Thermal behavior of malonylglucoside isoflavones in soybean flour analyzed by RPHPLC/DAD and eletrospray ionization mass spectrometry

Soybean (Glycine max (Merrill) L) contains high content of aglycone isoflavones, as well as glucoside and malonylconjugates. In this work, the content of isoflavones in defatted soy flour was determined by reversed-phase high-performance liquid chromatography (RPHPLC) after alcoholic extraction in methanol/water mixture in the ratio 80:20 (v/v). It was observed that the heating treatment transformed the malonylglucosides into glucoside isoflavones. After heat treatment at 121 degrees C for 30 min, nearly all malonylisoflavones were converted into glucoside, but acetylisoflavones were not detected via RPHPLC analysis. Electrospray ionization mass spectrometry confirmed the presence of malonylisoflavones in heat-treated defatted soy flour by direct infusion analysis. (c) 2012 Elsevier Ltd. All rights reserved.

Further Withaphysalin Derivatives from Acnistus arborescens

Two new epimeric chlorinated withaphysalins, rel-(4 beta,5 beta,6 alpha,18S,22R)- and rel-(4 beta,5 beta,6 alpha,18R,22R)-6-chloro-18,20-epoxy-18-ethoxy-4,5-dihydroxy-1- oxowitha-2,24-diene-26,22-lactone (1 and 2 resp.), together with the new rel-(4 beta,5 beta,6a,18R,22R)-6-chloro-18,20-epoxy-4,5-dihydroxy-18-methoxy-1-oxowitha-2,24-diene-26,22-lactone (3) and rel-(3 beta,4 beta,5 beta,6 beta,18R,22R)-5,6:18,20-diepoxy-3,18-diethoxy-4-hydroxy-1-oxowith-24-ene-26,22-lactone (4) were isolated from the leaves of Acnistus arborescens and named withaphysalins TW, respectively. The final structures and the complete 1H- and 13C-NMR assignments of the three chlorowithaphysalins 13 were performed by means of HR-ESI-MS and 1D- and 2D-NMR experiments, including COSY, HSQC, and HMBC, beside comparison with spectral data of analogous compounds from the literature. The structure of 4 was also confirmed by means of a single-crystal X-ray diffraction analysis.

Validation of the Subjective Handicap of Epilepsy (SHE) in Brazilian patients with epilepsy

The objectives of the study were to translate and adapt the Subjective Handicap of Epilepsy (SHE) instrument to Brazilian Portuguese and to determine its psychometric properties for the evaluation of quality of life in patients with epilepsy. A sample of 448 adult patients with epilepsy with different clinical profiles (investigation, preoperative period, postoperative period, and drug treatment follow-up) was evaluated with the SHE and the Epilepsy Surgery Inventory (ESI-55). Exploratory factorial analysis demonstrated that four factors explained 60.47% of the variance and were sensitive to discriminate the different clinical groups, with the preoperative group having the poorest quality of life. Internal consistency ranged from 0.92 to 0.96, and concurrent validity with the ESI-55 was moderate/strong (0.32-0.70). Test-retest reliability was confirmed, with an ICC value of 0.54 (2 days), 0.91 (7 days), and 0.97 (30 days). The SHE had satisfactory psychometric qualities for use in the Brazilian population, similar to those of the original version. The instrument seems to be more adequate in psychometric terms for the postoperative and drug treatment follow-up groups, and its use should be encouraged. (c) 2012 Elsevier Inc. All rights reserved.

Oxidation of lysergic acid diethylamide (LSD) by peroxidases: a new metabolic pathway

Lysergic acid diethylamide (LSD) is a potent hallucinogen that is primarily metabolized to 2-oxo-3-hydroxy-LSD (O-H-LSD) and N-desmethyl-LSD (nor-LSD) by cytochrome P450 complex liver enzymes. Due to its extensive metabolism, there still is an interest in the identification of new metabolites and new routes of its metabolism in humans. In the present study, we investigated whether LSD could be a substrate for horseradish peroxidase or myeloperoxidase (MPO). Using liquid chromatography coupled to UV detection and electrospray ionization mass spectrometry (LC-UV-ESI-MS), we found that both peroxidases were capable of metabolizing LSD to the same compounds that have been observed in vivo (i.e., O-H-LSD and nor-LSD). In addition, we found another major metabolite, N,N-diethyl-7-formamido-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide (FOMBK), which is an opened indolic ring compound. Hydrolysis of FOMBK led to the deformylated compound 7-amino-N,N-diethyl-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide. The reactions of LSD with the peroxidases were chemiluminescent and sensitive to inhibition by reactive oxygen scavengers, which indicated that the classic peroxidase cycle is involved in this new alternative metabolic pathway. Considering that MPO is abundant in immune cells and also present in the central nervous system, the degradation pathway described in this study suggests a possible route of LSD metabolism that may occur concurrently with the in vivo reaction catalyzed by the cytochrome P450 system.

