Purification of alpha-toxin from Clostridium perfringens type A in PEG-phosphate aqueous two-phase systems: a factorial study

BACKGROUND: Purification of a-toxin produced by Clostridium perfringens type A in aqueous two-phase systems (ATPS) was studied with a full two-level factorial design on two factors (concentrations of 8000 g mol(-1) PEG and phosphate salt at pH 8.0), to estimate the influence of these factors on the purification results. RESULTS: The partition coefficient (K), purification factor (PF) and activity yield (Y) were strongly influenced by the PEG and phosphate concentrations. Raising the levels of the two factors increased these responses. The highest purification factor (5.7) was obtained with PEG and phosphate concentrations of 17.5% and 15%, respectively. CONCLUSION: These results support the proposal that polymer excluded volume and hydrophobic interactions are the factors that drive the alpha-toxin in PEG/phosphate aqueous two-phase systems. (c) 2008 Society of Chemical Industry

One-pot synthesis of alpha,beta-epoxy ketones by palladium-catalyzed epoxidation-oxidation of terminal allylic alcohols

Described herein is a one-pot synthesis of a,p-epoxy ketones using a palladium-catalyzed epoxidation-oxidation sequence. Functionalized terminal allylic alcohols are treated with m-CPBA Under mild reaction conditions to obtain the alpha,beta-epoxy ketones. The main benefit of this approach is that the epoxidation of the terminal double bond and the oxidation of the secondary alcohol occured in the same reaction under mild reactions and both electron-donating and electron-withdrawing functionalities are tolerated in the reaction sequence. (C) 2010 Elsevier Ltd. All rights reserved.

Alpha-tocopherol supplementation decreases electronegative low-density lipoprotein concentration [LDL(-)] in haemodialysis patients

Background. Oxidative stress is a significant contributor to cardiovascular diseases (CVD) in haemodialysis (HD) patients, predisposing to the generation of oxidized low-density lipoprotein (oxLDL) or electronegatively charged LDL subfraction. Antioxidant therapy such as alpha-tocopherol acts as a scavenger of lipid peroxyl radicals attenuating the oxidative stress, which decreases the formation of oxLDL. The present study was designed to investigate the influence of the alpha-tocopherol supplementation on the concentration of electronegative low-density lipoprotein [LDL(-)], a minimally oxidized LDL, which we have previously described to be high in HD patients. Methods. Blood samples were collected before and after 120 days of supplementation by alpha-tocopherol (400 UI/day) in 19 stable HD patients (50 +/- 7.8 years; 9 males). The concentrations of LDL(-) in blood plasma [using an anti-LDL- human monoclonal antibody (mAb)] and the anti-LDL(-) IgG auto-antibodies were determined by ELISA. Calculation of body mass index (BMI) and measurements of waist circumference (WC), triceps skin folds (TSF) and arm muscle area (AMA) were performed. Results. The plasma alpha-tocopherol levels increased from 7.9 mu M (0.32-18.4) to 14.2 mu M (1.22-23.8) after the supplementation (P = 0.02). The mean concentration of LDL(-) was reduced from 570.9 mu g/mL (225.6-1241.0) to 169.1 mu g/mL (63.6-621.1) (P < 0.001). The anti-LDL(-) IgG auto-antibodies did not change significantly after the supplementation. The alpha-tocopherol supplementation also reduced the total cholesterol and LDL-C levels in these patients, from 176 +/- 42.3 mg/dL to 120 +/- 35.7 mg/dL (P < 0.05) and 115.5 +/- 21.4 mg/dL to 98.5 +/- 23.01 mg/dL (P < 0.001), respectively. Conclusion. The oral administration of alpha-tocopherol in HD patients resulted in a significant decrease in the LDL(-), total cholesterol and LDL-C levels. This effect may favour a reduction in cardiovascular risk in these patients, but a larger study is required to confirm an effect in this clinical setting.

