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The Carnitine Palmitoyltranferase I (CPT1) catalyzes the rate-limiting step of long-chain fatty acid (LCFA) mitochondrial β-oxidation. The enzyme promotes the conjugation of LCFA with l-carnitine, which allows LCFA to enter the mitochondria matrix. The structural features involved in CPT1 and LCFA-CoA interactions have not been fully elucidated, mainly due to the absence of CPT1 crystallographic data. Previous studies reported important residues (Lys556, Lys560, and Lys561) crucial to the CPT1 mechanism. Nonetheless, these studies have not explored the LCFA bindings. Using molecular modeling strategies, we aimed to understand the conformational changes in CPT1 structure induced by LCFA-CoA. For this purpose, a tridimensional CPT1A model was built by homology modeling using CRAT protein (PBD:1t7q, resolution 1.8 Å) as a template. We simulated the CPT1 structure in the presence and absence of LCFA-CoA by molecular dynamics (MD). By applying a principal component analysis (PCA), two states of apostructure CPT1 based on CoA-Loop (688-711) were observed. In contrast, just one state was evidenced along with smaller conformational subspaces in ligand-complexed simulations using LCFA-CoA. The CoA moiety of ligands interacts with charged residues, namely Lys560, Lys556, Arg563, and Arg645. The frequency of interactions observed for each of these residues is60% of simulation time, suggesting a dynamic profile of interactions in synergy with long-chain carbon interactions over α-I (478-492). Collectively, these features may be associated with the catalytic conformation of LCFA-CoA to CPT1a. Further calculations of free-energy for different fatty acids, such as alpha-linolenic (ALA), gamma-linolenic (GLA), and arachidonic (ARA) acids, yielded energy values ranging from -76.9 ± 15.9 to -68.5 ± 10.0 kcal mol

Two commercial exogenous pulmonary surfactants, Curosurf and Survanta, are investigated. Their thermotropic behavior and associated structural changes for the samples in bulk are characterized and described. For Survanta, the obtained results of differential scanning calorimetry showed a thermogram with three peaks on heating and only a single peak on cooling. Curosurf on the other hand, presents calorimetric thermograms with only one peak in both the heating and cooling scans. This distinct thermotropic behavior between the two pulmonary surfactants, a consequence of their particular compositions, is associated with structural changes that were evaluated by simultaneous small- and wide-angle X-ray scattering experiments with in situ temperature variation. Interestingly, for temperatures below ∼35 °C for Curosurf and ∼53 °C for Survanta, the scattering data indicated the coexistence of two lamellar phases with different carbon chain organizations. For temperatures above these limits, the coexistence of phases disappears, giving rise to a fluid phase in both pulmonary surfactants, with multilamelar vesicles for Curosurf and unilamellar vesicles for Survanta. This process is quasi-reversible under cooling, and advanced data analysis for the scattering data indicated differences in the structural and elastic properties of the pulmonary surfactants. The detailed and systematic investigation shown in this work expands on the knowledge of the structure and thermodynamic behavior of Curosurf and Survanta, being relevant from both physiological and biophysical perspectives and also providing a basis for further studies on other types of pulmonary surfactants.

The Commelina erecta L. (C. erecta) also known as erva-de-santa-luzia is reported by local population to have medical properties against some pathological conditions. In this study, two extracts of C. erecta leaves (aqueous and ethanolic) were phytochemically analysed and evaluated for their in-vitro antioxidant activities by DPPH, TBARS, NO assays and cell viability assays. The ultra-high performance liquid chromatography followed by tandem mass spectrometry analysis showed the presence of rutin and caffeic acid in aqueous and ethanolic extract. The total polyphenols in aqueous and ethanolic extracts found were 142.7 ± 3.0 and 123.1 ± 5.8 μg/mL of GAE, respectively. The ethanolic extract (5 mg/mL) inhibits TBARS by 33.8%, and the aqueous extract (5 mg/mL) exhibited scavenger property against nitric oxide derivatives to an extent of 77.8%. In cell culture, both extracts improved cell survivability under H2O2 induced oxidative stress. Thus, C. erecta extract is a good candidate to become a phytotherapic medicine.

