Antioxidant effect of phenolic compounds (PC) at different concentrations in IEC-6 cells: A spectroscopic analysis.

Phenolic compounds (PC) have been proposed as natural antioxidant agents that protect cells against oxidative stress-related diseases. Nonetheless, their low bioavailability forecasts controversy about mechanisms on their in vivo scavenging activity against reactive oxygen species (ROS). It has been proposed that PC reduce directly ROS concentration. An alternative or complementary action of PC could be the activation of the cell's antioxidant pathway, involving the regulation of gene expression, like that initiated by the Nrf2 transcription factor. To date there is not enough experimental data to support or discard this possibility. In the present study, we evaluated the use of several PC to prevent peroxidation of macromolecules and to elicit the activation of the Nrf2 transcription factor in H2O2-stresed IEC-6 enterocytic cell line. Synchrotron microspectroscopy demonstrated that PC compounds protected proteins, lipids and nucleic acids against oxidation induced by H2O2. Immunofluorescence results showed that treatment with quercetin (Qc), catechin (Cat) and capsaicin (Cap) induced the translocation of Nrf2 into the nucleus, at the same level as did H2O2 treatment, thus mimicking the action of the endogenous cell response to peroxidation. Even though the detailed mechanism still needs to be elucidated, we demonstrated the activation of Nrf2 by PCs in response to oxidative stress.

Inhibition of α-amylase by flavonoids: Structure activity relationship (SAR).

Flavonoids are recognized to regulate animals' food digestion processes trough interaction with digestive enzymes. The binding capacity of hesperetin (HES), luteolin (LUT), quercetin (QUE), catechin (CAT) and rutin (RUT) with pancreatic α-amylase were evaluated, using UV-Vis spectroscopy, fluorescence and molecular docking. Using p-nitrophenyl-α-d-maltopentoside (pNPG5) as substrate analog, LUT showed the best inhibitory capacity, even better than that of the positive control, acarbose (ACA). A mixed-type inhibition was observed for HES, LUT and QUE, a competitive-type for ACA, while no inhibition was observed with CAT and RUT. In agreement with kinetic results, α-amylase presented a higher affinity for LUT, when analyzed by fluorescence quenching. The binding of flavonoids to amylase followed a static mechanism, where the binding of one flavonoid per enzyme molecule was observed. Docking analysis showed that flavonoids bound near to enzyme active site, while ACA bound in another site behind the catalytic triad. Extrinsic fluorescence analysis, together with docking analysis pointed out that hydrophobic interactions regulated the flavonoid-α-amylase interactions. The present study provides evidence to understand the relationship of flavonoids structure with their inhibition mechanism.

Dietary fiber and phenolic compounds as functional ingredients: interaction and possible effect after ingestion.

Dietary fiber and phenolic compounds are two recognized dietary factors responsible for potential effects on human health; therefore, they have been widely used to increase functionality of some foods. This paper focuses on showing the use of both substances as functional ingredients for enriching foods, and at the same time, describes the use of a single material that combines the properties of the two types of substances. The last part of the work describes some facts related to the interaction between dietary fiber and phenolic compounds, which could affect the bioaccessibility and absorption of phenolics in the gut. In this sense, the purpose of the present review is to compile and analyze evidence relating to the use of dietary fiber and phenolic compounds to enhance technological and nutritional properties of foods and hypothesize some of the possible effects in the gut after their ingestion.

Carbon paste electrodes modified with biosolids, soils and biocomposites utilized to study the interaction between organic matter and copper.

Carbon paste electrodes (CPEs) modified with a biosolid, two types of soils with different amounts of organic matter (OM), and two biocomposites (soils mixed with a biosolid) were used to assess and compare the Cu(II) ion retention properties of the organic matter contained in the samples. The accumulation of Cu(II) on the surface of the modified carbon paste electrodes (MCPEs) was performed under open-circuit conditions. When comparing the response of the MCPEs while assessing parameters such as pH, preconcentration time, and adsorption/desorption capacity, it was found that the reaction mechanism of the two soils is different between the soils and dissimilar from the biosolid; while the biocomposites show reaction mechanisms that are intermediate between those of the soils and the biosolid. This was proven with the use of infrared spectroscopy, since the FTIR spectra show similarities between the two soils and significant differences between the soils and the biosolid.

