An alternative method for purifying and detoxifying diphtheria toxin

Infections caused by Corynebacterium diphtheriae frequently induce situations in which very small doses of antigens injected intradermally can cause strong inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a virulence factor that can be lethal to the human organism at doses below 0.1 mu g/kg of body weight. The present work proposes alternative methods of DT purification using affinity chromatography and of DT detoxification through conjugating with the polymer methoxypolyethylene glycol activated (mPEG). Tests were performed to evaluate: the formation of edemas and the presence of dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with mPEG, and the immunogenic activity of the purified and modified toxin. The results indicated that purification with Blue Sepharose was an efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein nitrogen. The modification of the Purified Toxin with mPEG did not result in the formation of edema or necrosis although it was immunogenic and stimulated the formation of antibodies that could neutralize the Purified Toxin. The toxoid obtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however. (C) 2011 Elsevier Ltd. All rights reserved.

Analysis of plasma and erythrocyte zinc levels in premenopausal women with breast cancer

Introduction: Zinc deficiency has been associated with damage and oxidative changes in DNA that may increase an individual`s risk of cancer. Furthermore, zinc metabolism may be affected in cancer patients, leading to alterations in its distribution that would favor carcinogenesis. Plasma and erythrocyte zinc levels in women with breast cancer were evaluated in this cross-sectional, controlled study. Material and methods: Fifty-five premenopausal women of 25 to 49 years of age with and without breast cancer were divided into two groups: Group A, composed of women without breast cancer (controls, n = 26) and Group B, composed of women with breast cancer (cases, n = 29). Plasma and erythrocyte zinc levels were measured by flame atomic absorption spectrophotometry at gamma = 213.9 nm. Diet was assessed using the 3-day diet recall method and analyzed using the NutWin software program, version 1.5. Student`s t-test was used to compare means and significance was established at p <0.05. Results: Mean plasma zinc levels were 69.69 +/- 9.00 g/dt, in the breast cancer patients and 65.93 +/- 12.44 g/dt. in the controls (p = 0.201). Mean erythrocyte zinc level was 41.86 +/- 8.28 mu gZn/gHb in the cases and 47.93 +/- 7.00 mu gZn/gHb in the controls (p < 0.05). In both groups, dietary zinc levels were above the estimated average requirement. Conclusions: The present results suggest that zinc levels are lower in the erythrocyte compartment of premenopausal women with breast cancer.

Characterization of Brazilian lager and brown ale beers based on color, phenolic compounds, and antioxidant activity using chemometrics

BACKGROUND: Epidemiological studies have shown that beer has positive effects on inhibiting atherosclerosis, decreasing the content of serum low-density lipoprotein cholesterol and triglycerides, by acting as in vivo free radical scavenger. In this research, the antioxidant activity of commercial Brazilian beers (n = 29) was determined by the oxygen radical absorbance capacity (ORAC) and 1,1 -diphenyl-2-picrylhydrazyl (DPPH(center dot)) assays and results were analyzed by chemometrics. RESULTS: The brown ale samples (n = 11) presented higher (P < 0.05) flavonoids (124.01 mg L(-1)), total phenolics (362.22 mg L(-1)), non-flavonoid phenolics (238.21 mg L(-1)), lightness (69.48), redness (35.75), yellowness (55.71), color intensity (66.86), hue angle (59.14), color saturation (0.9620), DPPH(center dot) values (30.96% inhibition), and ORAC values (3,659.36 mu mol Trolox equivalents L(-1)), compared to lager samples (n = 18). Brown ale beers presented higher antioxidant properties (P < 0.05) measured by ORAC (1.93 times higher) and DPPH (1.65 times higher) compared to lager beer. ORAC values correlated well with the content of flavonoids (r = 0.47; P = 0.01), total phenolic compounds (r = 0.44; P < 0.01) and DPPH (r = 0.67; P < 0.01). DPPH values also correlated well to the content of flavonoids (r = 0.69; P < 0.01), total phenolic compounds (r = 0.60; P < 0.01), and non-flavonoid compounds (r = 0.46; P = 0.01). CONCLUSION: The results suggest that brown ale beers, and less significantly lager beers, could be sources of bioactive compounds with suitable free radical scavenging properties. (C) 2010 Society of Chemical Industry

Bioavailability of dietary sodium copper chlorophyllin and its effect on antioxidant defence parameters of Wistar rats

