Behavior and Electrophysiological Response of Gravid and Non-Gravid Lucilia cuprina (Diptera: Calliphoridae) to Carrion-Associated Compounds.

The Australian blow fly, Lucilia cuprina Wiedmann (Diptera: Calliphoridae), is a major cause of myiasis (flystrike) in Merino sheep in Australia and New Zealand and, as a primary colonizer of fresh carrion, also an important species in forensic investigations. Olfaction is considered the most important cue for insects to rapidly locate carrion over long distances, so the first carrion visitors are predicted to be very sensitive to carrion-related volatile compounds. We studied the responses of the Australian blow fly, Lucilia cuprina, to the carrion-associated compounds dimethyl trisulfide (DMTS), butyric acid, 1-octen-3-ol and indole. We also tested 2-mercaptoethanol, a compound commonly used in fly traps in Australia. We investigated whether responses of the flies are affected by their ovarian status by comparing responses of gravid and non-gravid L. cuprina in electroantennography (EAG) and two-choice laboratory bioassays. All four compounds evoked an EAG response, while only DMTS evoked responses in gas chromatography-mass spectrometry electroantennographic detection (GCMS-EAD) analyses and two-choice bioassays. Gravid flies detected lower doses of the test compounds than non-gravid flies. Our results indicate that DMTS is an important semiochemical for L. cuprina to locate carrion resources, and has potential for use in fly traps for flystrike control. Our observations also suggest that the greater sensitivity of gravid L. cuprina allows them to find fresh carrion quickly to maximize reproductive success by avoiding unsuitable degraded carrion.

Sensory classification of table olives using an electronic tongue: Analysis of aqueous pastes and brines.

Table olives are highly appreciated and consumed worldwide. Different aspects are used for trade category classification being the sensory assessment of negative defects present in the olives and brines one of the most important. The trade category quality classification must follow the International Olive Council directives, requiring the organoleptic assessment of defects by a trained sensory panel. However, the training process is a hard, complex and sometimes subjective task, being the low number of samples that can be evaluated per day a major drawback considering the real needs of the olive industry. In this context, the development of electronic tongues as taste sensors for defects' sensory evaluation is of utmost relevance. So, an electronic tongue was used for table olives classification according to the presence and intensity of negative defects. Linear discrimination models were established based on sub-sets of sensor signals selected by a simulated annealing algorithm. The predictive potential of the novel approach was first demonstrated for standard solutions of chemical compounds that mimic butyric, putrid and zapateria defects (≥93% for cross-validation procedures). Then its applicability was verified; using reference table olives/brine solutions samples identified with a single intense negative attribute, namely butyric, musty, putrid, zapateria or winey-vinegary defects (≥93% cross-validation procedures). Finally, the E-tongue coupled with the same chemometric approach was applied to classify table olive samples according to the trade commercial categories (extra, 1st choice, 2nd choice and unsuitable for consumption) and an additional quality category (extra free of defects), established based on sensory analysis data. Despite the heterogeneity of the samples studied and number of different sensory defects perceived, the predictive linear discriminant model established showed sensitivities greater than 86%. So, the overall performance achieved showed that the electrochemical device could be used as a taste sensor for table olives organoleptic trade successful classification, allowing a preliminary quality assessment, which could facilitate, in the future, the complex task of sensory panelists.

Effect of ß-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos / Efeito do ß-mercaptoetanol e da cisteína sobre a qualidade e a atividade oxidativa do sêmen ovino após o descongelamento e sobre a viabilidade de embriões ovinos vitrificados

The effects of ß-mercaptoethanol (BME) and cysteine on the viability and oxidative activity of ram sperm after thawing and on development in vitro and viability of vitrified sheep embryos were evaluated. Ejaculates from four rams were pooled and extended, composing six treatments: no antioxidants; 2mM BME; 5mM BME; 2mM BME and 5mM cysteine; 5mM BME and 5mM cysteine; and 5mM cysteine. Sperm motility, membrane and acrosome integrity, mitochondrial functionality, production of reactive oxygen species and total antioxidant capacity were similar across treatments (P>0.05). A medium with no antioxidant presented cleavage and blastocyst development rates (60.3% and 33.6%, respectively) similar (P>0.05) to those of a medium with 50µM BME and 600µM cysteine (64.3% and 36.6%, respectively). Post-thawing viability of vitrified embryos was similar between media (P>0.05). Cysteine and BME had no influence on the post-thawing viability and oxidative activity of ram sperm and on the viability of vitrified sheep embryos.(AU)

