Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses: therefore, the knowledge of the volumetric mass transfer coefficient (k(L)a) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k(L)a values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium. in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2(2) full factorial design. Both variables showed statistically significant effects on k(L)a, and the highest values of this parameter in both media for simple fermentation (241 s(-1)) and extractive fermentation with ATPS (70.3 s(-1)) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k(L)a values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N(3)D(2)) and superficial gas velocity (V(s)) determined in distilled water (alpha = 0.39 and beta = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (alpha=0.38 and beta=0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (alpha=0.50 and beta=1.0). A reasonable agreement was found between the experimental data of k(L)a for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions. (C) 2009 Elsevier B.V. All rights reserved.

Separation and partitioning of Green Fluorescent Protein from Escherichia coli homogenate in poly(ethylene glycol)/sodium-poly(acrylate) aqueous two-phase systems

The partitioning of Green Fluorescent Protein (GFP) in poly(ethylene glycol)/Na-poly(acrylate) aqueous two-phase systems (PEG/NaPA-ATPS) has been investigated. The aqueous two-phase systems are formed by mixing the polymers with a salt and a protein solution. The protein partitioning in the two-phase system was investigated at 25 degrees C. The concentration of the GFP was measured by fluorimetry. It was found that the partitioning of GFP depends on the salt type, pH and concentration of PEG. The data indicates that GFP partitions more strongly to the PEG phase in presence of Na2SO4 relative to NaCl. Furthermore, the GFP partitions more to the PEG phase at higher pH. The partition to the PEG phase is strongly favoured in systems with larger tie-line lengths (i.e. systems with higher polymer concentrations). The molecular weight of PEG is important since the partition coefficient (K) of GFP gradually decreases with increasing PEG size, from K ca. 300-400 for PEG 400 to K equal to 1.19 for PEG 8000. A separation process was developed where GFP was separated from a homogenate in two extraction steps: the GFP is first partitioned to the PEG phase in a PEG 3000/NaPA 8000 system containing 3 wt% Na2SO4, where the K value of GFP was 8. The GFP is then re-extracted to a salt phase formed by mixing the previous top-phase with a Na2SO4 solution. The K-value of GFP in this back-extraction was 0.22. The total recovery based on the start material was 74%. (c) 2008 Elsevier B.V. All rights reserved.

Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems

The partition of hemoglobin, lysozyme and glucose-6-phospate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na2SO4, pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively. (c) 2007 Elsevier B.V. All rights reserved.

Phenolic compounds content and antioxidant activity in pomace from selected red grapes (Vitis vinifera L. and Vitis labrusca L.) widely produced in Brazil

The phenolic compounds content and antioxidant activity of pomace from the vinification of grape varieties widely produced in Brazil (Cabernet Sauvignon, Merlot, Bordeaux and Isabel) were investigated with a view to their exploitation as a potential source of natural antioxidants. Cabernet Sauvignon grape pomace was found to have the highest content of total phenolic compounds (74.75 mg gallic acid equivalent (GAE)/g), the highest antioxidant activity (determined using the 2,2`-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging methods; 485.42 and 505.52 mu Mol Trolox equivalent antioxidant capacity (TEAC)/g, respectively), and the highest reducing power (determined using the FRAP method; 249.46 mu Mol TEAC/g). The Bordeaux variety showed the highest oxidation inhibition power (41.13%), determined using the beta-carotene/linoleic acid method and the highest content of total anthocyanins (HPLC; 29.17 mg/g). Catechin was the most abundant non-anthocyanic compound identified in the grape pomace (150.16 mg/100 g) for all varieties. In this study, pomaces of the red wine vinification of Cabernet Sauvignon and Bordeaux varieties showed the highest potential as a source of antioxidant compounds and natural colourants, respectively. (C) 2011 Elsevier Ltd. All rights reserved.

Modulation of human neutrophil oxidative metabolism and degranulation by extract of Tamarindus indica L. fruit pulp

The tamarind (Tamarindus indica L) is indigenous to Asian countries and widely cultivated in the American continents. The tamarind fruit pulp extract (ExT), traditionally used in spices, food components and juices, is rich in polyphenols that have demonstrated anti-atherosclerotic, antioxidant and immunomodulatory activities. This study evaluated the modulator effect of a crude hydroalcoholic ExT on some peripheral human neutrophil functions. The neutrophil reactive oxygen species generation, triggered by opsonized zymosan (OZ), n-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and assessed by luminol- and lucigenin-enhanced chemiluminescence (LumCL and LucCL, respectively), was inhibited by ExT in a concentration-dependent manner. ExT was a more effective inhibitor of the PMA-stimulated neutrophil function [IC(50) (in mu g/10(6)cells) = 115.7 +/- 9.7 (LumCL) and 174.5 +/- 25.9 (LucCL)], than the OZ- [IC(50) = 248.5 +/- 23.1 (LumCL) and 324.1 +/- 34.6 (LucCL)] or fMLP-stimulated cells [IC(50) = 178.5 +/- 12.2 (LumCL)]. The ExT also inhibited neutrophil NADPH oxidase activity (evaluated by O(2) consumption), degranulation and elastase activity (evaluated by spectrophotometric methods) at concentrations higher than 200 mu g/10(6) cells, without being toxic to the cells, under the conditions assessed. Together, these results indicate the potential of ExT as a source of compounds that can modulate the neutrophil-mediated inflammatory diseases. (C) 2008 Elsevier Ltd. All rights reserved.