Dimethylsphingosine increases cytosolic calcium and intracellular pH in human T lymphocytes.

N,N-Dimethyl-D-erythro-sphingosine (DMS) is the N-methyl derivative of sphingosine; both are activators of sphingosine-dependent protein kinases. The aim of this work was to study the effect of DMS on cytosolic calcium and intracellular pH (pHi) in human T lymphocytes. The variations of calcium and pH were determined by fluorescence digital imaging using Fura-2-AM and BCECF-AM, respectively. DMS increased both pHi and Ca(2+)-cytoslic in human T lymphocytes. These effects were dose-dependent. This drug induced a fast increase in pHi and a release of calcium from different intracellular calcium pools than thapsigargin. DMS also induced a Ca(2+)-influx different from the store-operated calcium channels, since drug effect was not modified by 30 microM SKF 96365. The influx of calcium induced by DMS was completely blocked by preincubation in the presence of nickel, or lanthanum, while the increase in pHi was no affected. However, the presence of cadmium reduced but does not block Ca(2+)-influx. The inhibition of G-protein by 100 ng/mL pertussis toxin, and the inhibition of tyrosine kinases by genistein significantly reduced the cytosolic calcium increase induced by DMS by an inhibition of both, release of calcium from intracellular pools and influx from extracellular medium. The inhibition of pools emptiness by these drugs was related with the inhibition that they induce in the DMS cytosolic alcalinization. In summary, DMS increases pHi and as consequence releases calcium from intracellular pools, and it increases calcium-influx through a channel different from store-operated channel (SOC). Both cytosolic calcium and pHi increase are modulated by G-proteins and tyrosine kinases.

Maitotoxin-induced calcium entry in human lymphocytes: modulation by yessotoxin, Ca(2+) channel blockers and kinases.

We have studied the effect of the ciguatera-related toxin maitotoxin (MTX) on the cytosolic free calcium concentration ([Ca(2+)]i) of human peripheral blood lymphocytes loaded with the fluorescent probe Fura2 and the regulation of MTX action by different drugs known to interfere in cellular Ca(2+) signalling mechanisms and by the marine phycotoxin yessotoxin (YTX). MTX produced a concentration-dependent elevation of [Ca(2+)]i in a Ca(2+)-containing medium. This effect was stimulated by pretreatment with YTX 1 microM and NiCl(2) 15 microM. The voltage-independent Ca(2+) channel antagonist 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl]-1H-imidazole hydrochloride (SKF96365) blocked the MTX-induced [Ca(2+)]i elevation, while the L-type channel blocker nifedipine had no effect. Pretreatment with NiCl(2) or nifedipine did not modify YTX-induced potentiation of MTX effect, and SKF96365-induced inhibition was reduced in the presence of YTX, which suggest different pathways to act on [Ca(2+)]i. Preincubation with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H-89) or genistein (10 microM) also had no effect on the MTX-induced [Ca(2+)]i increment. In contrast, the PKC inhibitor bisindolilmaleimide I (GF109203X 1 microM) potentiated the MTX effect, whereas phosphatidylinositol (PI) 3-kinase inhibition with wortmannin (10 nM) reduced the MTX-elicited Ca(2+) entry. In summary, MTX produced Ca(2+) influx into human lymphocytes through a SKF96365-sensitive, nifedipine-insensitive pathway. The MTX-induced [Ca(2+)]i elevation was stimulated by the marine toxin YTX through a mechanism insensitive to SKF96365, nifedipine or NiCl(2). It was also stimulated by the divalent cation Ni(2+) and PKC inhibition and was partially inhibited by PI 3-kinase inhibition.

