Nicorandil Affects Mitochondrial Respiratory Chain Function by Increasing Complex III Activity and ROS Production in Skeletal Muscle Mitochondria.

Adenosine triphosphate (ATP)-dependent potassium channels openers (KATP) protect skeletal muscle against function impairment through the activation of the mitochondrial KATP channels (mitoKATP). Previous reports suggest that modulators of the mitochondrial KATP channels have additional effects on isolated mitochondria. To determine whether the KATP channel opener nicorandil has non-specific effects that explain its protective effect through the mitochondrial function, chicken muscle mitochondria were isolated, and respiration rate was determined pollarographically. The activity of the electron transport chain (ETC) complexes (I-IV) was measured using a spectrophotometric method. Reactive oxygen species (ROS) levels and lipid peroxidation were assessed using flow cytometry and thiobarbituric acid assay, respectively. Both KATP channel opener nicorandil and KATP channel blocker 5-hydroxydecanoate (5-HD) decreased mitochondrial respiration; nicorandil increased complex III activity and decreased complex IV activity. The effects of nicorandil on complex III were antagonized by 5-HD. Nicorandil increased ROS levels, effect reverted by either 5-HD or the antioxidant N-2-mercaptopropionyl glycine (MPG). None of these drugs affected lipid peroxidation levels. These findings suggest that KATP channel opener nicorandil increases mitochondrial ROS production from complex III. This results by partially blocking electron flow in the complex IV, setting electron carriers in a more reduced state, which is favored by the increase in complex III activity by nicorandil. Overall, our study showed that nicorandil like other mitochondrial KATP channel openers might not act through mitoKATP channel activation.

Efecto de Microemulsiones de Aceites Esenciales Sobre el Eritrocito Humano y Bacterias Patógenas / Effect of Microemulsions of Essential Oils on Human Erythrocyte and Pathogens Bacteria

RESUMEN: El objetivo del presente trabajo fue evaluar el efecto de las microemulsiones de aceite esencial de romero (AER) y árbol de té (AET) sobre el eritrocito humano y microorganismos patógenos. Para ello, se elaboraron microemulsiones de AER y AET al 8.0% (v/v), 5.0% (v/v) y 2.5% (v/v). Las microemulsiones fueron probadas sobre el eritrocito humano para determinar el porcentaje de hemólisis, el porcentaje de inhibición de hemólisis y su actividad antibacterial contra E. coli O157:H7 y S. aureus. Las microemulsiones con AER no presentaron actividad hemolítica significativa, caso contrario con las microemulsiones de AET al 8.0% (≈70%) y 5.0% (33%) que presentaron mayor actividad hemolítica. Las microemulsiones de AER protegieron significativamente al eritrocito contra la presencia de radicales libres, en comparación con aquellas de AET (p< 0.05). Además, las emulsiones de AET al 8.0% mostraron efectos antibacterianos contra E. coli O157:H7 y S. aureus mientras que AER al 8.0% solo mostraron efecto contra E. coli O157:H7. La limitante del estudio fue que no utilizamos células nucleadas para establecer si los aceites esenciales dañan el material nuclear. Sin embargo, observamos que el tipo y la cantidad de aceite utilizado pueden tener implicaciones serias sobre la membrana eritrocitaria. Se concluye que las microemulsiones de AER presentaron mejor efecto protector eritrocitario, mientras que las microemulsiones de AET presentaron mejor actividad antibacterial contra las bacterias estudiadas, pero con mayor efecto tóxico sobre el eritrocito.