Neuropharmacological effects in mice of Lychnophora species (Vernonieae, Asteraceae) and anticonvulsant activity of 4,5-di-O-[E]-caffeoylquinic acid isolated from the stem of L-rupestris and L-staavioides

Aiming at contributing with the search for neuroactive substances from natural sources, we report for the first time antinociceptive and anticonvulsant effects of some Lychnophora species. We verify the protective effects of polar extracts (600 mg/kg, intraperitoneally), and methanolic fractions of L. staavioides and L. rupestris (100 mg/kg, intraperitoneally) in pentylenetetrazole-induced seizures on mice. Previously, a screening was accomplished, evaluating the antinociceptive central activity (hot plate test), with different extracts of L. rupestris, L. staavioides and L. diamantinana. It was possible to select the possible extracts of Lychnophora with central nervous system activity. Some of the active extracts were submitted to fractionation and purification process and the methanolic fractions of L. rupestris (stem) and L. staavioides (stem), with anticonvulsant properties (100 mg/kg, intraperitoneally), yielded 4,5-di-O-[E]-caffeoylquinic acid. This substance was injected intraperitoneally in mice and showed anticonvulsant effect against pentylenetetrazole-induced seizures at doses of 25 and 50 mg/kg. It has often been shown that seizures induced by pentylenetetrazole are involved in inhibition and/or attenuation of GABAergic neurotransmission. However, other systems of the central nervous system such as adenosinergic and glutamatergic could be involved in the caffeoylquinic acid effects. Further studies should be conducted to verify that the target receptor could be participating in this anticonvulsant property. Although other investigations have reported a series of biological activities from Lychnophora species, this is the first report of central analgesic and anticonvulsant activity in species of this genus.

Protective effects of Mangifera indica L extract (Vimang), and its major component mangiferin, on iron-induced oxidative damage to rat serum and liver

In vivo preventive effects of a Mangifera indica L extract (Vimang) or its major component mangiferin on iron overload injury have been studied in rats given respectively, 50, 100, 250 mg kg(-1) body weight of Vimang, or 40 mg kg(-1) body weight of mangiferin, for 7 days prior to, and for 7 days following the administration of toxic amounts of iron-dextran. Both Vimang or mangiferin treatment prevented iron overload in serum as well as liver oxidative stress, decreased serum and liver lipid peroxidation, serum GPx activity, and increased serum and liver GSH, serum SOD and the animals overall antioxidant condition. Serum iron concentration was decreased although at higher doses, Vimang tended to increase it; percent tranferrin saturation, liver weight/body mass ratios, liver iron content was decreased. Treatment increased serum iron-binding capacity and decreased serum levels of aspartate-amine transferase (ASAT) and alanine-amine transferase (ALAT), as well as the number of abnormal Kupffer cells in iron-loaded livers. It is suggested that besides acting as antioxidants, Vimang extract or its mangiferin component decrease liver iron by increasing its excretion. Complementing earlier in vitro results from our group, it appears possible to support the hypothesis that Vimang and mangiferin present therapeutically useful effects in iron overload related diseases. (C) 2007 Elsevier Ltd. All rights reserved.

Determination of beta-artemether and its main metabolite dihydroartemisinin in plasma employing liquid-phase microextraction prior to liquid chromatographic-tandem mass spectrometric analysis

A method for the determination of artemether (ART) and its main metabolite dihydroartemisinin (DHA) in plasma employing liquid-phase microextraction (LPME) for sample preparation prior to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed. The analytes were extracted from 1 nil, of plasma utilizing a two-phase LPME procedure with artemisinin as internal standard. Using the optimized LPME conditions, mean absolute recovery rates of 25 and 32% for DHA and ART, respectively, were achieved using toluene-n-octanol (1:1, viv) as organic phase with an extraction time of 30 min. After extraction, the analytes were resolved within 5 min using a mobile phase consisting of methanol-ammonium acetate (10 mmol L(-1) pH 5.0, 80:20. v/v) on a laboratory-made column based on poly(methyltetradecylsiloxane) attached to a zirconized-silica support. MS-MS detection was employed using an electrospray interface in the positive ion mode. The method developed was linear over the range of 5-1000 ng mL(-1) for both analytes. Precision and accuracy were within acceptable levels of confidence (<15%). The assay was applied to the determination of these analytes in plasma from rats treated with ART. The two-phase LPME procedure is affordable and the solvent consumption was very low compared to the traditional methods of sample preparation. (C) 2010 Elsevier B.V. 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.