Hepatic disorders such as steatosis and alcoholic steatohepatitis are common diseases that affect thousands of people around the globe. This study aims to identify the main phenol compounds using a new HPLC-ESI+-MS/MS method, to evaluate some oxidative stress parameters and the hepatoprotective action of green dwarf coconut water, caffeic and ascorbic acids on the liver and serum of rats treated with ethanol. The results showed five polyphenols in the lyophilized coconut water spiked with standards: chlorogenic acid (0.18 µM), caffeic acid (1.1 µM), methyl caffeate (0.03 µM), quercetin (0.08 µM) and ferulic acid (0.02 µM) isomers. In the animals, the activity of the serum γ-glutamyltranspeptidase (γ-GT) was reduced to 1.8 I.U/L in the coconut water group, 3.6 I.U/L in the ascorbic acid group and 2.9 I.U/L in the caffeic acid groups, when compared with the ethanol group (5.1 I.U/L, p

We sought to investigate how far the growth of Saccharomyces cerevisiae under full anaerobiosis is dependent on the widely used anaerobic growth factors (AGF) ergosterol and oleic acid. A continuous cultivation setup was employed and, even forcing ultrapure N2 gas through an O2 trap upstream of the bioreactor, neither cells from S. cerevisiae CEN.PK113–7D (a lab strain) nor from PE-2 (an industrial strain) washed out after an aerobic-to-anaerobic switch in the absence of AGF. S. cerevisiae PE-2 seemed to cope better than the laboratory strain with this extremely low O2 availability, since it presented higher biomass yield, lower specific rates of glucose consumption and CO2 formation, and higher survival at low pH. Lipid (fatty acid and sterol) composition dramatically altered when cells were grown anaerobically without AGF: saturated fatty acid, squalene and lanosterol contents increased, when compared to either cells grown aerobically or anaerobically with AGF. We concluded that these lipid alterations negatively affect cell viability during exposure to low pH or high ethanol titers.

The aim of this work was to evaluate the effect of Ouratea sp. butter (OB) on crystallinity of solid lipids used in nanostructured lipid carriers systems. Firstly, the composition of fatty acids in OB was evaluated by transesterification reaction for gas chromatography fatty acid methyl ester analysis. The solid lipids such as stearic acid (SA), beeswax (BW) and carnauba wax (CW) were submitted to recrystallization process (heating–cooling). Moreover, binary mixtures between solid lipids and OB were prepared in ratio 1:1 (w/w) by heating of the components above the melting point followed by cooling at room temperature. Thus, the samples were characterized by differential scanning calorimetry (DSC), thermogravimetry (TG), X ray diffraction (XRD) and hot-stage polarized optical microscopy (HSPOM). DSC curves showed a shift of the melting point to lower temperatures in the lipid mixtures with OB. TG data suggested a thermal stability reduction in the lipid mixtures containing SA and CW and an increase thermal stability in the mixture containing BW. XRD data confirmed DSC results, showing a reduction in intensity of main diffraction peaks of the lipid mixtures and a presence of the amorphous portion in angle 2θ: 22°. Finally, HSPOM demonstrated that the microstructures of solid lipids decreased in size and thickness in the mixtures containing OB at room temperature and slightly before the melting point, confirming previous characterizations. These results suggest that lipid mixtures with OB present a lower crystallinity, and it is expected that amorphous portion facilitates drug incorporation, for example.

Peroxiredoxins (Prxs) are Cys-based highly efficient peroxidases. Prxs can be classified as 2-Cys and 1-Cys, according to the number of cysteines residues involved in the catalysis. 1-Cys Prx enzymes are still poorly characterized and the identities of their biological reductants are still controversial. Some of the reductant candidates are thioredoxin (Trx) and ascorbate. A. fumigatus is a human pathogen fungus that possesses two 1-Cys Prxs (Prx1 and PrxC). We had shown before that these Prxs display high reactivity towards H2O2 (~107 M-1s-1). Here, we described our studies on the reduction step. Thus, the genes encoding for Trx system from this fungus were cloned, expressed and purified. The 4 Trxs were efficiently reduced by TrxR (kcat/Km ~106 M-1s-1). However, our preliminary data indicated that Prx1 and PrxC were poorly reduced by these oxidoreductases. We are now investigating the erythroascorbate (a 5-carbon ascorbate analogue) role in Prx reduction. Since erythroascorbate can be found glycosylated in several fungi species, we are trying to identify and quantitate this reducing agent by mass spectrometry. The biochemical characterization of these Prxs catalytic cycle can open new therapeutical strategies, since at least Prx1 is involved in fungus virulence.

Coconut water (CW) is a natural nutritious beverage, which contains several biologically active compounds that are traditionally used in the treatment of diarrhea and rehydration. Several works with CW have been related with antioxidant activity, which is very important in the diabetic state. To evaluate the hypoglycemic and nephroprotective activities of CW, alloxan-induced diabetic rats were pre- and post-treated by gavage with CW (3 mL/kg), caffeic acid (CA) (10 and 15 mg/kg), and acarbose (Acb) (714 μg/kg) during a period of 16 days. Body weight, blood glucose, glycated hemoglobin (HbA1c), and Amadori products in plasma and kidney homogenates were evaluated in all groups and used as parameters for the monitoring of the diabetic state. The results showed that rats of the CW+diabetic group had maintenance in blood glucose compared with the control group (P.05) in addition to a decrease of HbA1c levels and increase of body weight when compared with the diabetic group rats (P.05). The animals of the CA and CA+diabetic groups did not have significant variation of body weight (P.05) during the experiment; however, they showed decrease in their HbA1c and urea levels in plasma as well as Amadori products in kidney homogenates when compared with the diabetic group (P.05). Our results indicate that CW has multiple beneficial effects in diabetic rats for preventing hyperglycemia and oxidative stress caused by alloxan.

