Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis

Arthropods display different mechanisms to protect themselves against infections, among which antimicrobial peptides (AMPs) play an important role, acting directly against invader pathogens. We have detected several factors with inhibitory activity against Candida albicans and Micrococcus luteus on the surface and in homogenate of eggs of the tick Rhipicephalus (Boophilus) microplus. One of the anti-M. luteus factors of the egg homogenate was isolated to homogeneity. Analysis by electrospray mass spectrometry (ESI-MS) revealed that it corresponds to microplusin, an AMP previously isolated from the cell-free hemolymph of X (B.) microplus. Reverse transcription (RT) quantitative polymerase chain reactions (qPCR) showed that the levels of microplusin mRNA gradually increase along ovary development, reaching an impressive highest value three days after the adult females have dropped from the calf and start oviposition. Interestingly, the level of microplusin mRNA is very low in recently laid eggs. An enhance of microplusin gene expression in eggs is observed only nine days after the onset of oviposition, achieving the highest level just before the larva hatching, when the level of expression decreases once again. Fluorescence microscopy analysis using an anti-microplusin serum revealed that microplusin is present among yolk granules of oocytes as well as in the connecting tube of ovaries. These results, together to our previous data. suggest that microplusin may be involved not only in protection of adult female hemocele, but also in protection of the female reproductive tract and embryos, what points this AMP as a considerable target for development of new methods to control R. (B.) microplus as well as the vector-borne pathogens. (c) 2009 Elsevier Ltd. All rights reserved.

Tetrachlorocarbonyliridates: Water-Soluble Carbon Monoxide Releasing Molecules Rate-Modulated by the Sixth Ligand

A new family of compounds is presented as potential carbon monoxide releasing molecules (CORMs). These compounds, based on tetrachlorocarbonyliridate(III) derivatives, were synthesized and fully characterized by X-ray diffraction, electrospray mass spectrometry, IR. NMR, and density functional theory calculations. The rate of CO release was studied via the myoglobin assay. The results showed that the rate depends on the nature of the sixth ligand, trans to CO, and that a significant modulation on the release rate can be produced by changing the ligand. The reported compounds are soluble in aqueous media, and the rates of CO release are comparable with those for known CORMs, releasing CO at a rate of 0.03-0.58 mu M min(-1) in a 10 mu M solution of myoglobin and 10 mu M of the complexes.

Tryptophan oxidation by singlet molecular oxygen [O-2 ((1)Delta(g))]: Mechanistic studies using O-18-labeled hydroperoxides, mass spectrometry, and light emission measurements

Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O-2 ((1)Delta(g))]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using O-18-labeled hydroperoxides ((WOOH)-O-18-O-18), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from (WOOH)-O-18-O-18 decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species.

Proteome analysis of human dorsolateral prefrontal cortex using shotgun mass spectrometry

The prefrontal cortex executes important functions such as differentiation of conflicting thoughts, correct social behavior and personality expression, and is directly implicated in different neurodegenerative diseases. We performed a shotgun proteome analysis that included IEF fractionation, RP-LC, and MALDI-TOF/TOF mass spectrometric analysis of tryptic digests from a pool of seven human dorsolateral prefrontal cortex protein extracts. In this report, we present a catalog of 387 proteins expressed in these samples, identified by two or more peptides and high confidence search scores. These proteins are involved in different biological processes such as cell growth and/or maintenance, metabolism/energy pathways, cell communication/signal trarisduction, protein metabolism, transport, regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism, and immune response. This analysis contributes to the knowledge of the human brain proteome by adding sample diversity and protein expression data from an alternative technical approach. It will also aid comparative studies of different brain areas and medical conditions, with future applications in basic and clinical research.