Crystal and molecular structures of dichloro((Z)-2-chloro-2-phenylvinyl)(organo)tellurium(IV) for organo = n-butyl and phenyl

In each of the title compounds, R[Ph(Cl)C=(H)C]TeCl(2), R = nBu (1) and Ph (2), the primary geometry about the Te(IV) atom is a pseudo-trigonal-bipyramidal arrangement, with two Cl atoms in apical positions, and the lone pair of electrons and C atoms in the equatorial plane. As the Te(IV) is involved in two, an intra- and an inter-molecular, Te center dot center dot center dot Cl interactions the coordination geometry might be considered as a Psi-pentagonal bipyramid in each case. In addition, in (2) there is a hint of a Te center dot center dot center dot pi interaction (Te center dot center dot center dot C = 3.911(3) A). The key feature in the crystal structure of both compounds is the formation of supramolecular chains mediated by Te center dot center dot center dot Cl contacts. (1): C(12)H(15)Cl(3)Te, triclinic, P (1) over bar, a = 5.9471 (11), b = 10.7826(22), c = 11.7983(19) angstrom, alpha = 75.416(12), beta = 78.868(13), gamma = 80.902(14)degrees, V = 713.6(2) angstrom(3), Z = 2, R(1) = 0.021; (2): C14HIIC13Te, orthorhombic, Pcab, a=7.7189(10), b=17.415(2), c=21.568(3)angstrom, V = 2899.3(6) angstrom(3), Z = 8, R(1) = 0.027.

The semi-synthetic kaurane ent-16 alpha-methoxykauran-19-oic acid induces vascular relaxation and hypotension in rats

The present work investigates the mechanisms involved in the vasorelaxant effect of ent-16 alpha-methoxykauran-19-oic acid (KA-OCH(3)), a semi-synthetic derivative obtained from the kaurane-type diterpene ent-kaur-16-en-19-oic acid (kaurenoic acid). Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats using standard muscle bath procedures. The cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3 AM. Blood pressure measurements were performed in conscious rats. KA-OCH(3) (10,50 and 100 mu mol/l) inhibited phenylephrine-induced contraction in either endothelium-intact or endothelium-denuded rat aortic rings. KA-OCH(3) also reduced CaCl(2)-induced contraction in a Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 mu mol/l). KA-OCH(3) (0.1-300 mu mol/l) concentration-dependently relaxed endothelium-intact and endothelium-denuded aortas pre-contracted with either phenylephrine or KCl, to a greater extent than kaurenoic acid. Moreover, a Ca(2+) mobilisation study showed that KA-OCH(3) (100 mu mol/l) inhibited the increase in Ca(2+) concentration in smooth muscle and endothelial cells induced by phenylephrine or KCl. Pre-incubation of intact or denuded aortic rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mu mol/l), 7-nitroindazole (100 mu mol/l), wortmannin (0.5 mu mol/l) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 1 mu mol/l) produced a rightward displacement of the KA-OCH(3) concentration-response curve. Intravenous administration of KA-OCH(3) (1-10 mg/kg) reduced mean arterial blood pressure in normotensive rats. Collectively, our results show that KA-OCH(3) induces vascular relaxation and hypotension. The mechanisms underlying the cardiovascular actions of KA-OCH(3) involve blockade of Ca(2+) influx and activation of the NO-cGMP pathway. (C) 2011 Elsevier B.V. All rights reserved.

Isolation and functional characterization of proinflammatory acidic phospholipase A(2) from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated BI-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000 Da and pl was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9 U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-alpha, IL-1 beta and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. (C) 2011 Elsevier Inc. All rights reserved.

Purification and characterization of Ts15, the first member of a new alpha-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: alpha-, beta-, gamma- and kappa-KTx. These peptides display varying selectivity and affinity for K(v) channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Nay channels (Na(V)1.4; Na(V)1.5; Na(V)1.6; Na(V)1.8 and DmNa(V)1) and 12 different subtypes of Kv channels (K(V)1.1 - K(V)1.6; K(V)2.1; K(V)3.1; K(V)4.2; K(V)4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is crosslinked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K(V)1.2 and K(V)1.3 channels with an IC(50) value of 196 +/- 25 and 508 +/- 67 nM, respectively. No effect on Na(V) channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new alpha-Ktx21 subfamily and therefore was classified as alpha-Ktx21.1. (C) 2011 Elsevier Ltd. All rights reserved.

