Analysing deltamethrin susceptibility and pyrethroid esterase activity variations in sylvatic and domestic Triatoma infestans at the embryonic stage

The aim of the present work was to study the deltamethrin susceptibility of eggs from Triatoma infestans populations and the contribution of pyrethroid esterases to deltamethrin degradation. Insects were collected from sylvatic areas, including Veinte de Octubre and Kirus-Mayu (Bolivia) and from domiciliary areas, including El Palmar (Bolivia) and La Pista (Argentina). Deltamethrin susceptibility was determined by dose-response bioassays. Serial dilutions of deltamethrin (0.0005-1 mg/mL) were topically applied to 12-day-old eggs. Samples from El Palmar had the highest lethal dose ratio (LDR) value (44.90) compared to the susceptible reference strain (NFS), whereas the Veinte de Octubre samples had the lowest value (0.50). Pyrethroid esterases were evaluated using 7-coumaryl permethrate (7-CP) on individually homogenised eggs from each population and from NFS. The El Palmar and La Pista samples contained 40.11 and 36.64 pmol/min/mg protein, respectively, and these values were statistically similar to NFS (34.92 pmol/min/mg protein) and different from Kirus-Mayu and Veinte de Octubre (27.49 and 22.69 pmol/min/mg protein, respectively). The toxicological data indicate that the domestic populations were resistant to deltamethrin, but no statistical contribution of 7-CP esterases was observed. The sylvatic populations had similar LDR values to NFS, but lower 7-CP esterase activities. Moreover, this is the first study of the pyrethroid esterases on T. infestans eggs employing a specific substrate (7-CP).

Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca(2+) fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca(2+)-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα(+)/calbindin(+) cells were closely surrounded by NAPE-PLD(+) fiber varicosities. No pyramidal PPARα(+)/calbindin(+) cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD(+)/calretinin(+) cells were specifically detected in CA3. NAPE-PLD(+) puncta surrounded the calretinin(+) cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions.

Use of Hy's law and a new composite algorithm to predict acute liver failure in patients with drug-induced liver injury.

BACKGROUND & AIMS Hy's Law, which states that hepatocellular drug-induced liver injury (DILI) with jaundice indicates a serious reaction, is used widely to determine risk for acute liver failure (ALF). We aimed to optimize the definition of Hy's Law and to develop a model for predicting ALF in patients with DILI. METHODS We collected data from 771 patients with DILI (805 episodes) from the Spanish DILI registry, from April 1994 through August 2012. We analyzed data collected at DILI recognition and at the time of peak levels of alanine aminotransferase (ALT) and total bilirubin (TBL). RESULTS Of the 771 patients with DILI, 32 developed ALF. Hepatocellular injury, female sex, high levels of TBL, and a high ratio of aspartate aminotransferase (AST):ALT were independent risk factors for ALF. We compared 3 ways to use Hy's Law to predict which patients would develop ALF; all included TBL greater than 2-fold the upper limit of normal (×ULN) and either ALT level greater than 3 × ULN, a ratio (R) value (ALT × ULN/alkaline phosphatase × ULN) of 5 or greater, or a new ratio (nR) value (ALT or AST, whichever produced the highest ×ULN/ alkaline phosphatase × ULN value) of 5 or greater. At recognition of DILI, the R- and nR-based models identified patients who developed ALF with 67% and 63% specificity, respectively, whereas use of only ALT level identified them with 44% specificity. However, the level of ALT and the nR model each identified patients who developed ALF with 90% sensitivity, whereas the R criteria identified them with 83% sensitivity. An equal number of patients who did and did not develop ALF had alkaline phosphatase levels greater than 2 × ULN. An algorithm based on AST level greater than 17.3 × ULN, TBL greater than 6.6 × ULN, and AST:ALT greater than 1.5 identified patients who developed ALF with 82% specificity and 80% sensitivity. CONCLUSIONS When applied at DILI recognition, the nR criteria for Hy's Law provides the best balance of sensitivity and specificity whereas our new composite algorithm provides additional specificity in predicting the ultimate development of ALF.

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests. Organophosphate compounds are used in agro-systems, and in programs to control pathogen vectors. Because they are continuously applied, organophosphates often reach water sources and may have an impact on aquatic life. The effects of acute and chronic exposure to the organophosphate insecticide malathion on the midge Chironomus sancticaroli are evaluated. To that end, three biochemical biomarkers, acetylcholinesterase (AChE), alpha (EST-α) and beta (EST-β) esterase were used. Acute bioassays with five concentrations of malathion, and chronic bioassays with two concentrations of malathion were carried out. In the acute exposure test, AChE, EST-α and EST-β activities declined by 66, 40 and 37%, respectively, at 0.251 µg L-1 and more than 80% at 1.37, 1.96 and 2.51 µg L-1. In chronic exposure tests, AChE and EST-α activities declined by 28 and 15% at 0.251 µg L-1. Results of the present study show that low concentrations of malathion can influence larval metabolism, indicating high toxicity for Chironomus sancticaroli and environmental risk associated with the use of organophosphates.

