RNA-seq analysis of the response of plant-pathogenic oomycete Phytophthora parasitica to the fungicide dimethomorph / Análisis RNA-seq de la respuesta del oomiceto fitopatógeno Phytophthora parasitica al fungicida dimetomorf

Phytophthora parasitica is an important oomycete that causes disease in a variety of plants, dimethomorph fungicides being specific for oomycetes. The aim of this study was to use RNA-seq to rapidly discover the mechanism by which dimethomorph acts in the treatment of P. parasitica. We found that the expression of 832 genes changed significantly after the dimethomorph treatment, including 365 up-regulated genes and 467 down-regulated genes. According to the Gene Ontology (GO) enrichment analysis, pathway enrichment and verification test results, the following conclusions are obtained: (i) the treatment of P. parasitica with dimethomorph causes changes in the expression levels of genes associated with the cell wall and cell wall synthesis; (ii) dimethomorph treatment results in reduced permeability of the cell membrane and changes in the expression of certain transport-related proteins; (iii) dimethomorph treatment increased reactive oxygen species and reduced the expression of genes related to the control of oxidative stress.

Phytophthora parasitica es un importante oomiceto que origina enfermedades en una variedad de plantas; el fungicida dimetomorf es específico contra oomicetos. El objetivo de este estudio fue utilizar la tecnología de RNA-seq para descubrir rápidamente el mecanismo por el que el dimetomorf actúa en el tratamiento de P. parasitica. Descubrimos que la expresión de 832 genes se modificaba significativamente tras el tratamiento con dimetomorf, incluyendo 365 genes que son sobrerregulados y 467 genes que son subrregulados. El análisis de enriquecimiento de ontología de genes (GO), análisis de enriquecimiento de las vías y pruebas de verificación permitieron extraer las conclusiones siguientes: 1) el tratamiento de P. parasitica con dimetomorf origina cambios en los niveles de expresión de los genes relacionados con la pared celular y su síntesis; 2) el tratamiento con dimetomorf origina una reducción de la permeabilidad de la membrana celular, así como cambios en la expresión de ciertas proteínas relacionadas con el transporte, y 3) el tratamiento con dimetomorf incrementó las especies reactivas del oxígeno y redujo la expresión de los genes relacionados con el control del estrés oxidativo.

Terpinen-4-ol, tyrosol, and ß-lapachone as potential antifungals against dimorphic fungi

Abstract This study aimed to evaluate the in vitro antifungal activity of terpinen-4-ol, tyrosol, and β-lapachone against strains of Coccidioides posadasii in filamentous phase (n = 22) and Histoplasma capsulatum in both filamentous (n = 40) and yeast phases (n = 13), using the broth dilution methods as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of these compounds. The mechanisms of action of these compounds were also investigated by analyzing their effect on cell membrane permeability and ergosterol synthesis. The MIC and MFCf these compounds against C. posadasii, mycelial H. capsulatum, and yeast-like H. capsulatum, were in the following ranges: 350-5720 µg/mL, 20-2860 µg/mL, and 40-1420 µg/mL, respectively for terpinen-4-ol; 250-4000 µg/mL, 30-2000 µg/mL, and 10-1000 µg/mL, respectively, for tyrosol; and 0.48-7.8 µg/mL, 0.25-16 µg/mL, and 0.125-4 µg/mL, respectively for β-lapachone. These compounds showed a decrease in MIC when the samples were subjected to osmotic stress, suggesting that the compounds acted on the fungal membrane. All the compounds were able to reduce the ergosterol content of the fungal strains. Finally, tyrosol was able to cause a leakage of intracellular molecules.