Preparation and characterization of D, L-PLA loaded 17-beta-Estradiol valerate by emulsion/evaporation methods

PLA microparticles containing 17-beta-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718-880 nm (inert microparticles) and 3-4 mu m (drug loaded microparticles). The encapsulation efficiency was similar to 80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-beta-estradiol.

Novel reports of glands in Neotropical species of Indigofera L. (Leguminosae, Papilionoideae)

By considering controversial discussions in the literature with regard to gland denomination in Indigofera species, as well as the taxonomic value of secretory structures in Leguminosae, we aim to morphologically detail glands that had been previously observed in I. microcarpa and I. sabulicola, and to investigate the occurrence of glands in vegetative and reproductive organs of other six Neotropical species that belong to the genus. Glands analyzed through scanning electronic microscopy (SEM) in combination with anatomic analyses correspond to secretory trichomes that Lire classified into seven types. Main variations in relation to types occurred with regard to head shape and peduncle size. Trichome heads were multicellular, with a thin cuticle. Hollow heads with conspicuous inner space characterized only one type (type I); the other trichome types had massive heads. Peduncles, which varied from biseriate to multiseriate, had thick, pecto-cellulosic cell walls. Trichomes were found on sterns, stipules, petioles, rachis, petiolules, leaflets, bracteoles, sepals, standards and fruits, more commonly along the margins. Each of the eight Indigofera species analyzed had at least two different trichome types out of the seven types that occurred in reproductive and vegetative organs of these taxa. Various types of secretory trichomes were found in I. campestris, I. lespedezioides, I. microcarpa, I. spicata. I. Suffruticosa and I. truxillensis. Stems and rachis were the vegetative organs in which a greater variety of trichomes occurred, and sepals were parts of reproductive organs with the same status. Five out of the seven secretory trichome types occurred on both vegetative and reproductive organs. Distribution and gland types differed between species and these gland distribution patterns can be used as diagnostic characters. Reports of glands in Indigofera campestris, I. hirsuta, I. lepedezioides, I. suffruticosa, I. spicata and I. truxillensis, their recognition as secretory trichomes. and the morphological variety of types found for such trichomes are novel data for Indigofera. (C) 2008 Elsevier GmbH. All rights reserved.

Antimicrobial activity of crude extratcs of Petiveria alliacea L.

Antimicrobial activity of crude extratcs of Petiveria alliacea L.. Petiveria alliacea L. (Phytolaccacea) is an herbaceous plant of great importance in traditional medicine. This species have been widely used in several applications such as antirheumatic, anticarcinogenic, anti-flu, antitussive, analgesic, insecticidal, acaricidal, as well as bactericide and fungicide. Currently, the pathogenic microorganisms are acquiring resistence against the traditional antibiotics, and the search for new herbal antimicrobial agents has been intensified. The objective of this study was to evaluate the antifungal and antibacterial activity of several leaf crude extracts of P. alliacea against several strains of bacterias and yeasts namely Bacilus subtilis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Candida parapsilosis, Candida kefyr and Candida albicans, using microdilution method. Promising results were observed for the 70% v/v ethanolic extract which presented minimum inhibitory concentration (MIC) from 250 to 760 mu g/mL for yeast. For the bacteria strains tested the MIC ranged between 240 to 3960 mu g/mL, depending of the extractive solution tested.

Evidence of caspase-mediated apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom

The aim of this work was to investigate the involvement of caspases in apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAA0. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in ""Asp and Glu"" residues. It displays high specificity toward hydrophobic L-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H(2)O(2) production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances. Published by Elsevier Inc.

Antiviral and antiparasite properties of an L-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence (Retracted article. See vol. 80, pg. 288, 2010)

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pl of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. (C) 2008 Elsevier Inc. All rights reserved.

Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom

The present article describes an L-amino acid oxidase from Bothrops atrox snake venom as with antiprotozoal activities in Trypanosoma cruzi and in different species of Leishmania (Leishmania braziliensis, Leishmania donovani and Leishmania major). Leishmanicidal effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Leishmania spp. cause a spectrum of diseases, ranging from self-healing ulcers to disseminated and often fatal infections, depending on the species involved and the host`s immune response. BatroxLAAO also displays bactericidal activity against both Gram-positive and Gram-negative bacteria. The apoptosis induced by BatroxLAAO on HL-60 cell lines and PBMC cells was determined by morphological cell evaluation using a mix of fluorescent dyes. As revealed by flow cytometry analysis, suppression of cell proliferation with BatroxLAAO was accompanied by the significant accumulation of cells in the G0/G1 phase boundary in HL-60 cells. BatroxLAAO at 25 mu g/mL and 50 mu g/mL blocked G0-G1 transition, resulting in G0/G1 phase cell cycle arrest, thereby delaying the progression of cells through S and G2/M phase in HL-60 cells. This was shown by an accentuated decrease in the proportion of cells in S phase, and the almost absence of G2/M phase cell population. BatroxLAAO is an interesting enzyme that provides a better understanding of the ophidian envenomation mechanism, and has biotechnological potential as a model for therapeutic agents. (C) 2011 Elsevier Masson SAS. All rights reserved.