Quercetin in lyotropic liquid crystalline formulations: Physical, chemical and functional stability

The purpose of this study was to develop a lyotropic liquid crystalline formulation using the emulsifier vitamin E TPGS and evaluate its behavior after incorporation of a flavonoid, quercetin. The physical (macro and microscopic), chemical (determination of quercetin content by the HPLC method) and functional (determination of quercetin antioxidant activity by DPPH center dot assay) stability of the lamellar liquid crystalline formulation containing flavonoid was evaluated when stored at 4+/-2 degrees C; 30+/-2 degrees C/70+/-5% RH (relative humidity) and 40+/-2 degrees C/70+/-5% RH during 12 months. The lamellar liquid crystalline structure of the formulation was maintained during the experiment, however chemical and functional stability results showed a great influence of the storage period in all conditions tested. A significant decrease in quercetin content (approximately 40%) was detected during the first month of storage and a similar significant loss in antioxidant activity was detected after 6 months. The remaining flavonoid content was unchanged during the final 6 months of the experimental period. The results suggest possible interactions between quercetin and the liquid crystalline formulation, which could inhibit or reduce the quercetin activity incorporated in the system. In conclusion, the present study demonstrated that incorporation of quercetin (1%) did not affect the liquid crystalline structure composed of vitamin E TPGS/IPM/PG-H2O (1:1) at 63.75/21.25/15 (w/w/w). Nevertheless, of the total quercetin incorporated in the system only 60% was free to act as an antioxidant.

Evaluation of the antioxidant activity as an additional parameter to attain the functional quality of natural extracts

Due to differences in the functional quality of natural extracts, we have also faced differences in their effectiveness. So, it was intended to assess the antioxidant activity of natural extracts in order to attain their functional quality. It was observed that all the extracts (brown and green propolis, Ginkgo biloba and Isoflavin Beta (R)) and the standard used (quercetin) showed antioxidant activity in a dose-dependent manner with IC50 values ranging from 0.21 to 155.28 mu g mL(-1) (inhibition of lipid peroxidation and scavenging of the DPPH center dot assays). We observed a high correlation (r(2)= 0.9913) among the antioxidant methods; on the other hand, the antioxidant activity was not related to the polyphenol and flavonoid content. As the DPPH center dot assay is a fast method, presents low costs and even has a high correlation with other antioxidant methods, it could be applied as an additional parameter in the quality control of natural extracts.

In vitro evaluation of the antioxidant activity of liposomal flavonols by the HRP--H(2)O(2)--luminol system

Considering that antioxidant flavonols have been reported to be beneficial to human health, but that their low water solubility and bioavailability limit their administration through systemic route, the development of suitable flavonol-carriers is of great importance for clinical therapeutics. The aim of this study was to prepare liposomes containing flavonols or not and evaluate their antioxidant activity. Vesicles were obtained by ethanol injection method and characterized in terms of entrapment efficiency, size and zeta potential. Inhibitory activity of liposomal flavonols on reactive oxygen species generation was assessed in vitro using luminol--H(2)O(2)--horseradish peroxidase technique. Antioxidant activity of liposomal flavonols is dependent on concentration and chemical structure of active compound. Quercetin and myricetin are the most active flavonols (IC(50) == 0.6--0.9 mu A mu mol/L), followed by kaempferol (IC(50) == 3.0--4.5 mu A mu mol/L) and galangin (IC(50) == 4.0--7.0 mu A mu mol/L). Our results suggest that antioxidant-loaded liposomes may be promising tools for therapy of diseases where oxidative stress is involved.

A common matrix metalloproteinase (MMP)-2 polymorphism affects plasma MMP-2 levels in subjects environmentally exposed to mercury

Mercury (Hg) exposure is associated with disease conditions, including cardiovascular problems. Although the mechanisms implicated in these complications have not been precisely defined yet, matrix metalloproteinases (MMPs) may be involved. The gene encoding MMP-2 presents genetic polymorphisms which affect the expression and activity level of this enzyme. A common polymorphism of MMP-2 gene is the C(-1306)T (rs 243865), which is known to disrupt a Sp1-type promoter site (CCACC box), thus leading to lower promoter activity associated with the T allele. This study aimed at examining how this polymorphism affects the circulating MMP-2 levels and its endogenous inhibitor, the tissue inhibitor of metalloproteinase-2 (TIMP-2) in 210 subjects environmentally exposed to Hg. Total blood and plasma Hg concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS). MMP-2 and TIMP-2 concentrations were measured in plasma samples by gelatin zymography and ELISA, respectively. Genotypes for the C(-1306)T polymorphism were determined by Taqman (R) Allele Discrimination assay. We found a positive association (p = 0.0057) between plasma Hg concentrations and MMP-2/TIMP-2 (an index of net MMP-2 activity). The C(-1306)T polymorphism modified MMP-2 concentrations (p = 0.0465) and MMP-2/TIMP-2 ratio (p = 0.0060) in subjects exposed to Hg, with higher MMP-2 levels been found in subjects carrying the C allele. These findings suggest a significant interaction between the C(-1306)T polymorphism and Hg exposure, possibly increasing the risk of developing diseases in subjects with the C allele. (C) 2011 Elsevier B.V. All rights reserved.

