Secreted protein extract analyses present the plant pathogen Alternaria alternata as a suitable industrial enzyme toolbox.

Lignocellulosic plant biomass is the most abundant carbon source in the planet, which makes it a potential substrate for biorefinery. It consists of polysaccharides and other molecules with applications in pharmaceutical, food and feed, cosmetics, paper and textile industries. The exploitation of these resources requires the hydrolysis of the plant cell wall, which is a complex process. Aiming to discover novel fungal natural isolates with lignocellulolytic capacities, a screening for feruloyl esterase activity was performed in samples taken from different metal surfaces. An extracellular enzyme extract from the most promising candidate, the natural isolate Alternaria alternata PDA1, was analyzed. The feruloyl esterase activity of the enzyme extract was characterized, determining the pH and temperature optima (pH 5.0 and 55-60 °C, respectively), thermal stability and kinetic parameters, among others. Proteomic analyses derived from two-dimensional gels allowed the identification and classification of 97 protein spots from the extracellular proteome. Most of the identified proteins belonged to the carbohydrates metabolism group, particularly plant cell wall degradation. Enzymatic activities of the identified proteins (ß-glucosidase, cellobiohydrolase, endoglucanase, ß-xylosidase and xylanase) of the extract were also measured. These findings confirm A. alternata PDA1 as a promising lignocellulolytic enzyme producer. SIGNIFICANCE: Although plant biomass is an abundant material that can be potentially utilized by several industries, the effective hydrolysis of the recalcitrant plant cell wall is not a straightforward process. As this hydrolysis occurs in nature relying almost solely on microbial enzymatic systems, it is reasonable to infer that further studies on lignocellulolytic enzymes will discover new sustainable industrial solutions. The results included in this paper provide a promising fungal candidate for biotechnological processes to obtain added value from plant byproducts and analogous substrates. Moreover, the proteomic analysis of the secretome of a natural isolate of Alternaria sp. grown in the presence of one of the most used vegetal substrates on the biofuels industry (sugar beet pulp) sheds light on the extracellular enzymatic machinery of this fungal plant pathogen, and can be potentially applied to developing new industrial enzymatic tools. This work is, to our knowledge, the first to analyze in depth the secreted enzyme extract of the plant pathogen Alternaria when grown on a lignocellulosic substrate, identifying its proteins by means of MALDI-TOF/TOF mass spectrometry and characterizing its feruloyl esterase, cellulase and xylanolytic activities.

La docencia de Toxicología en la Universidad de León / No disponible

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Butyric acid-based feed additives help protect broiler chickens from Salmonella Enteritidis infection.

Sodium butyrate is a sodium salt of a volatile short-chain fatty acid (butyric acid) used to prevent Salmonella Enteritidis infection in birds. Three groups of fifty 1-d-old broilers each were fed the following diets: T0 = standard broiler diet (control); T1 = standard broiler diet supplemented with 0.92 g/kg of an additive with free sodium butyrate (Gustor XXI B92); and T2 = standard broiler diet supplemented with 0.92 g/kg of an additive with sodium butyrate partially protected with vegetable fats (Gustor XXI BP70). Twenty percent of the birds were orally infected with Salmonella Enteritidis at d 5 posthatching and fecal Salmonella shedding was assessed at d 6, 9, 13, 20, 27, 34, and 41 of the trial. At d 42, all birds were slaughtered and 20 of them dissected: crop, cecum, liver, and spleen were sampled for bacteriological analyses. Both butyrate-based additives showed a significant reduction (P < 0.05) of Salmonella Enteritidis infection in birds from d 27 onward. However, the partially protected butyrate additive was more effective at the late phase of infection. Partially protected butyrate treatment successfully decreased infection not only in the crop and cecum but also in the liver. There were no differences in the spleen. These results suggest that sodium butyrate partially protected with vegetable fats offers a unique balance of free and protected active substances effective all along the gastrointestinal tract because it is slowly released during digestion.

DNA topoisomerase I from parasitic protozoa: a potential target for chemotherapy.

