and cytokines, cancer cell growth or development of neurodegenerative diseases. In the present study, we identified natural compounds and analogues that can block or sensitize the ATP (1 mM)-induced Ca²⁺ response using a HEK293 cell line stably expressing human P2X7 and fluorometric imaging plate reader technology. For instance, teniposide potently blocked the human P2X7 at sub-miromolar concentrations, but not human P2X4 or rat P2X2. A marked block of ATP-induced Ca²⁺ entry and Yo-Pro-1 uptake was also observed in human A375 melanoma cells and mouse microglial cells, both expressing P2X7. On the other hand, agelasine (AGL) and garcinolic acid (GA) facilitated the P2X7 response to ATP in all three cell populations. GA also enhanced the YO-PRO-1 uptake, whereas AGL did not affect the ATP-stimulated intracellular accumulation of this dye. According to the pathophysiological role of P2X7 in various diseases, selective modulators may have potential for further development, e.g. as neuroprotective or antineoplastic drugs.     

Angiotensin (1–7) re-establishes heart cell communication previously impaired by cell swelling: Implications for myocardial ischemia

The influence of hypertonic solution on dye coupling was investigated in cell pairs isolated from the left ventricle of adult Sprague Dawley rats.The hypertonic solution together with Lucifer Yellow CH, were dialyzed into one cell of the pair using the whole cell clamp tecnique, and the diffusion of dye in the dialyzed as well as in non-dialyzed cell, was followed by measuring the intensity of fluorescence in both cells as a function of time.The results indicated that: (1) Lucifer Yellow CH dialyzed into one cell of the pair diffuses easily into the nondialyzed cell through gap junctions; (2) the intracellular dialysis of an hypertonic solution into one cell of the pair, increases the area of the dialyzed cell and reduced the area of the non-dialyzed cell suggesting intercellular movement of water; (3) the hypertonic solution dialyzed into one cell of the pair abolished the dye coupling; (4) the gap junction permeability (Pj) estimated before and after administration of hypertonic solution showed an appreciably decrease of Pj; (5) angiotensin (1–7) (Ang (1–7) (10–9 M) administered to the bath re-established the dye coupling abolished by hypertonic solution and reduced the cell area; (6) the effect of Ang (1–7) was related to the activation of Mas receptor and was dependent on the activation of PKA. Conclusions: the reestablishment of dye coupling elicited by Ang (1–7) seen in cell pairs dialyzed with hypertonic solution, might indicate that under similar conditions like that seen during myocardial ischemia, the peptide might be of benefit preventing the impairment of cell communication and impulse propagation associated with cardiac reentrant arrhytmias.     

A review of recent results on electrochemical determination of the density of electronic states of nanostructured metal-oxide semiconductors and organic hole conductors

The paper reviews recent results on electrochemical methods applied to the determination of the density of electronic states in several components of advanced nanostructured devices such as the dye solar cell (Grätzel cell), switchable displays, and sensors. We discuss the application of cyclic voltammetry and impedance spectroscopy to determine the chemical capacitance of mesoporous metal-oxide semiconductors (mainly TiO₂), organic hole transport materials, and molecular functionalizing elements.     

Negatively Charged,Dye Labeled-Dideoxynucleotides for “Direct-Load” DNA Sequencing

Abstract

A four-color set of negatively charged, single dye as well as energy transfer dye labeled-ddNTPs were synthesized and evaluated in combination with a novel polymerase in a “direct-load” DNA sequencing, obviating the laborious and time consuming post-reaction work-up.     

Conformational changes on ligand binding in wild-type and mutants from Spodoptera frugiperda midgut trehalase