STUDY BY MASS SPECTROMETRY OF SOLUTIONS OF [HYDROXY(TOSYLOXY)IODO]BENZENE: PROPOSED DISPROPORTIONATION MECHANISMS

STUDY BY MASS SPECTROMETRY OF SOLUTIONS OF [HYDROXY(TOSYLOXY)IODO]BENZENE: PROPOSED DISPROPORTIONATION MECHANISMS. Solutions of [hydroxy(tosyloxy)iodo]benzene (HTIB or Koser's reagent) in acetonitrile were analyzed using high resolution electrospray ionization mass spectrometry (ESI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) under different conditions. Several species were characterized in these analyses. Based on these data, mechanisms were proposed for the disproportionation of the iodine(III) compounds in iodine(V) and iodine(I) species.

Synthesis and characterization of a diruthenium(II,III)-ketoprofen compound and study of the in vitro effects on CRC cells in comparison to the naproxen and ibuprofen derivatives

A new diruthenium(II,III) complex, of formula [Ru2Cl(ket)(4)], Ruket, containing the non-steroidal anti-inflammatory drug ketoprofen was synthesized and mainly characterized by electrospray ionization mass spectrometry (ESI-MS), UV-Vis-IR electronic spectroscopy and FTIR and Raman vibrational spectroscopies. The four drug-carboxylato bridging ligands stabilize a Ru-2(II,III) mixed valent core in a paddlewheel type structure as confirmed by ESI mass spectra, electronic and vibrational spectroscopies and magnetic measurements. Ruket and the analogous compounds containing ibuprofen, Ruibp, and naproxen, Runpx, were tested for the biological effects in the human colon carcinoma cells HT-29 and Caco-2 expressing high and low levels of COX-2 respectively. All compounds only weakly affected the proliferation of the colorectal cancer cells HT-29 and Caco-2, and similarly only partially inhibited the production/activity of MMP-2 and MMP-9 by HT-29 cells, suggesting that COX-2 inhibition by these drugs can only partially be involved in the pharmacological effects of these derivatives. (c) 2012 Elsevier Ltd. All rights reserved.

Tuning the reaction products of ruthenium and ciprofloxacin for studies of DNA interactions

This work presents two potential metallo-drugs, the ionic (C17H19FN3O3)(3)[RuCl6]center dot 3H(2)O (1) and the coordination [Ru(C17H17FN3O3)(3)]center dot 4H(2)O (2) compounds, obtained by the combination of ruthenium(III) and ciprofloxacin in different synthetic conditions. The ESI MS spectrum of 1 displayed a main peak at m/z = 994.6, assigned to the gaseous phase adduct (ciprofloxacin)(3)center dot H+, while 2 featured peaks at m/z 1093.3 and 547.1 ascribed to [Ru(C17H17FN3O3)(3)center dot H+-4H(2)O](+) and [Ru(C17H17FN3O3)(3)center dot 2H(+)-4H(2)O](2+). Thermal analysis corroborated the proposed water content for both complexes. Absorption spectra of the compounds in aqueous medium are dominated by ciprofloxacin transitions in the UV region but displayed weak bands in the visible region, assigned to ligand field transitions. The cyclic voltammograms of 2 exhibited a quasi-reversible process ascribed to the Ru(II)/(III) redox pair at -0.25V (vs. SHE) while 1 displayed this process at -0.11 V, showing that the central ruthenium ion is stabilized in the (III) oxidation state by the coordination to the hard oxygen atoms of ciprofloxacin. The solubility of 1 is pH dependent (as well as free ciprofloxacin) while 2 is fully water soluble and stable under physiological pH for at least 48 h. The compounds are also stable under incubation conditions (stomach pH and 37 degrees C) without significant pH lowering. An interaction study of 2 with ct-DNA showed a value of K-b = 2.47 (+/- 0.89) x 10(4) mol(-1) L for the intrinsic binding constant.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56-4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% alpha-helix, 39% beta-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys(52) residue and the amino acids Pro(45), Thr(49) and Arg(128) are conserved as in other 2-Cys-Prx. General significance: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. (C) 2012 Elsevier B.V. All rights reserved.

Purification and characterization of a surfactin-like molecule produced by Bacillus sp. H2O-1 and its antagonistic effect against sulfate reducing bacteria