BACKGROUND: There has been growing interest in sodium copper chlorophyllin (Cu-Chl) as a food colourant and supplement owing to its beneficial biological activities. Studies have revealed that this green pigment inhibits experimental carcinogenesis and interacts with proteins and genotoxic agents. Health-related activities have also been associated with the prevention of lipid peroxidation. However, intestinal absorption of this pigment has been considered insignificant, raising questions of whether eventual biological properties are related to pre- or post-absorptive actions. In this study, intestinal absorption of Cu-Chl and its appearance in serum and organs were estimated by high-performance liquid chromatography analysis in rat feeding experiments. The effect of ingested Cu-Chl on lipid peroxidation was analysed by measuring thiobarbituric acid-reactive substances and antioxidant enzyme activities in hepatic and brain tissues of oxidative stress-induced rats. RESULTS: The two main components of commercial Cu-Chl, namely Cu-chlorin e(6) and Cu-chlorin e(4), showed different digestive behaviours, and only Cu-chlorin e4 was found in serum, liver and kidneys. Antioxidant activity in vivo could be observed in brain and seemed to be related to in situ protection but not to antioxidant enzyme modulation. CONCLUSION: As at least one of the major components of Cu-Chl is effectively absorbed, further pharmacolkinetic studies are encouraged to access absorption rates and the role of ingested copper chlorophyllins in mammals. (C) 2009 Society of Chemical Industry

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. one hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p < 0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p < 0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p = 0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4 +/- 12.4%) and apolipoprotein B (apoB) (17.0 +/- 31.3%) when compared with the higher quartile (>0.085: LDL-c = 40.3 +/- 14.3%; apoB = 32.5 +/- 10.7%; p < 0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism. and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin. (C) 2008 Elsevier Inc. All rights reserved.

Bioequivalence study of two oral formulations of cefadroxil in healthy volunteers

Two different cefadroxil (CAS 50370-12-2) formulations were evaluated for their relative bioavailability in 24 healthy volunteers who received a single 500 mg oral dose of each preparation. An open, randomized clinical trial designed as a two-period crossover study with a 7-day washout period between doses was employed. Plasma samples for assessments of their cefadroxil concentration by HPLC-UV were obtained over 8 h after administration. Values of 48.94 +/- 10.18 mu g . h/ml for test, and 48.51 +/- 9.02 mu g . h/ml for the reference preparation AUC(0-t) demonstrate a nearly identical extend of drug absorption. Maximum plasma concentration C-max of 16.04 +/- 4.94 mu g/ml and 16.01 +/- 4.02 mu g/ml achieved for the test and reference preparations did not differ significantly. The parametric 90% confidence intervals (CI) of the mean of the difference (test-reference) between log-transformed values of the two formulations were 96.80% to 104.51% and 92.01% to 107.00% for AUC(0-t) and C-max, respectively. Since for both AUC(0-t) or C-max the 90% CI values are within the interval proposed by the Food and Drug Administration, the test product is bioequivalent to the reference product for both the rate and extent of absorption after single dose administration.

Electronegative LDL and lipid abnormalities in patients undergoing hemodialysis and peritoneal dialysis

Background: Oxidative modification of low-density lipoprotein (LDL) has been demonstrated in patients with end-stage renal disease, where it is associated with oxidative stress and plays a key role in the pathogenesis of atherosclerosis. In this context, the generation of minimally oxidized LDL, also called electronegative LDL [ LDL(-)], has been associated with active disease, and is a detectable sign of atherogenic tendencies. The purpose of this study was to evaluate serum LDL(-) levels and anti-LDL(-)IgG autoantibodies in end-stage renal disease patients on dialysis, comparing patients on hemodialysis (HD), peritoneal dialysis (PD) and a control group. In addition, the serum lipid profile, nutritional status, biochemical data and parameters of mineral metabolism were also evaluated. Methods: The serum levels of LDL(-) and anti-LDL(-) IgG autoantibodies were measured in 25 patients undergoing HD and 11 patients undergoing PD at the Centro Integradode Nefrologia, Rio de Janeiro, Brazil. Ten healthy subjects served as a control group. Serum levels of albumin, total cholesterol, triglycerides and lipoproteins were measured. Calculations of subjects` body mass index and measurements of waist circumference, triceps skin fold and arm muscle area were performed. Measurements of hematocrit, serum blood urea nitrogen, creatinine, parathyroid hormone, phosphorus and calcium were taken. Results: Levels of LDL(-) were higher in HD patients (575.6 +/- 233.1 mu g/ml) as compared to PD patients (223.4 +/- 117.5 mu g/ml, p < 0.05), which in turn were higher than in the control group (54.9 +/- 33.3 mu g/ml, p < 0.01). The anti-LDL(-) IgG autoantibodies were increased in controls (0.36 +/- 0.09 mu g/ ml) as compared to PD (0.28 +/- 0.12 mu g/ml, p < 0.001) and HD patients (0.2 +/- 0.1 mu g/ml, p < 0.001). The mean values of total cholesterol and LDL were considered high in the PD group, whereas the mean triceps skin fold was significantly lower in the HD group. Conclusion: Levels of LDL(-) are higher in renal patients on dialysis than in normal individuals, and are reciprocally related to IgG autoantibodies. LDL(-) may be a useful marker of oxidative stress, and this study suggests that HD patients are more susceptible to cardiovascular risk due to this condition. Moreover, autoantibodies reactive to LDL(-) may have protective effects in chronic kidney disease. Copyright (C) 2008 S. Karger AG, Basel.

