A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells^3-5. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells⁶, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies¹. Originally published by Naal et al.¹, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease^7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function². In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε₂₈₀ = 4,200 L/M/cm)¹². This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.     

Resveratrol Binding to Human Serum Albumin

Abstract

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419–426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclo- oxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents μM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure.

Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K_Res = 2.56× 10⁵ M^?1. The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of α-helix from 57% (free HSA) to 62% and a decrease of β-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.     

Stabilization of Helical Peptides by Mixed Spaced Salt Bridges

Abstract

Whether or not surface salt bridges have a strong stabilizing effect on the native structure in proteins remains uncertain. Previous studies of model peptides have shown that salt bridges spaced at i,i+4 along the chain are more stabilizing than those spaced at i,i+3, with a preference for the order acid-base rather than base-acid from N to C terminus. An analysis of the effect of spacing the ion pairs in short helical peptides is presented, in which acidic and basic side chains spaced two or three residues apart alternate along the chain. The mixed spacing proves to be stabilizing relative to pure spacings. A control peptide in which salt bridges were spaced uniformly three residues apart proved to form a β-sheet structure rather than a-helix. This is due to formation of a silk-like apolar face consisting of alanine side chains; the mesoscopic structure formed by these sheets can be imaged by scanning microscopy.     

Heme coordination states of unfolded ferrous cytochrome C

The structural changes of ferrous Cyt-c that are induced by binding to SDS micelles, phospholipid vesicles, DeTAB, and GuHCl as well as by high temperatures and changes in the pH have been studied by RR and UV-Vis absorption spectroscopies. Four species have been identified in which the native methionine-80 ligand is removed from the heme iron. This coordination site is either occupied by a histidine (His-33 or His-26) to form a 6cLS configuration, which is the prevailing species in GuHCl at pH 7.0 and ambient temperature, or remains vacant to yield a 5cHS configuration. The three identified 5cHS species differ with respect to the hydrogen-bond interactions of the proximal histidine ligand (His-18) and include a nonhydrogen-bonded, a hydrogen-bonded, and a deprotonated imidazole ring. These structural motifs have been found irrespective of the unfolding conditions used. An unambiguous spectroscopic distinction of these 5cHS species is possible on the basis of the Fe-N(imidazole) stretching vibrations, the RR bands in the region between 1300 and 1650 cm(-1), and the electronic transitions in the Soret- and Q-band regions. In acid and neutral solutions, the species with a hydrogen-bonded and a nonhydrogen-bonded His-18 prevail, whereas in alkaline solutions a configuration with a deprotonated His-18 ligand is also observed. Upon lowering the pH or increasing the temperature in GuHCl solutions, the structure on the proximal side of the heme is perturbed, resulting in a loss of the hydrogen-bond interactions of the His-18 ligand. Conversely, the hydrogen-bonded His-18 of ferrous Cyt-c is stabilized by electrostatic interactions which increase in strength from phospholipid vesicles to SDS micelles. The results here suggest that unfolding of Cyt-c is initiated by the rupture of the Fe-Met-80 bond and structural reorganizations on the distal side of the heme pocket, whereas the proximal part is only affected in a later stage of the denaturation process.     

Anthocyanin from strawberry (Fragaria ananassa) with the novel aglycone, 5-carboxypyranopelargonidin

An anthocyanin, 1, with the novel 4-substituted aglycone, 5-carboxypyranopelargonidin, was isolated in small amounts from the acidified, methanolic extract of strawberries, Fragaria ananassa Duch., by preparative HPLC after purification by partition against ethyl acetate, Amberlite XAD-7 and Sephadex LH-20 column chromatography. It was identified mainly by 2D NMR spectroscopy and electrospray LC-MS as the 3-O-beta-glucopyranoside of 5-carboxy-2-(4-hydroxyphenyl)-3,8-dihydroxy-pyrano[4,3,2-de]-1-benzopyrylium, an anthocyanidin which is homologous to 5-carboxypyranomalvidin (vitisidin A) reported in red wines and 5-carboxypyranocyanidin recently isolated from red onions. By comparison of UV-Vis absorption spectra, 1 showed in contrast to 2, pelargonidin 3-O-beta-glucopyranoside, a local absorption peak around 360 nm, a hypsochromic shift (8 nm) of the visible absorption maximum, and lack of a distinct UV absorption peak around 280 nm. The similarities between the absorption spectra of 1 in various acidic and neutral buffer solutions implied restricted formation of the instable colourless equilibrium forms, which are typical for most anthocyanins in weakly acidic solutions. The molar absorptivity (epsilon) of 1 varied little with pH contrary to similar values of for instance the major anthocyanin in strawberry, 2. However, 2 revealed higher epsilon-values than 1 at all pH values except 5.1. At pH 5.1, the epsilon-value of 1 (6250) was nearly four times the corresponding value of 2 (1720), which showed the potential of 5-carboxypyranopelargonidin derivatives as colorants in solutions with pH around 5. The colours of 1 and 2 in buffered solutions with pH 1.1 and pH 6.9 have been described by the CIELAB coordinates h(ab) (hue angle), C* (chroma), and L* (lightness).     

1,4-dihydropyridines: reactivity of nitrosoaryl and nitroaryl derivatives with alkylperoxyl radicals and ABTS radical cation

In the present paper, a direct quenching of radical species by a number of synthesized nitrosoaryl 1,4-dihydropyridines and their parent nitroaryl 1,4-dihydropyridines was determined in aqueous media at pH 7.4. These two series of compounds were compared with the C-4 unsubstituted 1,4-dihydropyridines derivatives and the corresponding C-4 aryl substituted 1,4-dihydropyridines derivatives. Kinetic rate constants were assessed by UV-Vis spectroscopy. Nitrosoaryl derivatives were more reactive than the parent nitroaryl 1,4-dihydropyridines.