On-line mass spectrometry of the electro-oxidation of methanol in acidic media on tungsten carbide

The electro-oxidation of methanol at supported tungsten carbide (WC) nanoparticles in sulfuric acid solution was studied using cyclic voltammetry, potentiostatic measurements, and differential electrochemical mass spectroscopy (DEMS). The catalyst was prepared by a sonochemical method and characterized by X-ray diffraction. Over the WC catalyst, the oxidation of methanol (1 M in a sulfuric acid electrolyte) begins at a potential below 0.5 V/RHE during the anodic sweep. During potentiostatic measurements, a maximum current of 0.8 mA mg(-1) was obtained at 0.4 V. Measurements of DEMS showed that the methanol oxidation reaction over tungsten carbide produces CO2 (m/z=44); no methylformate (m/z=60) was detected. These results are discussed in the context of the continued search for alternative materials for the anode catalyst of direct methanol fuel cells.

Quantitative ester analysis in cachaca and distilled spirits by gas chromatography-mass spectrometry (GC-MS)

An analytical procedure for the separation and quantification of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl lactate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, isoamyl octanoate, and ethyl laurate in cachaca, rum, and whisky by direct injection gas chromatography-mass spectrometry was developed. The analytical method is simple, selective, and appropriated for the determination of esters in distilled spirits. The limit of detection ranged from 29 (ethyl hexanoate) to 530 (ethyl acetate) mu g L-1, whereas the standard deviation for repeatability was between 0.774% (ethyl hexanoate) and 5.05% (isoamyl octanoate). Relative standard deviation values for accuracy vary from 90.3 to 98.5% for ethyl butyrate and ethyl acetate, respectively. Ethyl acetate was shown to be the major ester in cachaca (median content of 22.6 mg 100 mL(-1) anhydrous alcohol), followed by ethyl lactate (median content of 8.32 mg 100 mL(-1) anhydrous alcohol). Cachaca produced in copper and hybrid alembic present a higher content of ethyl acetate and ethyl lactate than those produced in a stainless-steel column, whereas cachaca produced by distillation in a stainless-steel column present a higher content of ethyl octanoate, ethyl decanoate, and ethyl laurate. As expected, ethyl acetate is the major ester in whiskey and rum, followed by ethyl lactate for samples of rum. Nevertheless, whiskey samples exhibit ethyl lactate at contents lower or at the same order of magnitude of the fatty esters.

Determination of fluoxetine in plasma by gas chromatography-mass spectrometry using stir bar sorptive extraction

This article presents a method employing stir bar sorptive extraction (SBSE) with in situ derivatization, in combination with either thermal or liquid desorption on-line coupled to gas chromatography-mass spectrometry for the analysis of fluoxetine in plasma samples. Ethyl chloroformate was employed as derivatizing agent producing symmetrical peaks. Parameters such as solvent polarity, time for analyte desorption, and extraction time, were evaluated. During the validation process, the developed method presented specificity, linearity (R-2 > 0.99), precision (R.S.D. < 15%), and limits of quantification (LOQ) of 30 and 1.37 pg mL(-1), when liquid and thermal desorption were employed, respectively. This simple and highly sensitive method showed to be adequate for the measurement-of fluoxetine in typical and trace concentration levels. (c) 2008 Elsevier B.V. All rights reserved.

Evaluation of the QuEChERS Method and Gas Chromatography-Mass Spectrometry for the Analysis Pesticide Residues in Water and Sediment

A method for the determination of pesticide residues in water and sediment was developed using the QuEChERS method followed by gas chromatography - mass spectrometry. The method was validated in terms of accuracy, specificity, linearity, detection and quantification limits. The recovery percentages obtained for the pesticides in water at different concentrations ranged from 63 to 116%, with relative standard deviations below 12%. The corresponding results from the sediment ranged from 48 to 115% with relative standard deviations below 16%. The limits of detection for the pesticides in water and sediment were below 0.003 mg L(-1) and 0.02 mg kg(-1), respectively.