The growing occurrence of drug resistant strains of unicellular prokaryotic parasites, along with insecticide-resistant vectors, are the factors contributing to the increased prevalence of tropical diseases in underdeveloped and developing countries, where they are endemic. Malaria, cryptosporidiosis, African and American trypanosomiasis and leishmaniasis threaten human beings, both for the high mortality rates involved and the economic loss resulting from morbidity. Due to the fact that effective immunoprophylaxis is not available at present; preventive sanitary measures and pharmacological approaches are the only sources to control the undesirable effects of such diseases. Current anti-parasitic chemotherapy is expensive, has undesirable side effects or, in many patients, is only marginally effective. Under this point of view molecular biology techniques and drug discovery must walk together in order to find new targets for chemotherapy intervention. The identification of DNA topoisomerases as a promising drug target is based on the clinical success of camptothecin derivatives as anticancer agents. The recent detection of substantial differences between trypanosome and leishmania DNA topoisomerase IB with respect to their homologues in mammals has provided a new lead in the study of the structural determinants that can be effectively targeted. The present report is an up to date review of the new findings on type IB DNA topoisomerase in unicellular parasites and the role of these enzymes as targets for therapeutic agents.

Cytotoxic effects of two antimolting insecticides in mammalian CHO-K1 cells.

Cytotoxicity of two insect growth regulators, diflubenzuron, a benzoylphenylurea derivative that inhibits the synthesis of new chitin in target organisms, and pyriproxyfen, an insect juvenile hormone analogue, were tested on CHO-K1 cultures, using the neutral red incorporation assay. Both compounds displayed cytotoxic effects that rise with time exposure. The presence of either fetal calf serum or bovine serum albumin diminished significantly the cytotoxicity of both compounds, thus pointing to a strong protein binding. In addition, extensive metabolization with rat liver submitochondrial fraction gave rise to metabolites less toxic than the parent compounds, implying the relative safety of both diflubenzuron and pyriproxyfen in mammals.

Changes in glutathione-redox balance induced by hexachlorocyclohexane and lindane in CHO-K1 cells.

1. The basal cytotoxic effect of the organochlorine pesticides hexachlorocyclohexane and lindane on CHO-K1 cultures was assessed at fractions of their lethal doses as determined by the neutral red incorporation (NRI) assay (NRI(6.25), NRI(12.5) and NRI(25)). The sulphur-redox cycle enzymes glutathione peroxidase, glutathione reductase and glutathione S-transferase, and total and oxidized glutathione were evaluated at several points during the standard growth curve of the cultures. 2. After incubation with each compound for 24 h, both glutathione peroxidase and reductase showed a substantial increase at the lowest exposure doses (NRI(6.25))--more significantly for lindane than for 1,2,3,4,5,6-hexachlorocyclohexane (HCH)--and dropped at higher doses of both compounds. The reduced and oxidized glutathione content was greatly diminished at the lower exposures, whereas the total glutathione content was higher at NRI(12.5) values. 3. Changes in cell membrane integrity were assessed for a wide range of pesticide concentrations with the lactate dehydrogenase release assay and lipid peroxidation. Membrane leakage and peroxide production were significantly enhanced at concentrations of HCH 50 microg ml(-1), although this effect was not significant at lindane concentrations < 200 microg ml(-1). 4. Lipid peroxidation increased with exposure to HCH at concentrations as low as NRI(6.25), whereas in the case of lindane, this increase was only significant at doses of NRI(25) and above.

Alterations of the glutathione-redox balance induced by metals in CHO-K1 cells.

The effects of cadmium (Cd(2+)), mercury (Hg(2+)), lead (Pb(2+)), copper (Cu(2+)) and nickel (Ni(2+)) on the glutathione (GSH)-redox cycle were assessed in CHO-K1 by the neutral red uptake inhibition (NR) assay (NR(6.25), NR(12.5) and NR(25)). Mercury proved to be the most and lead the least toxic of the metals tested. The effects on GSH content and intracellular specific activities of enzymes involved in the GSH-redox balance were measured after a 24-h exposure. Total GSH content increased significantly in cultures exposed to the lowest metal concentration assayed (NR(6.25)), but fell to below control values when exposed to concentrations equivalent to NR(25). Oxidised glutathione content dropped significantly at NR(6.25), while somewhat higher values were obtained for cultures exposed to higher doses. Glutathione peroxidase (Gpx) activities were 1.2-, 1.5-, 1.6-, 2.0- and 2.5-fold higher than untreated controls for cadmium, copper, mercury, nickel and lead, respectively, at concentrations equivalent to NR(6.25). Gpx activity declined at metal concentrations equivalent to NR(12.5) and NR(25). Glutathione reductase activity remained almost unchanged except at low doses of mercury, nickel and lead. Glutathione-S-transferase activity decreased at rising metal concentrations. The results suggest that a homeostatic defence mechanism was activated when cells were exposed to doses equivalent to NR(6.25) while the ability of the cells to respond weakened as the dose increased. A close relationship was also observed between metal cytotoxicity, total GSH content and the dissociation energy of the sulphur-metal bonds. These facts confirm the involvement of antioxidant defence mechanisms in the toxic action of these ions.