Glutamine treatment attenuates hyperglycemia-induced mitochondrial stress and apoptosis in umbilical vein endothelial cells

OBJECTIVE: The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis, mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical vein endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells were grown and subjected to glutamine and insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and the production of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicity assays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. RESULTS: Glutamine maintains the integrity of the mitochondria by reducing the cell permeability and cytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level was significantly (p<0.005) reduced when the cells were treated with glutamine. An apoptosis assay revealed significantly reduced apoptosis (p<0.005) in the glutamine-treated cells. Moreover, glutamine alone or in combination with insulin modulated inflammatory cytokine levels. Interleukin-10, interleukin-6, and vascular endothelial growth factor were up-regulated while tumor necrosis factor-α was down-regulated after treatment with glutamine. CONCLUSION: Glutamine, either alone or in combination with insulin, can positively modulate the mitochondrial stress and cell permeability in umbilical vein endothelial cells. Glutamine regulates the expression of inflammatory cytokines and maintains the balance of the mitochondria in a cytoprotective manner. .

Citotoxicidad de ciclozonarona sobre líneas celulares de fibroblastos y de cáncer de próstata / Cytotoxicity of Cyclozonarone on fibroblasts and prostate cancer cellular lines

INTRODUCCIÓN: El cáncer es una importante causa de mortalidad a nivel mundial, lo que ha motivado la búsqueda de compuestos que ayuden a su remisión, incluyendo compuestos naturales. En este contexto, las sesquiterpén quinonas son evaluadas como posibles sustancias antitumorales por sus propiedades citotóxicas. Una de ellas es Ciclozonarona...

INTRODUCTION: Cancer is a major cause of death in the world, which has motivated the search of compounds that could help to its remission, including natural compound. In this context sesquiterpene quinones are evaluated as possible antitumor substances for its cytotoxic properties, one of them is Ciclozonarone...

Vimentin and laminin are altered on cheek pouch microvessels of streptozotocin-induced diabetic hamsters

OBJECTIVE: Normal endothelial cells respond to shear stress by elongating and aligning in the direction of fluid flow. Hyperglycemia impairs this response and contributes to microvascular complications, which result in deleterious effects to the endothelium. This work aimed to evaluate cheek pouch microvessel morphological characteristics, reactivity, permeability, and expression of cytoskeleton and extracellular matrix components in hamsters after the induction of diabetes with streptozotocin. METHODS: Syrian golden hamsters (90-130 g) were injected with streptozotocin (50 mg/kg, i.p.) or vehicle either 6 (the diabetes mellitus 6 group) or 15 (the diabetes mellitus 15 group) days before the experiment. Vascular dimensions and density per area of vessels were determined by morphometric and stereological measurements. Changes in blood flow were measured in response to acetylcholine, and plasma extravasation was measured by the number of leakage sites. Actin, talin, α-smooth muscle actin, vimentin, type IV collagen, and laminin were detected by immunohistochemistry and assessed through a semiquantitative scoring system. RESULTS: There were no major alterations in the lumen, wall diameters, or densities of the examined vessels. Likewise, vascular reactivity and permeability were not altered by diabetes. The arterioles demonstrated increased immunoreactivity to vimentin and laminin in the diabetes mellitus 6 and diabetes mellitus 15 groups. DISCUSSION: Antibodies against laminin and vimentin inhibit branching morphogenesis in vitro. Therefore, laminin and vimentin participating in the structure of the focal adhesion may play a role in angiogenesis. CONCLUSIONS: Our results indicated the existence of changes related to cell-matrix interactions, which may contribute to the pathological remodeling that was already underway one week after induction of experimental diabetes.

Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-á) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-á (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-á treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-á decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-á did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-á increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80 percent and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 æM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Na+i, K+i and Cl-i regulation of exocytosis in guinea-pig antral mucous cells

Effects of intracellular Na+, K+ and Cl- on Ca(2+)-regulated exocytosis activated by 10 microM acetylcholine (ACh) were studied in guinea-pig antral mucous cells which are permeabilized by nystatin treatment. Ca(2+)-regulated exocytotic events were modulated by [Na+]i, [K+]i and [Cl-]i via mediation of PTX-sensitive G proteins. Increases in [Na+]i and PTX inhibit G protein (G(Na)), which suppressed the exocytosis. Increases in [K+]i caused the exchange of G proteins (from G(Na) to G(K)) to increase, and GK evoked activation of the exocytosis and was inhibited by PTX. Increases in [Cl-]i and PTX inhibit G protein (G(Cl)), which stimulates exocytotic events. Based on these observations, the exocytosis in antral mucous cells were modulated by intracellular ions, concentration of which were increased or decreased by cell volume changes caused by Ach.