Coconut water contains several uncharacterized substances and is widely used in the human consumption. In this paper we detected and quantified ascorbic acid and caffeic acid and total phenolics in several varieties of coconut using HPLS/MS/MS (25.8 ± 0.6 µg/mL and 1.078 ± 0.013 µg/mL and 99.7 µg/mL, respectively, in the green dwarf coconut water, or 10 mg and 539 µg and 39.8 mg for units of coconut consumed, 500 ± 50 mL). The antioxidant potential of four coconut varieties (green dwarf, yellow dwarf, red dwarf and yellow Malaysian) was compared with two industrialized coconut waters and the lyophilized water of the green dwarf variety. All varieties were effective in scavenging the DPPH radical (IC50=73 µL) and oxide nitric (0.1 mL with an IP of 29.9%) as well as in inhibiting the in vitro production of thiobarbituric acid reactive substances (1 mL with an IP of 34.4%), highlighting the antioxidant properties of the green dwarf which it is the most common used. In cell culture, the green dwarf water was efficient in protecting against oxidative damages induced by hydrogen peroxide. A água de coco contém várias substâncias não caracterizadas e é amplamente utilizada na alimentação humana. Neste estudo, foram detectados e quantificados ácido ascórbico e ácido cafeico e polifenóis totais em diversas variedades de coco, e usando HPLC/MS/MS (25,8 ± 0,6 µg/mL, 1,078 ± 0,013 µg/mL and 99,7 µg/mL, respectivamente, em água de coco do anão verde, ou 10 mg, 539 µg e 39,8 mg por unidades de coco consumido, 500 ± 50 mL). O potencial antioxidante da água de coco das quatro variedades de coqueiro (anão verde, anão amarelo, anão vermelho e amarelo malasiano) foi comparada com água de coco industrializada e água liofilizada da variedade anão verde. Todas as variedades foram eficazes - com destaque para as propriedades antioxidantes do anão verde, uma vez que é o mais comum usado – no sequestro do radical DPPH (IC50=73 µL), óxido nítrico (0,1 mL com IP de 29,9%) e inibindo a produção de espécies reativas com o ácido tiobarbitúrico (1 mL com IP de 34,4%) in vitro. Em cultura de células, a água de coco da variedade anão verde foi eficiente na proteção contra danos oxidativos induzidos por peróxido de hidrogênio.

Uric acid is the final product of purine metabolism in humans and accumulates in plasma in the order of hundred micromolar. The one electron oxidation of uric acid by peroxidases generates urate free radical and urate hydroperoxide. Urate hydroperoxide is a strong oxidant and might contribute to oxidative imbalance in inflammatory states. Urate hydroperoxide efficiently reacts with glutathione and thiol proteins. In this study, we investigated the oxidative metabolism of uric acid in inflammatory cells and whether it contributes to an oxidative environment. Human leukemic (HL-60) were differentiated in neutrophils (dHL-60) and activated with phorbol myristate acetate (PMA, 100 ng/mL). The activation of cells greatly increased oxygen consumption and superoxide production. The pre-incubation with uric acid (0.05 – 0.5 mM) increased even further the oxygen consumption and superoxide production (51%). Conversely, uric acid (0.2 – 2 mM) significantly decreased hypochlorous acid (HOCl) production. The overall oxidative status of the cell was evaluated by GSH/GSSG ratio using LC-MS/MS. Uric acid significantly decreased GSH/GSSG ratio in activated dHL-60 cells, showing its pro-oxidant capability. Data by LC-MS/MS revealed that activated dHL-60 cells efficiently oxidized uric acid to urate hydroperoxide. This oxidation was dependent on the activity of myeloperoxidase and on the addition of superoxide to the urate free radical. These results show that uric acid increases superoxide but decreases HOCl formation in activated dHL-60 cells. The decrease in HOCl might be due to a competition between chloride and uric acid by myeloperoxidase catalysis. In agreement, uric acid was largely oxidized to urate hydroperoxide and significantly increased the oxidation of GSH to GSSG in these cells. Therefore, the oxidation of uric acid in inflammatory cells can lead to an oxidative environment and exacerbate tissue damage. Acknowledgment FAPESP, INCT/NAP/CEPID-Redoxoma