Effective Tityus serrulatus anti-venom produced using the Ts1 component

Scorpion stings are a public health problem in Brazil, with most incidents involving the species Tityus serrulatus. Some T serrulatus toxins may act as immunogens for the production of a specific anti-venom, but many of the component toxins remain poorly characterized. Here, we describe the immunological characteristics of the toxin Ts1 (also known as TsVII and Ts-gamma) and evaluate production of neutralizing antibodies against the crude venom of T serrulatus. Recombinant Ts1 with one copy (Ts1((1))) or two copies in tandem (Ts1((2))) was expressed in BL21 (DE3) cells. Rabbits and mice were immunized with the recombinant proteins (inclusion bodies) and then tested for production of neutralizing antibodies. Neutralization assays showed that anti-Ts1((1)) and anti-Ts1((2)) protected animals challenged with T serrulatus crude venom and native Ts1 Thus, Ts1 could be used in a mixed ""cocktail"" of immunogens for T serrulatus anti-venom production. (C) 2008 Elsevier Ltd. All rights reserved.

Detailed H-1 and C-13 NMR structural assignment and relative stereochemistry determination for three closely related sesquiterpene lactones

A complete analysis of H-1 and C-13 NMR spectra of the trypanocidal sesquiterpene lactone eremantholide C and two of its analogues is described. These structurally similar sesquiterpene lactones were submitted to H-1 NMR, C-13 (H-1) NMR, gCOSY, gHSQC, gHMBC, J-resolved and DPFGSE-NOE NMR techniques. The detailed analysis of those results, correlated to some computational calculations (molecular mechanics), led to the total and unequivocal assignment of all H-1 and C-13 NMR data. The determination of all H-1/H-1 coupling constants and all signal multiplicities, together with the elimination of previous ambiguities were also achieved. Copyright (C) 2008 John Wiley & Sons, Ltd.

Characterization of interactions between polyphenolic compounds and human serum proteins by capillary electrophoresis

The interaction of ten natural polyphenolic compounds (chlorogenic acid, apigenin, catechin, epicatechin, flavanone, flavone, quercetin, rutin, vicenin-2 and vitexin) with human serum albumin and mixtures of human serum albumin and alpha(1)-acid glycoprotein under near physiological conditions is studied by capillary electrophoresis-frontal analysis. Furthermore, the binding of these polyphenolic compounds to total plasmatic proteins is evaluated using ultrafiltration and capillary electrophoresis. In spite of the relatively small differences in the chemical structures of the compounds studied, large differences were observed in their binding behaviours to plasmatic proteins. The hydrophobicity, the presence/absence of some functional groups, steric hindrance and spatial arrangement seem to be key factors in the affinity of natural polyphenols towards plasmatic proteins.

Genetic analysis of complement C1s deficiency associated with systemic lupus erythernatosus highlights alternative splicing of normal C1s gene

Deficiencies of complement proteins of the classical pathway are strongly associated with the development of autoimmune diseases. Deficiency of Clr has been observed to occur concomitantly with deficiency in Cls and 9 out of 15 reported cases presented systemic lupus erythernatosus (SLE). Here, we describe a family in which all four children are deficient in Cls but only two of them developed SLE. Hemolytic activity mediated by the alternative and the lectin pathways were normal, but classical pathway activation was absent in all children`s sera. Cls was undetectable, while in the parents` sera it was lower than in the normal controls. The levels of Clr observed in the siblings and parents sera were lower than in the control, while the concentrations of other complement proteins (C3, C4, MBL and MASP-2) were normal in all family members. Impairment of Cls synthesis was observed in the patients` fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the Cls cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of Cls mRNA transcripts in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3` splice site within intron I which increases the size of exon 2 by 87 nucleotides. (c) 2007 Elsevier Ltd. All rights reserved.