Comparative responses to salinity in wheat genotypes differing in salt tolerance 2-cell permeability, osmotic potential and cytoplasmic viscosity

Caryopses of two Triticum estivum genotypes, differing in salt tolerance [Sakha 8, tolerant, Giza 162, sensitive], were hydroponically grown under natural environmental conditions [in the laboratory] in presence and absence of different concentrations of NaCl added to the growth medium. NaCl stress increased nonelectrolyte permeability of leaf sheath subepidermal cells in Giza 162 [sensitive], whereas no such effect of salt was found in Sakha 8 [tolerant]. Psi s of leaf sheath subepidermal cells of both cultivars was decreased with rising NaCl concentration in the external solution. At 100 mM NaCl, the decrease in psi s in Giza 162 was greater than that found in Sakha 8. Cytoplasmic viscosity of Giza 162 was decreased by NaCl addition to the growth solution, although viscosity was higher in Giza 162 than in Sakha 8 under control condition [O mM NaCl]. Cytoplasmic viscosity in Sakha 8, however, did not change by salt stress. The results confirmed and extended earlier data that cytoplasmic characteristics are different in wheat salt sensitive and tolerant genotypes, they may correlate with salt tolerance

Biochemical bases of the pharmacological action of the flavonoid silymarin and of its structural isomer silibinin

The flavonoid silymarin and one its structural components, silibinin, have been well characterized as hepato-protective substances. However, little is known about the biochemical mechanisms of action of these substances. This review deals with recent investigations to elucidate the molecular action of the flavonoid. Three levels of action have been proposed for silymarin in experimental animals: a) as an antioxidant, by scavenging prooxidant free radicals and by increasing the intracellular concentration of the tripeptide glutathione; b) regulatory action of the cellular membrane permeability and increase of its stability against xenobiotic injury; c) at the nuclear expression, by increasing the synthesis of ribosomal RNA by stimulating DNA polymerase I and by exerting a steroid-like regulatory action on DNA transcription. The specific hepatoprotective action of silibinin against the toxicity of ethanol, phenylhydrazine and acetaminophen is also discussed. It is suggested that the biochemical effects observed for the flavonoid in experimental models may settle the basis for understanding the pharmacological action of silymarin and silibinin

Estudos espectroscopicos da agregacao de ferriprotoporfirina IX em meio aquoso e em membranas. Implicacoes para o mecanismo de lise celular / Spectroscopic studies of ferriprotoporphyrin IX in aqueous media in membranes. Implications for the mechanism of cell lysis

A tese descreve o estudo da agregacao de hemina (ferriprotoporfirina IX) em meio aquoso e em presenca de membranas. Foi estudada a dependencia de efeitos causados por hemina em bicamadas lipidicas (catalise de peroxidacao lipidica, alteracoes estruturais e de permeabilidade) do estado de agregacao desse composto. Foram empregadas tecnicas de espectroscopia optica e de ressonancia paramagnetica eletronica (e marcadores de spin). Foram utilizados lipossomos multilamelares, vesiculas unilamelares e membranas planas como modelos de membranas. Verificou-se que a agregacao de hemina em meio aquoso aumenta com o abaixamento do pH e com o aumento da forca ionica em presenca de membranas. A catalise de peroxidacao lipidica e efetuada por hemina monomerica ou dimerica , enquanto que agregados maiores se intercalam na bicamada aumentando a desordem e, consequentemente, a permeabilidade. Os resultados obtidos fornecem uma hipotese para o mecanismo, a nivel molecular da lise celular causada por hemina

Effect of cyclosporin A and trifluoperazine on rat liver mitochondria swelling and lipid peroxidation