Foram avaliados os efeitos do ß-mercaptoetanol (BME) e da cisteína sobre a viabilidade e a atividade oxidativa após o descongelamento do sêmen ovino e sobre o desenvolvimento in vitro e a viabilidade de embriões ovinos vitrificados. Ejaculados de quatro carneiros foram agrupados e diluídos, compondo seis tratamentos: sem antioxidantes; com BME 2mM; com BME 5mM; com BME 2mM e cisteína 5mM; com BME 5mM e cisteína 5mM; e com cisteína 5mM. Motilidade, integridade da membrana e do acrossoma, função mitocondrial, produção de espécies reativas de oxigênio e capacidade antioxidante total foram semelhantes entre os tratamentos (P>0,05). Em um meio sem antioxidantes, as taxas de clivagem e de desenvolvimento embrionário até blastocisto (60,3%, e 33,6%, respectivamente) foram semelhantes (P>0,05) às obtidas em um meio com BME 50µM e cisteína 600µM (64,3% e 36,6%, respectivamente). A viabilidade pós-descongelamento dos embriões vitrificados não diferiu entre os meios (P>0,05). O BME e a cisteína não influenciaram a viabilidade e a atividade oxidativa do sêmen ovino após o descongelamento e a viabilidade de embriões ovinos vitrificados.(AU)

Improved method for the determination of anatoxin-a and two of its metabolites in blue-green algae using liquid chromatography with fluorescence detection.

Anatoxin-a, a neurotoxin produced by blue-green algae (BGA) species, can cause death to exposed organisms. In North America, BGA are harvested and sold as food supplements, some of which contain elevated levels of other algal toxins, such as microcystins. Concern that elevated levels of anatoxin-a also may be present in BGA food supplements has led to the development of a simple method to determine the presence of anatoxin-a in BGA. Some researchers have successfully analyzed this compound using liquid chromatography with fluorescence detection by forming a fluorescent derivative with 4-fluoro-7-nitrobenzofurazan (NBD-F) in water and phytoplankton extracts. With this method, the background noise is high in BGA extracts due to the presence of co-extractives. Addition of o-phthaldialdehyde (OPA) and mercaptoethanol to the extract before addition of the NBD-F resulted in the successful removal of primary amines from the background noise when the NBD-F derivatives were detected with fluorescence. Improved chromatograms were obtained when extracts were cleaned up in this manner, leading to a lower detection limit (approximately 50 microg/kg) for anatoxin-a. The detection limits obtained for the 2 degradation products dihydroanatoxin-a and epoxyanatoxin-a in BGA extracts were similarly low (55 and 65 microg/kg, respectively).

[Determination of L-cysteine in enzymatic reaction mixture with pre-column derivatization by high performance liquid chromatography].

A new sensitive high performance liquid chromatographic method for the determination of L-cysteine in an enzymatic reaction mixture using ultra violet spectrometric detection was developed. The sample reacted with 5,5'-dithio-bis-nitrobenzoic acid (DTNB) and was analyzed on a Shimadzu VP-ODS column at room temperature, using gradient elution with detection at 330 nm. The L-cysteine chromatographic peak was determined in comparison with derivatives of 2-mercapto ethanol and dithiothreitol. The linear range was 5-950 micromol/L. The recoveries were 99.7%-100.5% and the relative standard deviations (RSDs) were less than 1.3%. The detection limit was 0.8 micromol/L. The method is simple and accurate.

Influence of some technological parameters on the formation of dimethyl sulfide, 2-mercaptoethanol, methionol, and dimethyl sulfone in port wines.

Volatile sulfur compounds of 15 young port wines and 12 old port wines were determined. As there is a great difference in the pool of sulfur compounds between the two groups of wines, an experimental protocol was performed to determine which technological parameter (dissolved O(2), free SO(2) levels, pH, and time/temperature) was related with the formation/consumption of these compounds. Four sulfur compounds were selected for this purpose: dimethyl sulfide, 2-mercaptoethanol, dimethyl sulfone, and methionol. The synergistic effects of increasing temperature and O(2) at lower pH had the largest impact. Dimethyl sulfide was formed during the experimental period in the presence of O(2). Dimethyl sulfone had the same behavior. Methionol decreased significantly in the presence of O(2), but no methional was formed. 2-Mercaptoethanol, considered to be an important "off-flavor" in dry wines, also decreased during the experimental period (54 days) in the presence of O(2), and the respective disulfide was formed. These results corroborate the fact that old port wine (barrel aged) never develops "off-flavors" associated with the presence of methionol (cauliflower), 2-mercaptoethanol (rubber/burnt), or methional (cooked potato). In fact, temperature and oxygen are the major factors in the consumption of these molecules. However, some notes of "quince" and "metallic" can appear during port wine aging, and these can be associated with the presence of dimethyl sulfide.