A functional matrix metalloproteinase (MMP)-9 polymorphism modifies plasma MMP-9 levels in subjects environmentally exposed to mercury

Mercury (Hg) exposure causes health problems including cardiovascular diseases. Although precise mechanisms have not been precisely defined yet, matrix metalloproteinases (MMPs) may be involved. The gene encoding MMP-9 presents genetic polymorphisms which affect the expression and activity level of this enzyme. Two polymorphisms in the promoter region [C(-1562)T and (CA)(n)] are functionally relevant, and are implicated in several diseases. This study aimed at examining how these polymorphisms affect the circulating MMP-9 levels and its endogenous inhibitor, the tissue inhibitor of metalloproteinase-1 (TIMP-1) in 266 subjects environmentally exposed to Hg. Blood and plasma Hg concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS). MMP-9 and TIMP-1 concentrations were measured in plasma samples by gelatin zymography and ELISA, respectively. Genotypes for the C(-1562)T and the microsatellite (CA)(n) polymorphisms were determined. We found a positive association (P<0.05) between plasma Hg concentrations and MMP-9/TIMP-1 ratio (an index of net MMP-9 activity). When the subjects were divided into tertiles with basis on their plasma Hg concentrations, we found that the (CA)(n) polymorphism modified MMP-9 concentrations and MMP-9/TIMP-1 ratio in subjects with the lowest Hg concentrations (first tertile), with the highest MMP-9 levels being found in subjects with genotypes including alleles with 21 or more CA repeats (H alleles) (P<0.05). Conversely, this polymorphism had no effects on subjects with intermediate or high plasma Hg levels (second and third tertiles, respectively). The C(-1562)T polymorphism had no effects on MMP-9 levels. These findings suggest a significant interaction between the (CA)(n) polymorphism and low levels of Hg exposure, possibly increasing the risk of developing diseases in subjects with H alleles. (c) 2010 Elsevier B.V. All rights reserved.

Mercury Exposure Increases Circulating Net Matrix Metalloproteinase (MMP)-2 and MMP-9 Activities

Mercury (Hg) exposure causes health problems that may result from increased oxidative stress and matrix metalloproteinase (MMP) levels. We investigated whether there is an association between the circulating levels of MMP-2, MMP-9, their endogenous inhibitors (the tissue inhibitors of metalloproteinases; TIMPs) and the circulating Hg levels in 159 subjects environmentally exposed to Hg. Blood and plasma Hg were determined by inductively coupled plasma-mass spectrometry (ICP-MS). MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA respectively. Thiobarbituric acid-reactive species (TBARS) were measured in plasma to assess oxidative stress. Selenium (Se) levels were determined by ICP-MS because it is an antioxidant. The relations between bioindicators of Hg and the metalloproteinases levels were examined using multivariate regression models. While we found no relation between blood or plasma Hg and MMP-9, plasma Hg levels were negatively associated with TIMP-1 and TIMP-2 levels, and thereby with increasing MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios, thus indicating a positive association between plasma Hg and circulating net MMP-9 and MMP-2 activities. These findings provide a new insight into the possible biological mechanisms of Hg toxicity, particularly in cardiovascular diseases.

Comparison of oxidation efficiency of disperse dyes by chemical and photoelectrocatalytic chlorination and removal of mutagenic activity

Degradation of Disperse Orange 1, Disperse Red 1 and Disperse Red 13 dyes has been performed using electrochemical oxidation on Pt electrode, chemical chlorination and photoelectrochemical oxidation on Ti/TiO(2) thin film electrodes in NaCl or Na(2)SO(4) medium. 100% discoloration was obtained for all tested methods after 1 h of treatment. Faster color removal was obtained by photoelectrocatalytic oxidation in 0.1 mol L(-1) NaCl pH 4.0 under UV light and an applied potential of +1.0V (vs SCE reference electrode), which indicates also values around 60% of TOC removal. The conventional chlorination method and electrochemical oxidation on Pt electrode resulted in negligible reduction of TOC removal. All dyes showed positive mutagenic activity in the Salmonella/microsome assay with the strain TA98 in the absence and presence of S9 (exogenous metabolic activation). Nevertheless, there is complete reduction of the mutagenic activity after 1 h of photoelectrocatalytic oxidation, suggesting that this process would be good option to remove disperse azo dyes from aqueous media. (C) 2008 Elsevier Ltd. All rights reserved.