Pharmacodynamic, pharmacokinetic and pharmacogenetic aspects of drugs used in the treatment of Alzheimer's disease.

With the aging population and its rapidly increasing prevalence, dementia has become an important public health concern in developed and developing countries. To date, the pharmacological treatment is symptomatic and based on the observed neurotransmitter disturbances. The four most commonly used drugs are donepezil, galantamine, rivastigmine and memantine. Donepezil, galantamine and rivastigmine are acetylcholinesterase inhibitors with different pharmacodynamic and pharmacokinetic profiles. Donepezil inhibits selectively the acetylcholinesterase and has a long elimination half-life (t½) of 70 h. Galantamine is also a selective acetylcholinesterase inhibitor, but also modulates presynaptic nicotinic receptors. It has a t½ of 6-8 h. Donepezil and galantamine are mainly metabolised by cytochrome P450 (CYP) 2D6 and CYP3A4 in the liver. Rivastigmine is a so-called 'pseudo-irreversible' inhibitor of acetylcholinesterase and butyrylcholinesterase. The t½ of the drug is very short (1-2 h), but the duration of action is longer as the enzymes are blocked for around 8.5 and 3.5 h, respectively. Rivastigmine is metabolised by esterases in liver and intestine. Memantine is a non-competitive low-affinity antagonist of the NMDA receptor with a t½ of 70 h. Its major route of elimination is unchanged via the kidneys. Addressing the issue of inter-patient variability in treatment response might be of special importance for the vulnerable population taking anti-dementia drugs. Pharmacogenetic considerations might help to avoid multiple medication changes due to non-response and/or adverse events. Some pharmacogenetic studies conducted on donepezil and galantamine reported an influence of the CYP2D6 genotype on the pharmacokinetics of the drugs and/or on the response to treatment. Moreover, polymorphisms in genes of the cholinergic markers acetylcholinesterase, butyrylcholinesterase, choline acetyltransferase and paraoxonase were found to be associated with better clinical response to acetylcholinesterase inhibitors. However, confirmation studies in larger populations are necessary to establish evidence of which subgroups of patients will most likely benefit from anti-dementia drugs. The aim of this review is to summarize the pharmacodynamics and pharmacokinetics of the four commonly used anti-dementia drugs and to give an overview on the current knowledge of pharmacogenetics in this field.

In rats, oral oleoyl-DHEA is rapidly hydrolysed and converted to DHEA-sulphate

Background: Dehydroepiandrosterone (DHEA) released by adrenal glands may be converted to androgens and estrogens mainly in the gonadal, adipose, mammary, hepatic and nervous tissue. DHEA is also a key neurosteroid and has antiglucocorticoid activity. DHEA has been used for the treatment of a number of diseases, including obesity; its pharmacological effects depend on large oral doses, which effect rapidly wanes in part because of its short half-life in plasma. Since steroid hormone esters circulate for longer periods, we have studied here whether the administration of DHEA oleoyl ester may extend its pharmacologic availability by keeping high circulating levels. Results: Tritium-labelled oleoyl-DHEA was given to Wistar male and female rats by gastric tube. The kinetics of appearance of the label in plasma was unrelated to sex; the pattern being largely coincident with the levels of DHEA-sulfate only in females, and after 2 h undistinguishable from the results obtained using labelled DHEA gavages; in the short term, practically no lipophilic DHEA label was found in plasma. After 24 h only a small fraction of the label remained in the rat organs, with a different sex-related distribution pattern coincident for oleoyl- and free- DHEA gavages. The rapid conversion of oleoyl-DHEA into circulating DHEA-sulfate was investigated using stomach, liver and intestine homogenates; which hydrolysed oleoyl-DHEA optimally near pH 8. Duodenum and ileum contained the highest esterase activities. Pure hog pancreas cholesterol-esterase broke down oleoyl-DHEA at rates similar to those of oleoyl-cholesterol. The intestinal and liver esterases were differently activated by taurocholate and showed different pH-activity patterns than cholesterol esterase, suggesting that oleoyl-DHEA can be hydrolysed by a number of esterases in the lumen (e.g. cholesterol-esterase), in the intestinal wall and the liver. Conclusion: The esterase activities found may condition the pharmacological availability (and depot effect) of orally administered steroid hormone fatty acid esters such as oleoyl-DHEA. The oral administration of oleoyl-DHEA in order to extend DHEA plasma availability has not been proved effective, since the ester is rapidly hydrolysed, probably in the intestine itself, and mainly converted to DHEA-sulfate at least in females.