The effect of cyclosporin A (CsA) or trifluoperazine (TFP) on lipid peroxidation and mitochondrial swelling was determined using liver mitochondria incubated with 30 microM Ca2+ and 250 microM t-butylhydroperoxide or 5 mM inorganic phosphate (P(i)). Lipid peroxidation was not modified by either 1 microM CsA or 40 microM TFP. These compounds presented a distinct effect on mitochondrial permeability. Under oxidative conditions, CsA only showed a transient protective effect whereas TFP completely inhibited mitochondrial swelling. Conversely, CsA was very efficient when Ca2+ and P(i) were used, a condition under which TFP was unable to prevent the swelling. These data are consistent with our previous results (M.F. Nepomuceno, D.V. Macedo and L. Pereira-da-Silva (1991). Brazilian Journal of Medical and Biological Research, 24: 833-836) showing that lipid peroxidation is one among other different components of the permeabilization process. The data suggest that lipid peroxidation is independent of swelling, occurring later than swelling, presumably when the mitochondrial reductant systems are depleted. The differential effects of CsA and TFP suggest that these compounds can be used as specific probes in the elucidation of the two distinct mechanisms responsible for mitochondrial swelling

Calcium homeostasis in Trypanosoma cruzi

By using the fluorescent Ca2+ indicator fura 2, submicromolar levels of intracellular Ca2+ have been detected in Trypanosoma cruzi different stages. The intracellular transport mechanisms involved in maintaining Ca2+ homeostasis in T. cruzi have been characterized by measuring Ca2+ transport in digitonin-permeabilized cells. Two intracellular calcium transport systems have been detected. Ca2+ uptake by the mitochondria occurs by an electrophoretic mechanism, is inhibited by antimycin A, FCCP, and ruthenium red, and stimulated by respiratory substrates, phosphate and acetate. This pool has a high capacity and low affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.6-0.7 microM. Ca2+ uptake by the endoplasmic reticulum is inhibited by high concentrations of vanadate and anticalmodulin agents, and stimulated by ATP. This pool has a low capacity and a high affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.05-1.0 microM. In addition, calmodulin has been purified from T. cruzi epimastigotes and shown to stimulate the homologous plasma membrane Ca(2+)-ATPase and cyclic-AMP phosphodiesterase. The gene encoding this protein has been cloned and sequenced and shown to have a great homology to mammalian calmodulin. The role of the plasma membrane of T. cruzi in the regulation of [Ca2+]i has been studied using fura 2-loaded epimastigotes or plasma membrane vesicles prepared from epimastigotes. Plasma membrane vesicles transport Ca2+ in the presence of Mg2+ and have a high affinity, vanadate-sensitive (Ca(2+)-Mg2+)-ATPase with an apparent Km for free Ca2+ of 0.3 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrokinetic energy conversion studies of urine-oxalic acid systems across urinary bladder membrane.

Electrokinetic studies of urine-oxalic acid systems with increasing concentration of oxalic acid in urine have been carried out across urinary bladder membranes. It has been found that electro-osmotic flux and streaming current decrease with increase in concentration of oxalic acid in urine while hydrodynamic flux and streaming potential increase with increase in concentration. Kinetic energy term (alpha 1) and polarizability term (alpha 2) have been computed for these systems and it has been found that polarizability decreases much faster with increase in concentration of oxalic acid in urine. Electrokinetic energy conversion of these systems have been computed and it has been found that electrokinetic energy conversion is maximum for urine and it decreases with increase in concentration of oxalic acid in urine. Poor energy conversion may lead to sluggish flushing action which may ultimately lead to formation of urinary calculi in the bladder and so present study may be of some use in predicting electrophysiology of the bladder.

Age related toxic effects of endosulfan on certain enzymes of rat erythrocytes.