Strongyloides venezuelensis: The antigenic identity of eight strains for the immunodiagnosis of human strongyloidiasis

The antigens of eight strains of Strongyloides venezuelensis were identified by means of the indirect immunofluorescence antibody (IFAT), enzyme-linked immunosorbent assay (ELISA) and immunoblot (IB) tests. Infective larvae (L3) from these strains were obtained from Rattus norvegicus feces cultures. For IFAT, sections of U were used while the ELISA and IB, tests were conducted with alkaline extract. Ninety serum samples were tested: 30 from patients with S. stercoralis, 30 from patients with other parasitic diseases, and 30 from healthy subjects (free of parasites). Average sensitivity and specificity among all eight strains, both for IFAT and ELISA, were, respectively, 93% and 100%. In the IB, anti-S. stercoralis IgG recognized a single antigenic fraction with 45 kDa. Serum samples from patients with S. stercoralis revealed antigens from different strains of S. venezuelensis, indicating antigenic identity for possible use in the synthesis of recombinant antigen that could be useful in immunodiagnosis and vaccine against this parasite. (C) 2008 Elsevier Inc. All rights reserved.

The azo dye Disperse Orange 1 induces DNA damage and cytotoxic effects but does not cause ecotoxic effects in Daphnia similis and Vibrio fischeri

Azo dyes constitute the largest group of colorants used in industry and can pass through municipal waste water plants nearly unchanged due to their resistance to aerobic treatment, which potentially exposes humans and local biota to adverse effects. Unfortunately, little is known about their environmental fate. Under anaerobic conditions, some azo dyes are cleaved by microorganisms forming potentially carcinogenic aromatic amines. In the present study, the azo dye Disperse Orange 1, widely used in textile dyeing, was tested using the comet, Salmonella/microsome mutagenicity, cell viability, Daphnia similis and Microtox (R) assays. The human hepatoma cell line (HepG2) was used in the comet assay and for cell viability. In the mutagenicity assay. Salmonella typhimurium strains with different levels of nitroreductase and o-acetyltransferase were used. The dye showed genotoxic effects with respect to HepG2 cells at concentrations of 0.2, 0.4, 1.0, 2.0 and 4.0 mu g/mL. In the mutagenicity assay, greater responses were obtained with the strains TA98 and YG1041, suggesting that this compound mainly induces frameshift mutations. Moreover, the mutagenicity was greatly enhanced with the strains overproducing nitroreductase and o-acetyltransferase, showing the importance of these enzymes in the mutagenicity of this dye. In addition, the compound induced apoptosis after 72 h in contact with the HepG2 cells. No toxic effects were observed for either D. similis or Vibrio fischeri. (C) 2011 Elsevier B.V. All rights reserved.

The azo dyes Disperse Red 1 and Disperse Orange 1 increase the micronuclei frequencies in human lymphocytes and in HepG2 cells

The use of azo dyes by different industries can cause direct and/or indirect effects oil human and environmental health due to the discharge of industrial effluents that contain these toxic compounds. Several studies have demonstrated the genotoxic effects of various azo dyes, but information on the DNA damage caused by Disperse Red 1 and Disperse Orange 1 is unavailable, although these dyes are used in dyeing processes in many countries. The aim of the present study was to evaluate the mutagenic activity of Disperse Red 1 and Disperse Orange 1 using the micronucleus (MN) assay in human lymphocytes and in HepG2 cells. In the lymphocyte assay. it was found that the number of MN induced by the lowest concentration of each dye (0.2 mu g/mL) was similar to that of the negative control. At the other concentrations, a dose response MN formation was observed up to 1.0 mu g/mL. At higher dose levels, the number of MN decreased. For the HepG2 cells the results were similar. With both dyes a dose dependent increase in the frequency of MN was detected. However for the HepG2, the threshold for this increase was 2.0 mu g/mL, while at higher doses a reduction in the MN number was observed. The proliferation index was also calculated in order to evaluate acute toxicity during the test. No differences were detected between the different concentrations tested and the negative control. (C) 2009 Elsevier B.V. All rights reserved.

Deconstructing Tick Saliva NON-PROTEIN MOLECULES WITH POTENT IMMUNOMODULATORY PROPERTIES

Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-alpha while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of similar to 110pmol/mu l) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E(2) (PGE(2) similar to 100 nM) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE(2) inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE(2) were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE(2), but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE(2) in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.

