An assessment of the transport mechanism and intraneuronal stability of L-carnosine.

L-Carnosine (ß-alanyl-L-histidine) is a well-known antioxidant and neuroprotector in various models on animals and cell cultures. However, while there is a plethora of data demonstrating its efficiency as a neuroprotector, there is a distinct lack of data regarding the mechanism of its take up by neurons. According to literature, cultures of rat astrocytes, SKPT cells and rat choroid plexus epithelial cells take up carnosine via the H+-coupled PEPT2 membrane transporter. We've assessed the effectiveness and mechanism of carnosine transport, and its stability in primary rat cortical culture neurons. We demonstrated that neurons take up carnosine via active transport with Km = 119 µM and a maximum velocity of 0.289 nmol/mg (prot)/min. Passive transport speed constituted 0.21∙10-4 nmol/mg (prot)/min (with 119 µM concentration in the medium)-significantly less than active transport speed. However, carnosine concentrations over 12.5 mM led to passive transport speed becoming greater than active transport speed. Using PEPT2 inhibitor zofenopril, we demonstrated that PEPT2-dependent transport is one of the main modes of carnosine take up by neurons. Our experiments demonstrated that incubation with carnosine does not affect PEPT2 amount present in culture. At the same time, after removing carnosine from the medium, its elimination speed by culture cells reached 0.035 nmol/mg (prot)/min, which led to a decrease in carnosine quantity to control levels in culture within 1 h. Thus, carnosine is taken up by neurons with an effectiveness comparable to that of other PEPT2 substrates, but its elimination rate suggests that for effective use as a neuroprotector it's necessary to either maintain a high concentration in brain tissue, or increase the effectiveness of glial cell synthesis of endogenous carnosine and its shuttling into neurons, or use more stable chemical modifications of carnosine.

Deuterated Arachidonic Acids Library for Regulation of Inflammation and Controlled Synthesis of Eicosanoids: An In Vitro Study.

The synthesis of signal lipids, including eicosanoids, is not fully understood, although it is key to the modulation of various inflammatory states. Recently, isotopologues of essential polyunsaturated fatty acids (PUFAs) deuterated at bis-allylic positions (D-PUFAs) have been proposed as inhibitors of non-enzymatic lipid peroxidation (LPO) in various disease models. Arachidonic acid (AA, 20:4 n-6) is the main precursor to several classes of eicosanoids, which are produced by cyclooxygenases (COX) and lipoxygenases (LOX). In this study we analyzed the relative activity of human recombinant enzymes COX-2, 5-LOX, and 15-LOX-2 using a library of arachidonic acids variably deuterated at the bis-allylic (C7, C10, and C13) positions. Kinetic parameters (KM, Vmax) and isotope effects calculated from kH/kD for seven deuterated arachidonic acid derivatives were obtained. Spectroscopic methods have shown that deuteration at the 13th position dramatically affects the kinetic parameters of COX-2 and 15-LOX-2. The activity of 5-LOX was evaluated by measuring hydroxyeicosatetraenoic acids (8-HETE and 5-HETE) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Deuteration at the seventh and 10th positions affects the performance of the 5-LOX enzyme. A flowchart is proposed suggesting how to modulate the synthesis of selected eicosanoids using the library of deuterated isotopologues to potentially fine-tune various inflammation stages.

Physicochemical and functional properties of Cucurbita maxima pumpkin pectin and commercial citrus and apple pectins: A comparative evaluation.

The physicochemical characteristics and functional properties of pumpkin (Cucurbita maxima D. var. Cabello de Ángel) pectin obtained by cavitation facilitated extraction from pumpkin pulp have been evaluated and compared with commercial citrus and apple pectins. C. maxima pectin had an Mw value of 90 kDa and a high degree (72%) of esterification. The cytoprotective and antioxidant effects of citrus, apple and pumpkin pectin samples with different concentrations were studied in vitro in cell lines HT-29 (human colon adenocarcinoma) and MDCK1 (canine kidney epithelium). All pectin samples exhibited cytoprotective effect in HT-29 and MDCK1 cells after incubation with toxic concentrations of cadmium and mercury for 4 h. Pumpkin pectin increased the proliferation of cadmium-treated MDCK1 cells by 210%. The studied pectins also inhibited oxidative stress induced by 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) in cell cultures, as determined by measuring the production of intracellular reactive species using dihydrochlorofluorescein diacetate (DCFH-DA). Pectin from pumpkin pomace had the highest (p < 0.05) protective effect against reactive oxygen species generation in MDCK1 cells induced by AAPH. Distinctive features of pumpkin pectin were highly branched RG-I regions, the presence of RG-II regions and the highest galacturonic acid content among the studied samples of pectins. This correlates with a considerable protective effect of C. maxima pectin against oxidative stress and cytotoxicity induced by heavy metal ions. Thus, C. maxima pectin can be considered as a source of new functional foods of agricultural origin.

Prostaglandin H synthase kinetics in the two-phase aqueous-micellar system.

Reaction mixture for PGHS (prostaglandin-H-synthase) is a two-phase system including micellar hydrophobic phase and hydrophilic aqueous phase. Reagents added to the mixture are distributed between phases, thus concentrations of reagents dissolved in phases can differ significantly from their overall contents. Using dynamic light scattering we found that the hydrophobic phase produced by tween-20 consists of micelles, which radius (4-5nm) does not depend on either tween-20 overall content (0.1%-1% v/v) or arachidonic acid (AA) addition (10-1000µM) or PGHS addition (1µM). Tween-20 overall content changing from 0.1% to 2% v/v dramatically affected COX kinetic, but accounting AA distribution between phases allowed us to estimate "true" parameters, independent of the tween-20 overall content and the concentration of another substrate: KM(Ox) equals 9.8µM O2 in the aqueous phase or 0.0074bar in the gaseous phase, KM(AA) equals 5400µM AA in the phase of tween-20 micelles and 5400/PµM AA in the aqueous phase (P is the distribution ratio for the AA between the aqueous phase and the hydrophobic phase (P≫1000)). This approach allowed to evaluate PS, the distribution ratio for the AA between the hydrophobic phase and the PGHS active center (PS ~310). This coefficient indicates the AA selectivity toward the cyclooxygenase active center.