Our results strongly support the assumption that the reactivity between the synthesized 1,4-dihydropyridines derivatives with alkylperoxyl radicals involves electron transfer reactions, which is documented by the presence of pyridine as final product of reaction and the complete oxidation of the nitroso group to give rise the nitro group in the case of the nitrosoaryl 1,4-dihydropyridines derivatives.     

Anthocyanin 3-galactosides from Cornus alba 'Sibirica' with glucosidation of the B-ring

The three anthocyanins, delphinidin 3-O-beta-galactopyranoside-3',5'-di-O-beta-glucopyranoside (1), delphinidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (2) and cyanidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (3), and the 3-O-beta-galactopyranosides of delphinidin (4) and cyanidin (5) were isolated from the bluish white berries and compound umbel of Siberian dogwood, Cornus alba 'Sibirica'. The ornamental autumn leaves and the characteristic purplish red bark of this variety were found to contain only pigment 5.     

An improved synthesis, crystal structures, and metallochromism of salts of [Ru(tolyl-terpy)(CN)3]

The previously reported complex [Ru(ttpy)(CN)₃]⁻ [ttpy = 4′(p-tolyl)-2,2′:6′,2″-terpyridine] is conveniently synthesised by reaction of ttpy with Ru(dmso)₄Cl₂ to give [Ru(ttpy)(dmso)Cl₂], which reacts in turn with KCN in aqueous ethanol to afford [Ru(ttpy)(CN)₃]⁻ which was isolated and crystallographically characterised as both its (PPN)⁺ and K⁺ salts. The K⁺ salt contains clusters containing three complex anions and three K⁺ cations connected by end-on and side-on cyanide ligation to the K⁺ ions. The solution speciation behaviour of [Ru(ttpy)(CN)₃]⁻ was investigated with both Zn²⁺ and K⁺ salts in MeCN, a solvent sufficiently non-competitive to allow the added metal cations to associate with the complex anion via the externally-directed cyanide lone pairs. UV-Vis spectroscopic titration of (PPN)[Ru(ttpy)(CN)₃] with Zn(ClO₄)₂ showed a blue shift of 2900 cm⁻¹ in the ¹MLCT absorption manifold due to the ‘metallochromism’ effect; a series of distinct binding events could be discerned corresponding to formation of 4:1, 1:1 and then 1:3 anion:cation adducts, all with high formation constants, as the titration proceeded. In contrast titration of (PPN)[Ru(ttpy)(CN)₃] with the more weakly Lewis-acidic KPF₆ resulted in a much smaller blue-shift of the ¹MLCT absorptions, and the titration data corresponded to formation of 1:1 and then 2:1 cation:anion adducts with weaker stepwise association constants of the order of 10⁴ and then 10³ M⁻¹. Although association of [Ru(ttpy)(CN)₃]⁻ resulted in a blue-shift of the ¹MLCT absorptions, the luminescence was steadily quenched, as raising the ³MLCT level makes radiationless decay via a low-lying ³MC state possible.     

Structural and spectroscopic characterisation of the holmium double-decker partially oxidised and bi-radical phthalocyanine complexes with iodine

Two crystals of holmium(III) double-decker iodine doped phthalocyanines, HoPc₂I_5/3 (I) and HoPc₂I (II), were grown directly in the reaction of holmium chips with 1,2-dicyanobenzene under versatile quantity of iodine at 180-160 °C. The complex I crystallises in the P4/mcc space group of tetragonal system, while the complex II crystallises in the P2/c space group of monoclinic system. The space group of P4/mcc and z = 1 requires that the Ho(III) atom is statistically disordered in the HoPc₂I_5/3 structure. The iodine atoms form linear symmetrical triiodide ions in I, while the I⁻ ions in II. Assignment of iodine species as in the HoPc₂I_5/3 and I⁻ in HoPc₂I complexes point to the +5/9 and +1 oxidation state of the HoPc₂ unit in these complexes. Thus one Pc macrocycle of the double-decker HoPc₂ units has a non-integer oxidation state of −1.222 in I, while both Pc-rings are one-electron oxidised radical Pc⁻ in II. Magnetic susceptibilities of HoPc₂I_5/3 and HoPcI at room temperature are 4.56 × 10⁻² and 5.12 × 10⁻² emu/mol and the calculated magnetic moments are 10.46 and 11.08 μ_B, respectively. UV-Vis spectroscopic measurement of I and II in benzene solution were carried out and discussed.     

Study on structure-property-reactivity-function relationship of human neuronal growth inhibitory factor (hGIF)

Human metallothionein-3 (hMT3), also named as human neuronal growth inhibitory factor (hGIF), can inhibit the outgrowth of embryonic cortical neurons in the presence of brain extracts. In order to systematically study the structure-property-reactivity-function relationship of hGIF, our laboratory designed a series of mutants and studied their structure, property, reactivity and functions by a series of chemical and biological tools including UV spectroscopy, CD spectroscopy, NMR, chemical reaction and primary neuronal culture assays. In summary, we concluded that the bioactivity of hGIF was regulated by multiple factors, including the ⁶CPCP⁹ motif, an additional threonine insert at sequence position 5, domain-domain interactions, the structure and stability of the metal-thiolate cluster and the linker. Our studies provide more and more evidences which revealed that the bioactivity of hGIF is mainly related to the essential metal release and its characteristic conformation.