Determinación de residuos de siete insecticidas organofosforados en frutas mediante cromatografía de gases con detector de nitrógenofósforo y confirmación por espectrometría de masas / Residue analysis of seven organophosphorous insecticides in fruit by GC/NPD and confirmation by mass spectrometry

Se ha estudiado la presencia de residuos de siete insecticidas organofosforados en peras y manzanas adquiridas en la provincia de León. Se ha utilizado un sistema de extracción para matrices acuosas y determinación analítica mediante cromatografía de gases (GC) y detección con un detector de nitrógeno-fósforo (NPD). Las muestras positivas se confirmaron mediante cromatografía de gases y espectrometría de masas (GC/MS). Los insecticidas analizados mediante una técnica de extracción de multiresiduos fueron: diclorvós, diazinón, metilparatión, metil-pirimifós, paratión, malatión y fentión. Se han analizado 54 muestras obtenidas en la cesta de la compra de la ciudad de León, 28 manzanas (13 de la variedad Reineta y 15 de la variedad Golden) y 26 peras (15 de la variedad Conferencia y 11 de la variedad Blanquilla), de las que aparecieron 6 (11 por ciento) unidades (1 manzana de la variedad Reineta, 3 peras de la variedad Conferencia y 2 peras de la variedad Blanquilla) contaminadas con diazinón. Ninguna de las muestras sobrepasó el límite máximo de residuos (LMR) establecido para este compuesto (0.5 ppm) por el RD 280/94 en productos vegetales. Los datos de consumo medio por habitante de Castilla y León de peras y manzanas nos han permitido conocer la ingesta diaria estimada (IDE) del diazinón (rango 0,004 0,045 µg/kg/día), que al compararlo con la ingesta diaria admisible (IDA) (2 µg/kg/día), permite estimar un margen de seguridad comprendido entre 44 y 500 (AU)

Citotoxicidad del fungicida mancozeb en cultivos de CHO-K1 / Cytotoxicity of the fungicide mancozeb on CHOK1 cultures

Se ha determinado la citotoxicidad del fungicida ditiocarbámico mancozeb, en cultivos celulares de ovario de hámster (CHO-K1), usando los bioensayos estandarizados de incorporación de rojo neutro (RN) y del contenido total de proteínas (PT). Las dos técnicas mostraron ser comparables en la determinación del efecto citotóxico, mostrando valores de RN50 menores de 15 mg/ml después de 24 h de exposición al plaguicida. La citotoxicidad fue mayor cuanto mayor fue el tiempo de exposición al mancozeb, en ausencia de suero fetal bovino en el medio de cultivo. La preincubación del mancozeb con diferentes concentraciones de fracción submitocondrial de hígado de rata, originó metabolitos menos tóxicos que el compuesto de origen, lo que indica una cierta protección metabólica proporcionada por la fracción S9. Igualmente, el metabolito final de su degradación, la etilentiourea (ETU) mostró menor citotoxicidad que el compuesto original a los tiempos de exposición cortos (AU)

Cyclodiene organochlorine insecticide-induced alterations in the sulfur-redox cycle in CHO-K1 cells.

The effect of the cyclodiene organochlorine pesticides aldrin, dieldrin and endosulfan was assessed on CHO-K1 cultures at fractions of their lethal doses, determined by the neutral red (NRI) incorporation assay (NRI6.25, NRI12.5 and NRI25). Glutathione peroxidase, reductase and S-transferase, and total and oxidised glutathione were evaluated along the standard growth curve of the cultures. After a 24-h incubation with each insecticide, glutathione peroxidase incurred a large increase, while glutathione reductase and S-transferase activities were slightly higher than untreated controls. Unlike oxidised glutathione, the content of total glutathione declined significantly after exposure to cyclodiene insecticides. Changes in cell membrane integrity were assessed by the lactate dehydrogenase (LDH) release assay and lipid peroxidation for a wide range of pesticide concentrations. Membrane leakage and peroxide production were significantly enhanced at concentrations of aldrin and as low as 12.5 microg/ml, whereas dieldrin and endosulfan increased membrane fragility at much higher concentrations.