On the mechanisms of phenothiazine-induced mitochondrial permeability transition: Thiol oxidation, strict Ca(2+) dependence, and cyt c release

Phenothiazines (PTZ) are drugs widely used in the treatment of schizophrenia. Trifluoperazine, a piperazinic PTZ derivative, has been described as inhibitor of the mitochondrial permeability transition (MPT). We reported previously the antioxidant activity of thioridazine at relatively low concentrations associated to the inhibition of the MPT (Brit. J. Pharmacol., 2002;136:136-142). In this study, it was investigated the induction of MPT by PTZ derivatives at concentrations higher than 10 mu M focusing on the molecular mechanism involved. PTZ promoted a dose-response mitochondrial swelling accompanied by mitochondrial transmembrane potential dissipation and calcium release, being thioridazine the most potent derivative. PTZ-induced MPT was partially inhibited by CsA or Mg(2+) and completely abolished by the abstraction of calcium. The oxidation of reduced thiol group of mitochondrial membrane proteins by PTZ was upstream the VIP opening and it was not sufficient to promote the opening of PTP that only occurred when calcium was present in the mitochondrial matrix. EPR experiments using DMPO as spin trapping excluded the participation of reactive oxygen species on the PTZ-induced MPT. Since 117 give rise to cation radicals chemically by the action of peroxidases and cyanide inhibited the PTZ-induced swelling, we propose that VIZ bury in the inner mitochondrial membrane and the chemically generated 117 cation radicals modify specific thiol groups that in the presence of Ca(2+) result in MPT associated to cytochrome c release. These findings contribute for the understanding of mechanisms of MET induction and may have implications for the cell death induced by PTZ. (C) 2010 Elsevier Inc. All rights reserved.

LC-MS-MS Identification and Determination of the Flavone-C-Glucoside Vicenin-2 in Rat Plasma Samples Following Intraperitoneal Administration of Lychnophora Extract

The flavone C-glucoside, vicenin-2, in semi-purified extracts of the leaves of Lychnophora ericoides was quantified in rat plasma samples using a method based on reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry. Vicenin-2 was analyzed on a LiChrospher (R) RP18 column using an isocratic mobile phase consisting of a mixture of methanol: water (30:70, v/v) plus 2.0% glacial acetic acid at a flow rate of 0.8 mL min(-1). Genistein was used as internal standard. The mass spectrometer was operated in positive ionization mode and analytes were quantified by multiple reaction monitoring at m/z 595 > 457 for vicenin-2 and m/z 271 > 153 for internal standard. Prior to the analysis, each rat plasma sample was acidified with 200 mu L of 50 mmol L(-1) acetic acid solution and extracted by solid-phase extraction using a C18 cartridge. The absolute recoveries were reproducible and the coefficients of variation values were lower than 5.2%. The method was linear over the 12.5 - 1500 ng mL(-1) concentration range and the quantification limit was 12.5 ng mL(-1). Within-day and between-day assay precision and accuracy were studied at three concentration levels (40, 400 and 800 ng mL(-1)) and were lower than 15%. The developed and validated method seems to be suitable for analysis of vicenin-2 in plasma samples obtained from rats that receive a single i.p. dose of 200 mg kg(-1) vicenin-2 extract.

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM`s inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 mu M Ca(2+), DHM (50-250 mu M) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca(2+) efflux and cytochrome c release. DHM (50-250 mu M) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline. (C) 2009 Elsevier Ltd. All rights reserved.

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm x 4.6 mm, 5 mu m) at 35 degrees C under isocratic conditions using 5 mM KH(2)PO(4) buffer solution pH 6.0: methanol: diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1). UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL, plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5,4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether: n-hexane: methanol (50:30:20). In both methods the solvent was evaporated at 40 degrees C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL(-1) plasma (r >= 0.9927), limits of quantification of 50 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL(-1) plasma (r >= 0.9900), limits of quantification of 30 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis. (c) 2008 Elsevier B.V. All rights reserved.