Development of a Multicommutated Flow System with Chemiluminometric Detection for Quantification of Gentamicin in Pharmaceuticals

A new flow procedure based on multicommutation with chemiluminometric detection was developed to quantify gentamicin sulphate in pharmaceutical formulations. This approach is based on gentamicin's ability to inhibit the chemiluminometric reaction between luminol and hypochlorite in alkaline medium, causing a decrease in the analytical signal. The inhibition of the analytical signal is proportional to the concentration of gentamicin sulphate, within a linear range of 1 to 4 mu g mL(-1) with a coefficient variation <3%. A sample throughput of 55 samples h(-1) was obtained. The developed method is sensitive, simple, with low reagent consumption, reproducible, and inexpensive, and when applied to the analysis of pharmaceutical formulations (eye drops and injections) it gave results with RSD between 1.10 and 4.40%.

Singlet molecular oxygen trapping by the fluorescent probe diethyl-3,3 '-(9,10-anthracenediyl)bisacrylate synthesized by the Heck reaction

Singlet molecular oxygen O(2)((1)Delta(g)) is a potent oxidant that can react with different biomolecules, including DNA, lipids and proteins. Many polycyclic aromatic hydrocarbons have been studied as O(2)((1)Delta(g)) chemical traps. Nevertheless, a suitable modification in the polycyclic aromatic ring must be made to increase the yield of O(2)((1)Delta(g)) chemical trapping. With this goal, an anthracene derivative, diethyl-3,3 '-(9,10-anthracenediyl)bisacrylate (DADB), was obtained from the reaction of 9,10-dibromoanthracene and ethyl acrylate through the Heck coupling reaction. The coupling of ethyl acrylate with the anthracene ring produced a new lipophilic, esterified, fluorescent probe reactive toward O(2)((1)Delta(g)). This compound reacts with O(2)((1)Delta(g)) at a rate of k(r) = 1.69 x 10(6) M(-1) s(-1) forming a stable endoperoxide (DADBO(2)), which was characterized by UV-Vis, fluorescence, HPLC/MS and (1)H and (13)C NMR techniques. The photophysical, photochemical and thermostability features of DADB were also evaluated. Furthermore, this compound has the potential for great application in biological systems because it is easily synthetized in large amount and generates specific endoperoxide (DADBO(2)), which can be easily detected by HPLC tandem mass spectrometry (HPLC/MS/MS).

Identification of new ozonation disinfection byproducts of 17 beta-estradiol and estrone in water

Estrogens are a class of micro-pollutants found in water at low concentrations (in the ng L(-1) range), but often sufficient to exert estrogenic effects due to their high estrogenic potency. Disinfection of waters containing estrogens through oxidative processes has been shown to lead to the formation of disinfection byproducts, which may also be estrogenic. The present work investigates the formation of disinfection byproducts of 17 beta-estradiol (E2) and estrone (E1) in the treatment of water with ozone. Experiments have been carried out at two different concentrations of the estrogens in ground water (100 ng L(-1) and 100 mu g L(-1)) and at varying ozone dosages (0-30 mg L(-1)). Detection of the estrogens and their disinfection byproducts in the water samples has been performed by means of ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a triple quadrupole (QqQ) and a quadrupole-time of flight (QqTOF) instrument. Both E2 and El have been found to form two main byproducts, with molecular mass (MM) 288 and 278 in the case of E2, and 286 and 276 in the case of El, following presumably the same reaction pathways. The E2 byproduct with MM 288 has been identified as 10epsilon-17beta-dihydroxy-1,4-estradieno-3-one (DEO), in agreement with previously published results. The molecular structures and the formation pathways of the other three newly identified byproducts have been suggested. These byproducts have been found to be formed at both high and low concentrations of the estrogens and to be persistent even after application of high ozone dosages. (C) 2011 Elsevier Ltd. All rights reserved.