Analysis of pyridoquinoline derivatives by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry.

A method using liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) has been developed for the characterization and determination of pyridoquinoline derivatives 4,6-bis(dimethylaminoethylamino)-2,8,10-trimethylpyrido[3,2-g]quinoline, 4,6-bis(dimethylaminoethoxy)-2,8,10-trimethylpyrido[3,2-g]quinoline and 4,6-bis[(dimethylaminoethyl)thio]-2,8,10-trimethylpyrido[3,2-g] quinoline, all with potential antitumor properties. LC separation was performed on a conventional C18 column using a binary mobile phase composed of acetonitrile and 50 mM aqueous ammonium formate at pH 3. The APCI mass spectra obtained showed that proton addition giving [M + H]+ was the common mode of ionization to the amino- and thiopyridoquinolines, whereas the alkoxypyridoquinoline was identified by the main formation of the [M - (C2H3)N(CH3)2 + H]+, followed by the [M + H]+ ion. The LC separation conditions and MS detection parameters were optimized for the determination. The analytical method was also applied to the determination of these pyridoquinoline derivatives in fetal calf serum using liquid-liquid extraction with dichloromethane. Acceptable recovery values were obtained, ranging between 45 and 98%.

Efecto del Salbutamol a dosis dopantes en los depósitos grasos de ratas entrenadas en condiciones aeróbicas / Effect of aerobic exercise and salbutamol treatment on fatty depots in wistar rats

El objeto de este trabajo ha sido probar si la reducción en los depósitos de grasa en ratas tratadas simultáneamente con salbutamol y condiciones de entrenamienot físico era debida al ejercicio o al tratamiento con agonistas adrenérgicos. Las ratas fueron tratadas con salbutamol a dos dosis distintas: terapéutica (16mg/kf peso corporal, dos veces al día), y de dopaje (3 mg/kg de peso corporal, dos veces al día), y los animales fueron entrenados siguiendo un protocolo aeróbico durante el experimento (90 días). algunos animales fueron tratados con propanolol (10mg/kg peso corporal, dos veces al día, 30 minutos antes del tratamiento con salbutamol), un Beta-antagonista no específico. Los niveles de grasa perirrenal decayeron sustancialmente sin camios en el peso corporal. Esta reducción en los depósitos de grasa ocurrió tanto por el entrenamiento como por el tratamiento con salbutamol, pero no fue revertido cuando se administró propanolol. La reducción en los depósitos de grasa en ratas fue una consecuencia del ejercicio sin implicación del sistema adrenérgico. De la misma manera, se observó una disminución significativa de los niveles plasmáticos de ácidos grasos y triglicéridos sanguíneos como consecuencia de la administración del salbutamol a las dosis de dopaje, que no pudo ser revertida por propranolol (AU)

Muscle and serum changes with salbutamol administration in aerobically exercised rats.

Treatment of experimental animals subjected to 90 days physical training programme plus repeated doses of salbutamol, a beta-adrenergic agonist, administered under two different regimes: therapeutic (16 microg/kg body weight, twice a day) and doping (3 mg/kg body weight, twice a day), caused a marked increase in size of skeletal (soleus, gastrocnemius and plantaris) leg muscles. Adrenergic involvement of salbutamol-linked hypertrophy was demonstrated by co-administration of the non-specific beta-adrenergic antagonist D,L-propranolol (10 mg/kg body weight twice a day). The salbutamol-induced muscle hypertrophy was associated with an early increase in creatine phosphokinase (CK) and its myocardial isozyme (CKmb), without significant changes in lactate dehydrogenase (LDH), alanine aminotransferase (AAT) and aspartate aminotransferase (DAT). The induction of muscle-injury biomarkers was completely abolished by co-administration of propranolol, thus suggesting the adrenergic involvement of these alterations.

Alterations on polyamine content and glutathione metabolism induced by different concentrations of paraquat in CHO-K1 cells.