Trehalase specifically hydrolyses trehalose into two glucose units and is most important in insects and fungi. Previous evidence suggested that Spodoptera frugiperda midgut trehalase (wild type, WT) has substantial conformational changes on binding different substances. Our goal is to understand this mobility. For this, two deletion mutants were produced, lacking regions supposed to be the cause of mobility [(102 residues from the N-terminus (NT) and this portion plus 31 residues from the C-terminus (NCT)]. Circular dichroism spectra before and after denaturation of the enzymes support the assertion that they are appropriately folded. The overall results show that the removal of 102 or 133 amino acids does not greatly change the interaction with the substrate and competitive inhibitors, but leads to a considerable decrease in kcat/Km values from WT 74,500 M^?1 s^?1 to NT 647 M^?1 s^?1 and NCT 1,044 M^?1 s^?1. Diethyl pyrocarbonate His modification only occurs in wild and truncated trehalases in the presence of some ligands. Looking for changes in folding WT, NT, and NCT were incubated with different compounds in the presence of Sypro Orange, that binds to hydrophobic regions increasing its fluorescence. The dye fluorescence is affected by 2 compounds when WT is present, and at least by 5 compounds when NT or NCT are present, suggesting that conformational changes caused by ligand binding occur only in the vicinity of the active site. These data provide physical evidence in favor of a change in folding around the active site caused by ligand binding, in agreement to prior chemical modification and other kinetic data and challenging the hypothesis that N- and C-terminal are the mobile regions.     

Evaluation of intrinsic spectroscopic properties of chromophore assemblies by shielding with cyclohexyl base pairs within a DNA duplex

Here, we investigated spectroscopic behaviors of tetramethylrhodamine (TMR) homo- and hetero-dimers within DNA duplex. In order to shield the chromophores from natural base pairs, we used cyclohexyl base pairs as ‘insulators’; these pairs were inserted between the chromophores and nucleobases. When a single TMR moiety was sandwiched between cyclohexyl base pairs, the emission intensity increased by fivefold relative to a TMR between natural base pairs, because electron transfer from nucleobases was suppressed. Next, we inserted two TMRs between the cyclohexyl base pairs and found that they facilitated H-dimer formation of TMR; a distinct hypsochromic shift was induced only when cyclohexyl base pairs were inserted. We further examined quenching behavior of a TMR paired with a quencher dye between cyclohexyl base pairs. Interestingly, fluorescence from TMR was quenched by nitro methyl red more efficiently in the presence of cyclohexyl base pairs than in their absence. This suggests that neighboring natural base pairs disturbed electron or hole transfer between the fluorophore and the quencher. The cyclohexyl base pairs shielded the chromophore pair from the natural base pairs and allowed intrinsic electron transfer.     

Enhancement of photovoltaic performance of a novel dye, “T18”, with ketene thioacetal groups as electron donors for high efficiency dye-sensitized solar cells

A new ruthenium polypyridine sensitizer, T18, for Dye-Sensitized Solar Cells (DSSCs) is proposed containing an electron rich ketene thioacetal modified bipyridyl group as an ancillary ligand. The T18 dye, (cis-bis(thiocyanato)(2,2′-bipyridyl-4,4′-dicarboxylato){4,4′-bis(1,3-dithian-2-ylidenemethyl)-2,2′-bipyridine}ruthenium(II) mono-tetrabutylammonium salt), has a red-shifted and larger MLCT absorption coefficient when compared to the current best performing dye, N719 {cis-bis(thiocyanato)bis-(2,2′-bipyridine-4,4′-dicarboxylato) ruthenium(II) bis-tetrabutylammonium salt}. We studied the photovoltaic performance of DSSCs made with T18 and N719 with different electrolytes. The results indicated that T18 surpassed the performance of N719 under some conditions, especially with an iodine-free electrolyte based on the cobalt(III/II) tris(4,4′-di-tert-butyl-2,2′-bipyridyl) complex. The main drawback in DSSCs incorporating T18 is the catalysis of recombination processes involving the injected electrons and the iodine-based electrolyte.     

Toxoplasma gondii: trypsin treatment of trophozoites for enhanced immune cytolysis in the dye test

The Sabin-Feldman dye test for the detection of toxoplasma antibodies has been modified by the use of fresh guinea pig serum and of trypsinized toxoplasma trophozoites. Guinea pig serum at a final concentration as low as 1.0–1.5% has been found sufficient to give satisfactory results. The use of trypsinized parasites has been observed to enhance the sensitivity of the titer. It is suggested that the organisms are coated with a trypsin labile substance which may be responsible for preventing access of antibodies to the parasite cytoplasm. The complement system in the serum may be the only essential accessory factor in this respect.     

SYNTHESIS OF NOVEL PIPERIDINYL LINKER BASED ENERGY TRANSFER TERMINATORS AND THEIR POTENTIAL USE IN DNA SEQUENCING

Synthesis of novel piperidinyl linker based ET cassettes and terminators is described. These novel terminators are evaluated in the DNA sequencing experiments using thermostable DNA polymerase.