Microcystins in South American aquatic ecosystems: Occurrence, toxicity and toxicological assays

The acute poisoning of chronic renal patients during hemodialysis sessions in 1996 in Caruaru City (Pernambuco State, Brazil) stimulated an intensive search for the cause of this severe complication. This search culminated in the identification of microcystins (MC), hepatotoxic cyclic heptapeptides produced by cyanobacteria, as the causative agents. More than ten years later, additional research data provides us with a better understanding of the factors related to cyanobacterial bloom occurrence and production of MC in Brazil and other South American countries. The contamination of water bodies and formation of toxic blooms remains a very serious concern, especially in countries in which surface water is used as the main source for human consumption. The purpose of this review is to highlight the discoveries of the past 15 years that have brought South American researchers to their current level of understanding of toxic cyanobacteria species and that have contributed to their knowledge of factors related to MC production, mechanisms of action and consequences for human health and the environment. (C) 2010 Elsevier Ltd. All rights reserved.

Enzymatic-spectrophotometric determination of paraquat in urine samples: A method based on its toxic mechanism

Paraquat is a broad-spectrum contact herbicide that has been encountered worldwide in several cases of accidental, homicidal, and suicidal poisonings. The pulmonary toxicity of this compound is related to the depletion of NADPH in the pneumocytes, which is continuously consumed by the reduction/oxidation of paraquat and reductase enzyme systems in the presence of O(2) (redox cycling). Based on this mechanism, an enzymatic-spectrophotometric method was developed for the determination of paraquat in urine samples. The velocity of NADPH consumption was monitored at 340 nm, every 10 s during 15 min. The velocity of NADPH oxidation correlated with the paraquat levels found in samples. The enzymatic-spectrophotometric method showed to be sensitive, making possible the detection of paraquat in urine samples at concentrations as low as 0.05 mg/L.

Bioequivalence assay between orally disintegrating and conventional tablet formulations in healthy volunteers

The purpose of this study was to evaluate bioequivalence of two commercial 8 mg tablet formulations of ondansetrona available ill the Brazilian market. In this study, a simple, rapid, sensitive and selective liquid chromarography-tandem mass spectrometry method is described for the determination of ondansetron in human plasma samples. The method was validated over a concentration range of 2.5-60 ng/ml and used in a bioequivalence trial between orally disintegrating and conventional tablet ondansetron formulations, to assess its usefulness in this kind of Study. Vonau flash (R) (Biolab Sanus Farmaceutica, Brazil, as test formulations) and Zofran (R) (GlaxoSmithKline, Brazil, as reference formulation) were evaluated following a single 8 mg close to 23 healthy volunteers of both genders. The dose was administered after an overnight fast according to a two-way crossover design. Bioequivalence between the products was determinated by Calculating 90% confidence interval (90% CI) for the ratio of C(max), AUC(0-t) and AUC(0-(sic)) values for the test and reference products, using logarithmically transformed data. The 90% confidence interval for the ratio of C(max) (87.5-103.8%), AUC(0-t) (89.3-107.2%) and AUC(0--(sic)) (89.7-106.0%) values for the test and reference products is Within the 80-125% interval, proposed by FDA, EMEA and ANVISA. It was concluded that two ondansetron formulations are bioequivalent ill their rate and extent of absorption. (C) 2008 Elsevier B.V. All rights reserved.

Development and validation of sensitive methods for determination of sibutramine hydrochloride monohydrate and direct enantiomeric separation on a protein-based chiral stationary phase

Sibutramine hydrochloride monohydrate, chemically 1-(4-chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl) hydrochloride monohydrate (SB center dot HCl center dot H2O), was approved by the U.S. Food and Drug Administration for the treatment of obesity. The objective of this study was to develop, validate, and compare methods using UV-derivative spectrophotometry (UVDS) and reversed-phase high-performance liquid chromatography (HPLC) for the determination of SB center dot HCl center dot H2O in pharmaceutical drug products. The UVDS and HPLC methods were found to be rapid, precise, and accurate. Statistically, there was no significant difference between the proposed UVDS and HPLC methods. The enantiomeric separation of SB was obtained on an alpha-1 acid glycoprotein column. The R- and S-sibutramine were eluted in < 5 min with baseline separation of the chromatographic peaks (alpha = 1.9 and resolution = 1.9).