The effect of herbicide paraquat has been assessed on CHO-K1 cultures at different concentrations. Glutathione peroxidase, reductase and S-transferase, as well as total and oxidized glutathione, were evaluated along the standard growth curve of the cultures. Paraquat was then administered during mid-log phase at concentrations that produced a calculated lethality of 6.25%, 12.5% and 25%, using the lysosomal dye assay, neutral red. After 24hr of incubation with paraquat, glutathione peroxidase suffered a large dose-response increase, unlike glutathione reductase and S-transferase, the activities of which were lower than untreated controls. The profile of total glutathione content was similar to that found for glutathione peroxidase, increasing with the administered doses of the herbicide. Polyamine content has been also studied at the same concentrations of paraquat, showing that intracellular spermidine and spermine pools were negatively affected with paraquat in a dose-response manner, unlike putrescine, which maintained elevated pools at the three concentrations assayed.

Early alterations of polyamine metabolism induced after acute administration of clenbuterol in mouse heart.

An acute treatment of mice with clenbuterol, a beta-adrenergic agonist, produced a marked increase of polyamines levels in heart, particularly during the early phase of administration of the drug. A single dose of 1.5 mg/kg caused as much as a 10 fold induction in activity of ornithine decarboxylase (ODC) and 3 to 4 fold increase in levels of putrescine, spermidine and spermine in mouse heart. Maximum changes were observed 3 to 4 hours post-administration of clenbuterol. This treatment did not produce any change in S-adenosylmethionine decarboxylase activity. The induction of cardiac ODC by clenbuterol was also dose dependent with a peak at about 5 micromol/kg. Co-administration of difluoromethylornithine, an irreversible inhibitor of ODC, or propranolol, a nonspecific beta-antagonist, with clenbuterol completely prevented the induction of ODC activity as well as the increase in polyamine levels in heart. However, pretreatment with alprenolol or metoprolol, the specific beta1 and beta2-antagonists, respectively, produced only partial prevention. The cardiac ODC from controls as well as clenbuterol treated mice exhibited similar affinity (Km) for its substrate, ornithine, while maximum enzyme activity (Vmax) was about 14 fold higher in clenbuterol treated mouse heart than in the control. Clenbuterol produced no change in the level of specific ODC mRNA or the protein, but the enzyme from the drug-treated mouse heart was considerably more stable than the control. Pretreatment of mice with either cycloheximide or actinomycin D followed by administration of clenbuterol could not prevent the induction in ODC activity suggesting that de novo biosynthesis of the enzyme protein or ODC mRNA was not responsible for induction of ODC activity. Post-translational changes in ODC may be responsible for an early increase of ODC activity due to clenbuterol treatment.

S-adenosylmethionine synthesis in Leishmania infantum promastigotes.

Methionine adenosyltransferase (MAT), S -adenosylmethionine (AdoMet), and S -adenosylhomocysteine (AdoHcy), have been analysed at different time-points during the growth curve of Leishmania infantum. MAT activity and AdoMet content peaked in the lag and early log phases, whereas higher levels of AdoHcy were found in stationary phase cells. MAT activity of cell extracts displayed hyperbolic kinetics for both its substrates, l -methionine and ATP, with km values of 35 microm and 5 m m, respectively. MAT has an absolute requirement for divalent cations, and is dependent on sulfydryl protective agents. Unlike other sources, L. infantum MAT activity seems to be transcriptionally regulated, with an accumulation of MAT-mRNA during rapid growth periods of promastigotes.

Plasma and muscle polyamine levels in aerobically exercised rats treated with salbutamol.

The induction of hypertrophy of cardiac and skeletal muscles has been studied after treatment with two different salbutamol dosages, therapeutic and doping. Treatment of rats subjected to a physical training schedule with repeated doses (16 microg kg(-1) per day or 3 mg kg(-1) per day) of salbutamol, a specific beta-adrenergic agonist, induced a marked increase in both skeletal and heart-muscle weight, whereas total body weight did not change significantly. Adrenergic involvement of salbutamol-linked muscle hypertrophy was demonstrated by co-administration of the non-specific beta-adrenergic antagonist, propranolol (20 mg kg(-1) per day). Salbutamol-induced muscle hypertrophy was associated with an increase in serum, skeletal-muscle and heart levels of the naturally occurring polyamines putrescine, spermidine and spermine. These observations suggest the involvement of polyamines in muscle hypertrophy and the possible role of blood polyamines as exposure biomarkers in beta-adrenergic-muscle hypertrophy.