Relaxation evoked by extracellular Ca(2+) in rat aorta is nerve-independent and involves sarcoplasmic reticulum and L-type Ca(2+) channell

The perivascular nerve network expresses a Ca(2+) receptor that is activated by high extracellular Ca(2+) concentrations and causes vasorelaxation in resistance arteries. We have verified the influence of perivascular nerve fibers on the Ca(2+)-induced relaxation in aortic rings. To test our hypothesis, either pre-contracted aortas isolated from rats after sensory denervation with capsaicin or aortic rings acutely denervated with phenol were stimulated to relax with increasing extracellular Ca(2+) concentration. We also studied the role of the endothelium on the Ca(2+)-induced relaxation, and we verified the participation of endothelial/nonendothelial nitric oxide and cyclooxygenise-arachidonic acid metabolites. Additionally, the role of the sarcoplasmic reticulum, K(+) channels and L-type Ca(2+) channels on the Ca(2+)-induced relaxation were evaluated. We have observed that the Ca(2+)-induced relaxation is completely nerve independent, and it is potentiated by endothelial nitric oxide (NO). In endothelium-denuded aortic rings, indomethacin and AH6809 (PGF(2 alpha) receptor antagonist) enhance the relaxing response to Ca(2+). This relaxation is inhibited by thapsigargin and verapamil, while was not altered by tetraethylammonium. In conclusion, we have shown that perivascular nervous fibers do not participate in the Ca(2+)-induced relaxation, which is potentiated by endothelial NO. In endothelium-denuded preparations, indomethacin and AH6809 enhance the relaxation induced by Ca(2+). The relaxing response to Call was impaired by verapamil and thapsigargin, revealing the importance of L-type Ca(2+) channels and sarcoplasmic reticulum in this response. (c) 2008 Elsevier Inc. All rights reserved.

Synthesis of indole derivatives by domino hydroformylation/indolization of 2-nitrocinnamaldehydes

The present work furnishes an innovative preparation of substituted indoles based on tandem hydroformylation, where the chemo- and the regio-selectivities are good, so the yield of the reaction. The novelty has been established in the four-step transformation of substituted alpha nitrocinnamaldehydes into desired indoles in a one-pot reaction. Under hydroformylation reaction conditions we have been able to trigger off a cascade of reactions, which gave substituted indoles in high yields. Useful intermediates are prepared by using this technique for the synthesis of well-known biologically active molecules. (C) 2008 Elsevier B.V. All rights reserved.

Novel and facile solution-phase synthesis of 2,5-diketopiperazines and O-glycosylated analogs

This work describes the synthesis in Solution of a series of related diketopiperazines with potential biological activities: cyclo(L-Pro-L-Ser), cyclo(L-Phe-L-Ser), cyclo(D-Phe-L-Ser) and the corresponding glycosylated analogs of the latter, cyclo[D-Phe-L-Ser(alpha GlcNAc)] and cyclo[D-Phe-L-Ser(beta GlcNAc)]. The synthetic approach involved coupling reactions of -OH or O-glycosylated serine benzyl esters with NFmoc-protected amino acids (Pro or Phe), followed by one-pot deprotection-cyclization reaction in the presence of 20% piperidine in DMF. (C) 2009 Elsevier Ltd. All rights reserved.

Effects of repetitive stress during the acute phase of Trypanosoma cruzi infection on chronic Chagas` disease in rats

The effect of repetitive stress during acute infection with Trypanosoma cruzi (T. cruzi) on the chronic phase of ensuing Chagas` disease was the focus of this investigation. The aim of this study was to evaluate in Wistar rats the influence of repetitive stress during the acute phase of infection (7 days) with the Y strain of T. cruzi on the chronic phase of the infection (at 180 days). Exposure to ether vapor for 1min twice a day was used as a stressor. Repetitive stress enhanced the number of circulating parasites and cardiac tissue disorganization, from a moderate to a severe diffuse mononuclear inflammatory process and the presence of amastigote burden in the cardiac fibers. Immunological parameters revealed that repetitive stress triggered a reduced concanavalin A induced splenocyte proliferation in vitro with major effects on the late chronic phase. Serum interleukin-12 concentration decreased in both stressed and infected rats in the early phase of infection although it was higher on 180 days post-infection. These results suggest that repetitive stress can markedly impair the host`s immune system and enhance the pathological process during the chronic phase of Chagas` disease.

Prostaglandin E(2)-loaded microspheres as strategy to inhibit phagocytosis and modulate inflammatory mediators release

PGE(2), an arachidonic acid metabolite produced by various type of cells regulates a broad range of physiological activities in the endocrine, cardiovascular, gastrointestinal, and immune systems, and is involved in maintaining the local homeostasis. In the immune system, PGE(2) is mainly produced by APCs and it can suppress the Th1-mediated immune responses. The aim of this study was to develop PGE(2)-loaded biodegradable MS that prolong and sustain the in vivo release of this mediator. An o/w emulsion solvent extraction-evaporation method was chosen to prepare the MS. We determined their diameters, evaluated the in vitro release of PGE(2), using enzyme immunoassay and MS uptake by peritoneal macrophages. To assess the preservation of biological activities of this mediator, we determined the effect of PGE(2) released from MS on LPS-induced TNF-alpha release by murine peritoneal macrophages. We also analyzed the effect of encapsulated PGE(2) on inflammatory mediators release from HUVECs. Finally, we studied the effect of PGE(2) released from biodegradable MS in sepsis animal model. The use of this formulation can provide an alternative strategy for treating infections, by modulating or inhibiting inflammatory responses, especially when they constitute an exacerbated profile. (C) 2008 Elsevier B.V. All rights reserved.