ABSTRACT: The aim of the study was to evaluate the effect of microemulsions of rosemary (AER) and tea tree (AET) essential oils on human erythrocyte and pathogen bacteria. Microemulsions of each oil were prepared at 8.0% (v/v), 5.0% (v/v) and 2.5% (v/v), and they were tested on human erythrocyte to determine the hemolysis percentage, hemolysis inhibition percentage and the antibacterial capacity against E. coli O157:H7 and S. aureus. All AER microemulsions showed no significant hemolytic activity. On the contrary, AET microemulsions showed hemolytic effect but those in concentrations of 8.0% (≈70 %) and 5.0% (33%) showed the highest effect. In addition, AER microemulsions showed protective effect against free radicals in comparison with the AET microemulsions (p< 0.05). On the other hand, the AET microemulsion at 8.0% showed antibacterial effect against E. coli O157:H7 and S. aureus, and the AER at 8.0% showed antibacterial effect against E. coli O157:H7. The limitation of this study was that nucleated cells were not used to observe the damage of the essential oils on nuclear material. However, the observed damage of erythrocyte's membrane is depending on type and amount of used oil. Therefore, it can be concluded that the AER microemulsions showed better protective effect of erythrocytes, while AET microemulsions showed better antibacterial effect against the tested bacteria, although with toxic effect on the erythrocytes.

Membrana de Quitosano con Aceites Esenciales de Romero y Árbol de Té: Potencial como Biomaterial / Membrane of chitosan with essential oils of Romero and Tree of Tea: Potential as biomaterial

RESUMEN: El quitosano (CH) tiene propiedades hemostáticas, cicatrizantes y estimulantes del sistema inmune del hospedero contra infecciones virales y bacterianas. Además es biodegradable, no tóxico, antimicrobiano, biocompatible y filmogénico. Estas propiedades han sido utilizadas para acarrear y liberar compuestos bioactivos para sus posibles usos en la salud humana. Por otro lado, los compuestos bioactivos como los aceites esenciales de gran volatilidad como romero (RMO) [Rosmarinus officinalis] y árbol de té (TTO) [Melaleuca alternifolia], pueden ser alternativa importante de uso como antioxidantes y antimicrobianos que podrían inducir cambios favorables en la membrana de CH. Por lo tanto, se incorporaron aceites esenciales de TTO y RMO (0.25, 0.50 y 1.00% v/v) en membranas de CH por el método de casting y se evaluaron su solubilidad, PVA, transparencia y capacidad de bloqueo de la luz ultravioleta, capacidad antioxidante y de protección del eritrocito humano. RMO y TTO redujeron la solubilidad (28-58%) y PVA (entre 28-58%) de las membranas de CH. Todas las membranas fueron amarillas, ligeramente opacas y con capacidad de bloquear la luz ultravioleta. También, las membranas mostraron relativa baja capacidad antioxidante por el radical ABTS (≈ 6 at 9 %), pero con excelente efecto protector sobre el eritrocito humano (> 63 %).

ABSTRACT: Chitosan (CH) has hemostatic properties and accelerated healing ability to stimulate the host immune system against viral and bacterial infections. It is also biodegradable, nontoxic, antimicrobial, biocompatible, and capable of forming membranes and to carry and to release bioactive compounds. These properties of CH can be useful to carry some volatile essential oils having bioactive compounds (antioxidants and antimicrobials) with potential application in human health as those as rosemary (RMO) [Rosmarinus officinalis] and tea tree (TTO) [Melaleuca alternifolia]. These essential oils were incorporated into the chitosan by the casting method in this study. The physical properties (solubility, PVA, transparency and blocking ultraviolet light) of these membranes with TTO and RMO (0.25, 0.50 and 1.00% v/v), antioxidant capacity and protection human erythrocyte were evaluated. TTO and RMO reduced solubility (28-58%) and PVA (between 28-58%) of CH membranes. All membranes were yellow, slightly opaque and they were able to block the ultraviolet light. Membranes also showed relatively low antioxidant capacity by the radical ABTS (≈ 6 at 9%), but it was accompanied by an excellent protective effect on the human erythrocyte (> 63%).

Substrate specificity of the 3-methylcrotonyl coenzyme A (CoA) and geranyl-CoA carboxylases from Pseudomonas aeruginosa.