Bixin and lycopene modulation of free radical generation induced by cisplatin-DNA interaction

This work evaluated the Modulation of reactive oxygen species (ROS) produced by the cisplatin-human DNA interaction in a cell-free experimental model by the carotenoids bixin and lycopene extracted from, natural dietary Sources and purified through luminol- and Cypridina luciferin methoxy-analogue (MCLA)- enhanced chemiluminescence assays. The results showed that the ROS generation by DNA-cisplatin interaction was inhibited by both lycopene and bixin in a concentration-dependent manner. At a concentration of 100 mu M, lycopene and bixin inhibited Superoxide anion (O center dot(2)) generation at 90% and 82%, respectively, and the total ROS generation at 44% and 42%, respectively. The formation of significant amounts of isomers or degradation products of both carotenoids was not observed after ROS scavenging, as evaluated by high-performance liquid chromatography. Taken together, these results Suggest that carotenoids can be helpful to Modulate the oxidative stress found in cancer therapy with cisplatin. (c) 2008 Elsevier Ltd. All rights reserved.

The Scientific Basis for High-Intensity Interval Training: Optimising Training Programmes and Maximising Performance in Highly Trained Endurance Athletes

While the physiological adaptations that occur following endurance training in previously sedentary and recreationally active individuals are relatively well understood, the adaptations to training in already highly trained endurance athletes remain unclear. While significant improvements in endurance performance and corresponding physiological markers are evident following submaximal endurance training in sedentary and recreationally active groups, an additional increase in submaximal training (i.e. volume) in highly trained individuals does not appear to further enhance either endurance performance or associated physiological variables [e.g. peak oxygen uptake (V-dot O2peak), oxidative enzyme activity]. It seems that, for athletes who are already trained, improvements in endurance performance can be achieved only through high-intensity interval training (HIT). The limited research which has examined changes in muscle enzyme activity in highly trained athletes, following HIT, has revealed no change in oxidative or glycolytic enzyme activity, despite significant improvements in endurance performance (p < 0.05). Instead, an increase in skeletal muscle buffering capacity may be one mechanism responsible for an improvement in endurance performance. Changes in plasma volume, stroke volume, as well as muscle cation pumps, myoglobin, capillary density and fibre type characteristics have yet to be investigated in response to HIT with the highly trained athlete. Information relating to HIT programme optimisation in endurance athletes is also very sparse. Preliminary work using the velocity at which V-dot O2max is achieved (Vmax) as the interval intensity, and fractions (50 to 75%) of the time to exhaustion at Vmax (Tmax) as the interval duration has been successful in eliciting improvements in performance in long-distance runners. However, Vmax and Tmax have not been used with cyclists. Instead, HIT programme optimisation research in cyclists has revealed that repeated supramaximal sprinting may be equally effective as more traditional HIT programmes for eliciting improvements in endurance performance. Further examination of the biochemical and physiological adaptations which accompany different HIT programmes, as well as investigation into the optimal HIT programme for eliciting performance enhancements in highly trained athletes is required.

Wolbachia infections are distributed throughout insect somatic and germ line tissues

Wolbachia are intracellular microorganisms that form maternally-inherited infections within numerous arthropod species. These bacteria have drawn much attention, due in part to the reproductive alterations that they induce in their hosts including cytoplasmic incompatibility (CI), feminization and parthenogenesis. Although Wolbachia's presence within insect reproductive tissues has been well described, relatively few studies have examined the extent to which Wolbachia infects other tissues. We have examined Wolbachia tissue tropism in a number of representative insect hosts by western blot, dot blot hybridization and diagnostic PCR. Results from these studies indicate that Wolbachia are much more widely distributed in host tissues than previously appreciated. Furthermore, the distribution of Wolbachia in somatic tissues varied between different Wolbachia/host associations. Some associations showed Wolbachia disseminated throughout most tissues while others appeared to be much more restricted, being predominantly limited to the reproductive tissues. We discuss the relevance of these infection patterns to the evolution of Wolbachia/host symbioses and to potential applied uses of Wolbachia.

Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens

The role of individual viral proteins in the immune response to bluetongue virus (BTV) is not clearly understood. To investigate the contributions of the outer capsid proteins, VP2 and VP5, and possible interactions between them, these proteins were expressed from recombinant vaccinia viruses either as individual proteins or together in double recombinants, or with the core protein VP7 in a triple recombinant. Comparison of the immunogenicity of the vaccinia expressed proteins with BTV expressed proteins was carried out by inoculation of rabbits and sheep. Each of the recombinants was capable of stimulating an anti-BTV antibody response, although there was a wide range in the level of response between animals and species. Vaccinia-expressed VP2 was poorly immunogenic, particularly in rabbits. VP5, on the whole, stimulated higher ELISA titers in rabbits and sheep and in some animals in both species was able to stimulate virus neutralizing antibodies. When the protective efficacy of VP2 and VP5 was tested in sheep, vaccinia-expressed VP2, VP5 and VP2 + VP5 were protective, with the most consistent protection being in groups immunized with both proteins. (C) 1997 Elsevier Science B.V.