Abstract Background Bacillus sp. H2O-1, isolated from the connate water of a Brazilian reservoir, produces an antimicrobial substance (denoted as AMS H2O-1) that is active against sulfate reducing bacteria, which are the major bacterial group responsible for biogenic souring and biocorrosion in petroleum reservoirs. Thus, the use of AMS H2O-1 for sulfate reducing bacteria control in the petroleum industry is a promising alternative to chemical biocides. However, prior to the large-scale production of AMS H2O-1 for industrial applications, its chemical structure must be elucidated. This study also analyzed the changes in the wetting properties of different surfaces conditioned with AMS H2O-1 and demonstrated the effect of AMS H2O-1 on sulfate reducing bacteria cells. Results A lipopeptide mixture from AMS H2O-1 was partially purified on a silica gel column and identified via mass spectrometry (ESI-MS). It comprises four major components that range in size from 1007 to 1049 Da. The lipid moiety contains linear and branched β-hydroxy fatty acids that range in length from C13 to C16. The peptide moiety contains seven amino acids identified as Glu-Leu-Leu-Val-Asp-Leu-Leu. Transmission electron microscopy revealed cell membrane alteration of sulfate reducing bacteria after AMS H2O-1 treatment at the minimum inhibitory concentration (5 μg/ml). Cytoplasmic electron dense inclusions were observed in treated cells but not in untreated cells. AMS H2O-1 enhanced the osmosis of sulfate reducing bacteria cells and caused the leakage of the intracellular contents. In addition, contact angle measurements indicated that different surfaces conditioned by AMS H2O-1 were less hydrophobic and more electron-donor than untreated surfaces. Conclusion AMS H2O-1 is a mixture of four surfactin-like homologues, and its biocidal activity and surfactant properties suggest that this compound may be a good candidate for sulfate reducing bacteria control. Thus, it is a potential alternative to the chemical biocides or surface coating agents currently used to prevent SRB growth in petroleum industries.

Characterization and optimization of ArtinM lectin expression in Escherichia coli

Background ArtinM is a D-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM) in Escherichia coli system. Results The ArtinM coding region was inserted in pET29a(+) vector and expressed in E. coli BL21(DE3)-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C) and shaking speeds (130, 200 or 220 rpm) during induction, concentrations of the induction agent IPTG (0.01-4 mM) and periods of induction (1-19 h). BL21-CodonPlus(DE3)-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h) resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized D-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM). The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises β-sheet structure. Conclusions Overall, the optimized process to express rArtinM in E. coli provided high amounts of soluble, correctly folded and active recombinant protein, compatible with large scale production of the lectin.

Estudo por espectrometria de massas de soluções de hidróxi(tosilóxi)iodobenzeno: propostas para mecanismos de desproporção

Solutions of [hydroxy(tosyloxy)iodo]benzene (HTIB or Koser's reagent) in acetonitrile were analyzed using high resolution electrospray ionization mass spectrometry (ESI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) under different conditions. Several species were characterized in these analyses. Based on these data, mechanisms were proposed for the disproportionation of the iodine(III) compounds in iodine(V) and iodine(I) species.

Antiproliferative activity of Eremanthus crotonoides extracts and centratherin demonstrated in brain tumor cell lines

The genus Eremanthus is recognized by the predominance of sesquiterpene lactones from the furanoheliangolide type, a class of substances extensively tested against cancer cell lines. Thus, the species E. crotonoides (DC.) Sch. Bip., Asteraceae, obtained on "restinga" vegetation was evaluated against U251 and U87-MG glioma cell lines using the MTT colorimetric assay. Dichloromethane fraction was cytotoxic to both glioblastoma multiforme cell lines. We then conducted UPLC-PDA-ESI-MS/MS analysis of the dichloromethane fraction, which allowed the identification of the sesquiterpene lactones centratherin and goyazensolide. The isolation of centratherin was performed using chromatographic techniques and the identification of this substance was confirmed according to NMR data. Cytotoxic activity of centratherin alone was also evaluated against both U251 and U87-MG cells, which showed IC50 values comparable with those obtained for the commercial anticancer drug doxorubicin. All the tested samples showed cytotoxic activity against glioblastoma multiforme cells which suggests that E. crotonoides extracts may be important sources of antiproliferative substances and that the centratherin may serve as prototype for developing new antiglioblastoma drugs.

A study of the anti-plasmodium activity of angiotensin II analogs

Controlling the dissemination of malaria requires the development of new drugs against its etiological agent, a protozoan of the Plasmodium genus. Angiotensin II and its analog peptides exhibit activity against the development of immature and mature sporozoites of Plasmodium gallinaceum. In this study, we report the synthesis and characterization of angiotensin II linear and cyclic analogs with anti-plasmodium activity. The peptides were synthesized by a conventional solid-phase method on Merrifield's resin using the t-Boc strategy, purified by RP-HPLC and characterized by liquid chromatography/ESI (+) MS (LC-ESI(+)/MS), amino acid analysis, and capillary electrophoresis. Anti-plasmodium activity was measured in vitro by fluorescence microscopy using propidium iodine uptake as an indicator of cellular damage. The activities of the linear and cyclic peptides are not significantly different (p < 0.05). Kinetics studies indicate that the effects of these peptides on plasmodium viability overtime exhibit a sigmoidal profile and that the system stabilizes after a period of 1 h for all peptides examined. The results were rationalized by partial least-square analysis, assessing the position-wise contribution of each amino acid. The highest contribution of polar amino acids and a Lys residue proximal to the C-terminus, as well as that of hydrophobic amino acids in the N-terminus, suggests that the mechanism underlying the anti-malarial activity of these peptides is attributed to its amphiphilic character.