Dehydroepiandrosterone increases resistance to experimental infection by Trypanosoma cruzi

Dehydroepiandrosterone (DHEA) enhances immune responses against a wide range of viral, bacterial, and parasitic pathogens. In a previous study, we reported that administration of DHEA significantly decreased the numbers of blood parasites in Trypanosoma cruzi experimental infection. The present study was undertaken to determine the effectiveness of DHEA in reducing the severity of acute phase T cruzi infection of male and female Wistar rats. Animals were treated subcutaneously with 40 mg/kg body weight/day of DHEA. The concentration of nitric oxide (NO) was determined in spleen peritoneal cavity. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were determined in the sera of uninfected and infected animals. DHEA treatment augments NO production for both sexes after in vitro LPS treatment for uninfected animals. Infection triggered enhanced NO levels although not significant. IL-2 and IFN-gamma were detectable in higher concentrations in treated and infected rats of both genders when compared to untreated controls. These data suggest that DHEA may have a potent immunoregulatory function that can affect the course of T cruzi infection. (c) 2008 Elsevier B.V. All rights reserved.

Enhanced protection by melatonin and meloxicam combination in experimental infection by Trypanosoma cruzi

P>The aim of this study was to evaluate a possible synergism between melatonin and meloxicam in up-regulating the immune response in male Wistar rats infected with Trypanosoma cruzi during immunosuppression phenomenon, which characterizes the acute phase of the Chagas` disease. Male Wistar rats were infected with the Y strain of T. cruzi. Experiments were performed on 7, 14 and 21 days post-infection. Several immunological parameters were evaluated including gamma-interferon (IFN-gamma), interleukin-2 (IL-2), nitric oxide (NO) and prostaglandin E(2) (PGE(2)). The combined treatment with melatonin and meloxicam significantly enhanced the release of IL-2 and INF-gamma into animals` serum, when compared with the infected control groups during the course of infection. Furthermore, the blockade of PGE(2) synthesis and the increased release of NO by macrophage cells from T. cruzi-infected animals contributed to regulate the production of Th1 subset cytokines significantly reducing the parasitaemia in animals treated with the combination of both substances. Therefore, our results suggest that the association of melatonin and meloxicam was more effective in protecting animals against the harmful actions of T. cruzi infection as compared with the treatments of meloxicam or melatonin alone.

Trypanosoma cruzi: Do different sylvatic strains trigger distinct immune responses?

Strains of Trypanosoma cruzi are multiclonal populations that can be classified in groups or genotypes, differing in pathogenicity, virulence, and histotropism. In this experiment the distinct behavior of two strains of T. cruzi, MORC-1 and MORC-2, was documented. Blood parasitemia, spleen proliferation, nitric oxide, histopathology of the spleen and heart were used as tools to evaluate parasite persistence. Groups of male mice were separated and divided in three groups: Control (C), Infected (IM-1) and Infected (IM-2). The peak of parasitemia occurred on 10 days post infection for both strains. LPS stimulated animals, infected MORC-2 group displayed significant higher concentrations of NO when compared to infected MORC-1 group (P < 0.05). For ConA stimulated lymphoproliferation, infected MORC-1 group displayed higher proliferation index as compared to infected MORC-2 group. An opposite behavior for IL-4 and TNF-alpha was observed according to the strain. For MORC-1 enhanced concentrations of IL-4 were present with concomitant reduced levels of TNF-alpha, while for MORC-2 enhanced concentrations of TNF-alpha and reduced levels of IL-4 were found. The histopathology of heart and spleen showed important differences in which MORC-1 displayed statistically enhanced number of amastigote in the heart and spleen as compared to MORC-2. Concluding, each strain triggered a distinct immune response with enhanced cytokine TH-1 profile for MORC-2 and TH-2 for MORC-1. (C) 2009 Elsevier Inc. All rights reserved.