Evaluación de las técnicas de aglutinación en lámina con el antígeno TR y el 2-mercaptoetanol: aplicación en el diagnóstico de la leptospirosis humana / Evaluatión of the technicals of the slide macroscopic agglutination reaction with the TR antigen and the 2-mercaptoetanol test: diagnosis of the human leptospirosis

Se evalúan las técnicas de aglutinación en lámina con antígeno TR y el 2-mercaptoetanol (2ME) para el diagnóstico de la leptospirosis humana, determinándose los parámetros cualitativos de sensibilidad (90 por ciento), valores predictivos de una prueba positiva (90 por ciento). Se detecto, por la prueba de 2ME, que de los cuarenta casos positivos por el TR cuantitativo, 36 presentaron anticuerpos de la clase IgM, 3 tenían IgM e IgG y un solo caso IgG. Se cuantificó el nivel de anticuerpos presentes en los sueros reactivos, presentándose el mayor número de casos para la dilución 1/10. Se puede considerar que los resultados obtenidos en este trabajo demuestran que ambas técnicas deberán ser utilizadas en el diagnóstico de la entidad

Electrophoretic artifacts arising from the use of thiol-containing reagents.

Thiol reagents migrate as a curtain behind the salt front when loaded with the sample solution onto disc-electrophoresis gels. In immobilized pH gradients (IPG) the same compounds are driven by electrophoresis and electroosmosis from the alkaline to the neutral and acidic regions of the gradients. In either case, a dose-dependent sideways spreading results in spurious reduction between adjacent lanes if samples with and without reducing agent are loaded side by side.

Separation of o-phthalaldehyde-mercaptoethanol derivatives of amino acids from blood plasma on reversed-phase Nova-Pak C18 cartridges.

A separation of 25 o-phthalaldehyde-mercaptoethanol derivatives of primary amino acids in plasma prepared from human blood has been developed for Waters 10 cm x 0.8 cm I.D., 4-microns Nova-Pak C18 Radial-Pak cartridges. A binary gradient system with solvent-switching capability for the A pump is required. Computer methodologies have been utilized to develop mobile phase mixtures of phosphate buffer (pH 6.9), water, methanol and tetrahydrofuran. Advantages of the method include simple sample preparation, fast turnover time (67 min including the pre-column Autotag derivatization procedure) and exceptional column durability (several hundred analyses).

Removal of artifactual bands associated with the presence of 2-mercaptoethanol in two-dimensional polyacrylamide gel electrophoresis.

A method for eliminating artifactual bands due to the presence of 2-mercaptoethanol in two-dimensional gels is described. The method is based on a modification of the procedure of application of the first dimension gel to the SDS slab gel. 2-Mercaptoethanol is removed during equilibration and replaced by iodoacetamide. The use of iodoacetamide improves the recovery of proteins and results in a better detection of them.

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins.

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.

Modified methods for measuring citrulline and carbamoyl-beta-alanine with reduced light sensitivity and sucrose interference.

Two modified procedures for the colorimetric quantitation of citrulline aimed primarily at the assay of ornithine transcarbamylase were developed. Both methods give highly reproducible results in a short period of time since color is developed in 25 min at 100 degrees C. One method is more sensitive than previous methods and the other is as sensitive but requires less than one-fifth as much acid for color development. The reduction in acid concentration results in the stability of the colored complex for at least 50 min under the laboratory lighting conditions and allows for the presence of 5 mumol sucrose and 0.03 mumol mercaptoethanol in the assay. A low-acid modification for quantitating carbamoyl-beta-alanine is described also and may be applicable to the assay of dihydropyrimidinase.

A rapid and sensitive high-performance liquid chromatographic assay for 6-mercaptopurine metabolites in red blood cells.

A highly sensitive and rapid assay for the detection of 6-mercaptopurine metabolites in the red blood cells of leukemic patients receiving the drug has been developed. The method employs a batch-chromatographic procedure using a mercurial cellulose resin to selectively absorb thiol compounds combined with separation by high-performance liquid chromatography using a Partisil-SAX column and uv detection. This method permits detection of 6-thioinosine monophosphate, 6-thiouric acid, and 6-thioguanosine mono-, di-, and triphosphates in patient samples with a sensitivity of 5-10 pmol. No 6-thioinosine di- or triphosphates were detected in patient samples. The results of our study indicate that 6-thioguanosine triphosphate is the major metabolite of 6-mercaptopurine retained by red blood cells after oral or iv administration of the drug.

Mercuri-nitrophenol as a reporter group for the conformational change of hemoglobin.

One mole of horse hemoglobin tetramer reacts with 2 moles of 2-chloromercuri-4-nitrophenol (MNP) at beta 93 cysteine. The difference spectra between NMP-bound hemoglobin and hemoglobin, measured with the aid of ascorbic acid and ascorate oxidase [EC 1.10.3.3] as deoxygenation reagents, indicate that the pK of the phenolic hydroxyl group of MNP increases by 0.6 to 0.8 pH unit on deoxygenation of the hemoglobin. The Hill constant of the modified hemoglobin changes with pH. It decreases from about 2.4 at pH 6.8 to about 1.0 at pH 9.0 This effect of the reagent is interpreted as inherent to the reporter groups.