Modulation of thapsigargin-induced calcium mobilisation by cyclic AMP-elevating agents in human lymphocytes is insensitive to the action of the protein kinase A inhibitor H-89.

Ca2+ mobilisation from internal stores and from the extracellular medium is one of the primary events involved in lymphocyte activation and proliferation. Regulation of these processes by adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) was studied in Fura2-loaded human peripheral blood lymphocytes. Cytosolic Ca2+ concentration ([Ca2+]i) was measured in single cells by the use of a ratio imaging fluorescence microscope and Ca2+ mobilisation was achieved by the use of the endoplasmic reticulum (ER) Ca2+ ATPase inhibitor, thapsigargin (Thg). Our results show that both activation and inhibition of PKA, with forskolin (FSK) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H-89), respectively, inhibited the Thg-induced Ca2+ entry. Furthermore, FSK also reduced the ability of Thg to release Ca2+ from internal stores. This reduction was inhibited by the adenylyl cyclase (AC) inhibitor 9-(tetrahydro-2-furanyl)-9-H-purin-6-amine (SQ22,536), but not by the PKA inhibitor H89, indicating that cAMP but not PKA is responsible for this effect. FSK effect was mimicked by dibutyryl cAMP (dbcAMP) and by inhibition of phosphodiesterases (PDEs) with rolipram (ROL) and milrinone (MIL). We also showed that a very high concentration of H-89 (100 microM) releases Ca2+ from an intracellular pool, although this action is probably independent of PKA inhibition. Neither 10 microM H-89 nor other cAMP/PKA-modulating drugs had any effect on the basal [Ca2+]i of human lymphocytes. We conclude that PKA may act as a fine modulator of capacitative Ca2+ entry, while cAMP has a PKA-independent interaction with the Ca2+ stores of human lymphocytes.

HCO(3)(-) ions modify the role of PKC isoforms in the modulation of rat mast cell functions.

PKC and the intracellular calcium signal are two well-known intracellular signaling pathways implicated in the induction of mast cell exocytosis. Both signals are modified by the presence or absence of HCO(3)(-) ions in the external medium. In this work, we studied the regulation of the exocytotic process by PKC isozymes and its relationship with HCO(3)(-) ions and PKC modulation of the calcium entry. The calcium entry, induced by thapsigargin and further addition of calcium, was inhibited by PMA, a PKC activator, and enhanced by 500 nM GF109203X, which inhibits Ca(2+)-independent PKC isoforms. PMA inhibition of the Ca(2+) entry was reverted by 500 and 50 nM GF109203X, which inhibit Ca(2+)-independent and Ca(2+)-dependent isoforms, respectively, and Gö6976, a specific inhibitor of Ca(2+)-dependent PKCs. Thus, activation of Ca(2+)-dependent and Ca(2+)-independent PKC isoforms inhibit Ca(2+) entry in rat mast cells, either in a HCO(3)(-)-buffered or a HCO(3)(-)-free medium. PMA, GF109203X, Gö6976 and rottlerin, a specific inhibitor of PKC delta, were also used to study the role of PKC isoforms in the regulation of exocytosis induced by thapsigargin, ionophore A23187 and PMA. The results demonstrate that Ca(2+)-dependent PKC isoforms inhibit exocytosis in a HCO(3)(-)-dependent way. Moreover, Ca(2+)-independent PKC delta was the main isoform implicated in promotion of Ca(2+)-dependent mast cell exocytosis in the presence or absence of HCO(3)(-). The role of PKC isoforms in the regulation of mast cell exocytosis depends on the stimulus and on the presence or absence of HCO(3)(-) ions in the medium, but it is independent of PKC modulation of the Ca(2+) entry.

Modulation of cytosolic calcium levels of human lymphocytes by yessotoxin, a novel marine phycotoxin.