The effect of oral administration of endosulfan (12.5 mg/kg body weight), daily for 4 days was investigated on erythrocytes of female rats of 4 different age groups i.e. 15, 30, 70 and 365 days old. Erythrocyte membrane Na+, K(+)-ATPase and Mg2(+)-ATPase activities were significantly inhibited in all the age groups of rats. However, percent inhibition was maximum in the youngest animals. A significant decrease in the activity of erythrocyte glutathione reductase was observed in 30 and 70 days old rats whereas a significant increase in the activity of glucose-6-phosphate dehydrogenase (G-6-PD) was observed in these groups. The increase in G-6-PD activity may be a physiological response to compensate for decrease in the reduced glutathione level which results from decrease in the activity of glutathione reductase.

Acumulacion de calcio en fibras hipermeabilizadas de musculo liso vascular, esqueletico y cardiaco de rata / Calcium accumulation in hypermeabilizated fibers of vascular smooth , skeletal and cardiac muscles of the rats

Este trabajo presenta resultados sobre la capacidad para acumular calcio de las preparaciones multicelulares hiperpermeabilizadas de músculo liso aórtico, músculo esquelético y músculo ventricular de rata. La supresión de la fundación de la membrana plasmática como barrera de permeabilidad a los iones, permitió exponer el retículo sarcoplásmico (RS) a soluciones conteniendo "buffers" de 45Ca-EGTA y medir la cantidad de 45Ca acumulado por las preparaciones durante un período de 30 minutos y a la temperatura ambiente. El 45Ca cargado fue atribuido a la actividad de la bomba de calcio del RS dado que aquél dependió de los valores de pCa en los medios de incubación y fue estimulado por oxalato de K. Este método permitió discriminar la capacidad para acumular calcio del RS en diferentes tipos musculares. Los valores de 45Ca acumulado en ausencia de oxalto de K fueron: 5.16 ñ 0.08, 7.02 ñ 0.38 y 2.59 ñ 0.15 µmoles Ca2+/gm de proteína tisular en músculo liso aórtico, músculo esquelético y músculo ventriuclar respectivamente. Los valores obtenidos en presencia de 10 mM de oxalato de K evidenciaron el siguiente orden creciente de l as capacidades de acumulación de calcio: músculo esquelético > músculo ventricular > ou = músculo liso aórtico. Teniendo en cuenta el contenido de proteína de cada tipo muscular, la cantidad de calcio acumulado por el RS que se puede calcular excede la necesaria para producir la activación máxima de la concentracción

Effect of 11 beta OH pregna-1,4-diene-3,20-dione (deltaHOP) on thymocyte plasma membranes

En trabajos anteriores se demostró que la acción inibitoria del esteroide delta HOP a altas concentraciones no era consecuencia de un efecto genómico como en el caso de los glucocorticoides. Para investigar si este efecto se producía a traves de la membrana plasmática, en este trabajo se estudió , en tromocitos de ratas, las alteraciones producidas por estos esteroides en la fuidez de la membrana por polarización de fluorescencia con 1,6-difenil-1, 3, 5-hexatrieno (DPH) y la movilidad de las proteínas de susperficie por la formación de "caps" con concanavalina A fluorescente (Con A-fl). La polarización de fluorescencia disminuyó con los glucocorticoides por aumento de la fluidez de la membrana, mientras que la alfa HOP no produjo ningún cambio. En los experimentos con Con A-fl se observó una disminución del inúmero de células con "cap" cuando se incubó con delta HOP y con los inhibidores de "caps" (citocalasina B y acida sódica), mientras que los glucocorticoides no tuvieron efecto inhibidor. El tratamiento "in vitro" con delta HOP o glucocorticoides produjo el mismo efecto que "in vitro". Estos resultados sugieren que la delta HOP actúa en forma superficial sobre la membrana plasmática, in hibiendo la movilidad de las proteínas de superficie, pero no alterando la fluidez de la bicapa lipídica

Liquid membrane phenomenon in the biological actions of benzodiazepines.

The liquid membrane phenomenon in benzodiazepines has been studied. Transport of glycine, GA-BA, noradrenaline, dopamine and serotonin in the presence of the liquid membranes generated by the benzodiazepines in association with lecithin and cholesterol has been studied. The data indicate that modification in permeabilities in the presence of the liquid membranes is likely to make a significant contribution to several biological actions of the benzodiazepines.