Biotin-containing 3-methylcrotonyl coenzyme A (MC-CoA) carboxylase (MCCase) and geranyl-CoA (G-CoA) carboxylase (GCCase) from Pseudomonas aeruginosa were expressed as His-tagged recombinant proteins in Escherichia coli. Both native and recombinant MCCase and GCCase showed pH and temperature optima of 8.5 and 37 degrees C. The apparent K(0.5) (affinity constant for non-Michaelis-Menten kinetics behavior) values of MCCase for MC-CoA, ATP, and bicarbonate were 9.8 microM, 13 microM, and 0.8 microM, respectively. MCCase activity showed sigmoidal kinetics for all the substrates and did not carboxylate G-CoA. In contrast, GCCase catalyzed the carboxylation of both G-CoA and MC-CoA. GCCase also showed sigmoidal kinetic behavior for G-CoA and bicarbonate but showed Michaelis-Menten kinetics for MC-CoA and the cosubstrate ATP. The apparent K(0.5) values of GCCase were 8.8 microM and 1.2 microM for G-CoA and bicarbonate, respectively, and the apparent K(m) values of GCCase were 10 microM for ATP and 14 microM for MC-CoA. The catalytic efficiencies of GCCase for G-CoA and MC-CoA were 56 and 22, respectively, indicating that G-CoA is preferred over MC-CoA as a substrate. The enzymatic properties of GCCase suggest that it may substitute for MCCase in leucine catabolism and that both the MCCase and GCCase enzymes play important roles in the leucine and acyclic terpene catabolic pathways.

The atu and liu clusters are involved in the catabolic pathways for acyclic monoterpenes and leucine in Pseudomonas aeruginosa.

Evidence suggests that the Pseudomonas aeruginosa PAO1 gnyRDBHAL cluster, which is involved in acyclic isoprenoid degradation (A. L. Díaz-Pérez, N. A. Zavala-Hernández, C. Cervantes, and J. Campos-García, Appl. Environ. Microbiol. 70:5102-5110, 2004), corresponds to the liuRABCDE cluster (B. Hoschle, V. Gnau, and D. Jendrossek, Microbiology 151:3649-3656, 2005). A liu (leucine and isovalerate utilization) homolog cluster was found in the PAO1 genome and is related to the catabolism of acyclic monoterpenes of the citronellol family (AMTC); it was named the atu cluster (acyclic terpene utilization), consisting of the atuCDEF genes and lacking the hydroxymethyl-glutaryl-coenzyme A (CoA) lyase (HMG-CoA lyase) homolog. Mutagenesis of the atu and liu clusters showed that both are involved in AMTC and leucine catabolism by encoding the enzymes related to the geranyl-CoA and the 3-methylcrotonyl-CoA pathways, respectively. Intermediary metabolites of the acyclic monoterpene pathway, citronellic and geranic acids, were accumulated, and leucine degradation rates were affected in both atuF and liuD mutants. The alpha subunit of geranyl-CoA carboxylase and the alpha subunit of 3-methylcrotonyl-CoA carboxylase (alpha-MCCase), encoded by the atuF and liuD genes, respectively, were both induced by citronellol, whereas only the alpha-MCCase subunit was induced by leucine. Both citronellol and leucine also induced a LacZ transcriptional fusion at the liuB gene. The liuE gene encodes a probable hydroxy-acyl-CoA lyase (probably HMG-CoA lyase), an enzyme with bifunctional activity that is essential for both AMTC and leucine degradation. P. aeruginosa PAO1 products encoded by the liuABCD cluster showed a higher sequence similarity (77.2 to 79.5%) with the probable products of liu clusters from several Pseudomonas species than with the atuCDEF cluster from PAO1 (41.5%). Phylogenetic studies suggest that the atu cluster from P. aeruginosa could be the result of horizontal transfer from Alphaproteobacteria. Our results suggest that the atu and liu clusters are bifunctional operons involved in both the AMTC and leucine catabolic pathways.