Yessotoxin (YTX) is a polyether toxin of marine origin that has been classified among the diarrheic shellfish poisoning (DSP) toxins group due to its lipophilic nature. However, unlike other DSP toxins, YTX does not produce diarrhea and its mechanisms of action are unknown. We studied the effect of YTX on the cytosolic calcium levels of freshly isolated human lymphocytes by means of fluorescence imaging microscopy. We showed that YTX produced a calcium influx through nifedipine and SKF 96365 (1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl]-1H-imidazole hydrochloride)-sensitive channels. This Ca2+ entry was not affected by the DSP toxin okadaic acid, which inhibits protein phosphatases. In addition, YTX also produced an inhibition of capacitative calcium entry activated by thapsigargin or by preincubation in a Ca2+-free medium. This capacitative calcium entry was not sensitive to nifedipine. Furthermore, the inhibitory effect of YTX was dependent on the time of addition of the toxin. We suggest that YTX may interact with calcium channels in a way similar to that described for other polyether marine compounds such as brevetoxins and maitotoxin, although an involvement of other second messengers is also likely.

HCO3(-) ions increase mast cell sensitivity to thapsigargin-induced Ca(2+) entry.

In rat mast cells Ca(2+) entry is modified by the presence or absence of other ions in the external medium. HCO3(-) ions, which modify mast cell degranulation, seemed to modulate the Ca(2+) entry elicited by the intracellular Ca(2+)-ATPase inhibitor thapsigargin. In this work we studied the regulation of the Ca(2+) entry by HCO3(-) and its relationship with exocytosis. The Ca(2+) entry was activated by thapsigargin and Ca(2+) in mast cells bathed by a HCO3(-)-buffered medium or a HCO3(-)-free medium. Both Ca(2+) entry and exocytosis were enhanced by the presence of HCO3(-) ions. Nondegranulated mast cells showed a low Ca(2+) entry either in the presence or absence of HCO3(-). Thus, mast cells with a high [Ca(2+)](i) increase in a HCO3(-)-buffered medium undergo degranulation. In the same cells a second Ca(2+) entry was significantly higher than the first Ca(2+) entry in a HCO3(-)-free medium, while in a HCO3(-)-buffered medium the first and second Ca(2+) entries reached similar [Ca(2+)](i) levels. Although the second Ca(2+) entry is high in a HCO3(-)-free medium, degranulation is still low. Our results demonstrate that HCO3(-) ions increase the capacitative Ca(2+) entry and the sensitivity of mast cells to intracellular Ca(2+) in order to induce degranulation.

Evidence for an electrogenic, negatively protein-kinase-A-modulated, Na+-dependent HCO3- transporter in human lymphocytes.

We studied the effects of external HCO3- on pHi regulation in human lymphocytes after an acid load. Cells were acidified by preincubation with NH4Cl and pHi recovery was measured with the fluorescent dye BCECF. Cells recovering in HCO3--containing medium reached a higher final pHi, the H+ efflux rate was increased and shifted to alkaline pHi compared to that of cells recovering in HCO3--free solution. The resting pHi was higher in a HCO3--containing solution. Experiments carried out in the presence of amiloride, DIDS and in the absence of external Na+ suggest the existence of two major mechanisms acting in the pHi recovery of lymphocytes after an acid load: an amiloride-sensitive Na+/H+ exchanger and a DIDS-sensitive Na+-dependent HCO3- transporter. The last mechanism could be a Na+/HCO3- cotransporter based on membrane potential changes determined with the potential-sensitive fluorescent probe bis-oxonol. Preincubation of cells with forskolin and H-89 showed protein-kinase-A-dependent downregulation of the amiloride-insensitive recovery of pHi in human lymphocytes. In summary, this paper provides functional evidence for the existence of a Na+/HCO3--dependent mechanism involved in pHi recovery in human lymphocytes following an acid load, that is electrogenic and downregulated by PKA.