The gnyRDBHAL cluster is involved in acyclic isoprenoid degradation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1 mutants affected in the ability to degrade acyclic isoprenoids were isolated with transposon mutagenesis. The gny cluster (for geranoyl), which encodes the enzymes involved in the lower pathway of acyclic isoprenoid degradation, was identified. The gny cluster is constituted by five probable structural genes, gnyDBHAL, and a possible regulatory gene, gnyR. Mutations in the gnyD, gnyB, gnyA, or gnyL gene caused inability to assimilate acyclic isoprenoids of the citronellol family of compounds. Transcriptional analysis showed that expression of the gnyB gene was induced by citronellol and repressed by glucose, whereas expression of the gnyR gene had the opposite behavior. Western blot analysis of citronellol-grown cultures showed induction of biotinylated proteins of 70 and 73 kDa, which probably correspond to 3-methylcrotonoyl-coenzyme A (CoA) carboxylase and geranoyl-CoA carboxylase (GCCase) alpha subunits, respectively. The 73-kDa biotinylated protein, identified as the alpha-GCCase subunit, is encoded by gnyA. Intermediary metabolites of the isoprenoid pathway, citronellic and geranic acids, were shown to accumulate in gnyB and gnyA mutants. Our data suggest that the protein products encoded in the gny cluster are the beta and alpha subunits of geranoyl-CoA carboxylase (GnyB and GnyA), the citronelloyl-CoA dehydrogenase (GnyD), the gamma-carboxygeranoyl-CoA hydratase (GnyH), and the 3-hydroxy-gamma-carboxygeranoyl-CoA lyase (GnyL). We conclude that the gnyRDBHAL cluster is involved in isoprenoid catabolism.

Interactions of chromium with microorganisms and plants.

Chromium is a highly toxic non-essential metal for microorganisms and plants. Due to its widespread industrial use, chromium (Cr) has become a serious pollutant in diverse environmental settings. The hexavalent form of the metal, Cr(VI), is considered a more toxic species than the relatively innocuous and less mobile Cr(III) form. The presence of Cr in the environment has selected microbial and plant variants able to tolerate high levels of Cr compounds. The diverse Cr-resistance mechanisms displayed by microorganisms, and probably by plants, include biosorption, diminished accumulation, precipitation, reduction of Cr(VI) to Cr(III), and chromate efflux. Some of these systems have been proposed as potential biotechnological tools for the bioremediation of Cr pollution. In this review we summarize the interactions of bacteria, algae, fungi and plants with Cr and its compounds.

The pseudomonas aeruginosa motR gene involved in regulation of bacterial motility.

A mini-Tn5-Hg insertion mutant derived from Pseudomonas aeruginosa W51D (W51M1) was isolated in which mini-Tn5 insertion disrupted the motR gene showing that it forms part of the cluster involved in bacterial motility and chemotaxis. Characterization of the W51M1 motility behavior, and also of a PAO1 motR::mini-Tn5-Hg mutant, suggests that the product of the motR gene is a negative regulator of bacterial motility which controls the number of flagella per cell.

The branched-chain dodecylbenzene sulfonate degradation pathway of Pseudomonas aeruginosa W51D involves a novel route for degradation of the surfactant lateral alkyl chain.

Pseudomonas aeruginosa W51D is able to grow by using branched-chain dodecylbenzene sulfonates (B-DBS) or the terpenic alcohol citronellol as a sole source of carbon. A mutant derived from this strain (W51M1) is unable to degrade citronellol but still grows on B-DBS, showing that the citronellol degradation route is not the main pathway involved in the degradation of the surfactant alkyl moiety. The structures of the main B-DBS isomers and of some intermediates were identified by gas chromatography-mass spectrometric analysis, and a possible catabolic route is proposed.

The Pseudomonas aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase which is specifically involved in rhamnolipid synthesis.

A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent beta-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-beta-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the rhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase absolutely required for the synthesis of the beta-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.

Purification and partial characterization of a chromate reductase from Bacillus.

A soluble NADH-dependent enzyme capable of reducing hexavalent chromium [Cr(VI)] to the trivalent form [Cr(III)] was purified from chromate-resistant Bacillus QC1-2. An enriched single protein band of 24 kDa was observed by SDS-PAGE following HPLC ion-exchange and size-exclusion procedures. In the latter step, the chromate reductase showed a molecular mass of 44 kDa, which suggested that the enzyme consists of two subunits of about 24 kDa. Purified chromate reductase displayed optimal activity at a temperature and pH of 37 degrees C and 7.0, respectively. The enzyme showed a Km of 0.35 mM for chromate and a Vmax of 50 nmol Cr(VI) reduced per minute per mg protein.