Degradation of pheromone and plant volatile components by a same Odorant-Degrading Enzyme in the cotton leafworm, Spodoptera littoralis

Background: Odorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant. Methodology: Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis , a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants. Conclusion: SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.

Lipase Improvement: Goals and strategies

Lipases have received great attention as industrial biocatalysts in areas like oils and fats processing, detergents, baking, cheese making, surface cleaning, or fine chemistry . They can catalyse reactions of insoluble substrates at the lipid-water interface, preserving their catalytic activity in organic solvents. This makes of lipases powerful tools for catalysing not only hydrolysis, but also various reverse reactions such as esterification, transesterification, aminolysis, or thiotransesterifications in anhydrous organic solvents. Moreover, lipases catalyse reactions with high specificity, regio and enantioselectivity, becoming the most used enzymes in synthetic organic chemistry. Therefore, they display important advantages over classical catalysts, as they can catalyse reactions with reduced side products, lowered waste treatment costs, and under mild temperature and pressure conditions. Accordingly, the use of lipases holds a great promise for green and economical process chemistry.

Nitric oxide and the release of lipoprotein lipase from white adipose tissue

Background/Aim: Lipoprotein lipase (LPL) is the main enzyme responsible for the distribution of circulating triacylglycerides in tissues. Its regulation via release from active sites in the vascular endothelium is poorly understood. In a previous study we reported that in response to acute immobilization (IMMO), LPL activity rapidly increases in plasma and decreases in white adipose tissue (WAT) in rats. In other stress situations IMMO triggers a generalized increase in nitric oxide (NO) production. Methods/Results: Here we demonstrate that in rats: 1) in vivo acute IMMO rapidly increases NO concentrations in plasma 2) during acute IMMO the WAT probably produces NO via the endothelial isoform of nitric oxide synthase (eNOS) from vessels, and 3) epididymal WAT perfused in situ with an NO donor rapidly releases LPL from the endothelium. Conclusion: We propose the following chain of events: stress stimulus / rapid increase of NO production in WAT (by eNOS) / release of LPL from the endothelium in WAT vessels. This chain of events could be a new mechanism that promotes the rapid decrease of WAT LPL activity in response to a physiological stimulus.

Combining phospholipases and a liquid lipase for one-step biodiesel production using crude oils

Background Enzymatic biodiesel is becoming an increasingly popular topic in bioenergy literature because of its potential to overcome the problems posed by chemical processes. However, the high cost of the enzymatic process still remains the main drawback for its industrial application, mostly because of the high price of refined oils. Unfortunately, low cost substrates, such as crude soybean oil, often release a product that hardly accomplishes the final required biodiesel specifications and need an additional pretreatment for gums removal. In order to reduce costs and to make the enzymatic process more efficient, we developed an innovative system for enzymatic biodiesel production involving a combination of a lipase and two phospholipases. This allows performing the enzymatic degumming and transesterification in a single step, using crude soybean oil as feedstock, and converting part of the phospholipids into biodiesel. Since the two processes have never been studied together, an accurate analysis of the different reaction components and conditions was carried out. Results Crude soybean oil, used as low cost feedstock, is characterized by a high content of phospholipids (900 ppm of phosphorus). However, after the combined activity of different phospholipases and liquid lipase Callera Trans L, a complete transformation into fatty acid methyl esters (FAMEs >95%) and a good reduction of phosphorus (P <5 ppm) was achieved. The combination of enzymes allowed avoidance of the acid treatment required for gums removal, the consequent caustic neutralization, and the high temperature commonly used in degumming systems, making the overall process more eco-friendly and with higher yield. Once the conditions were established, the process was also tested with different vegetable oils with variable phosphorus contents. Conclusions Use of liquid lipase Callera Trans L in biodiesel production can provide numerous and sustainable benefits. Besides reducing the costs derived from enzyme immobilization, the lipase can be used in combination with other enzymes such as phospholipases for gums removal, thus allowing the use of much cheaper, non-refined oils. The possibility to perform degumming and transesterification in a single tank involves a great efficiency increase in the new era of enzymatic biodiesel production at industrial scale.

Preparation of (S)-1-halo-2-octanols using ionic liquids and biocatalysts

Preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol was carried out by the enzymatic hydrolysis of halohydrin palmitates using biocatalysts. Halohydrin palmitates were prepared by various methods from palmitic acid and 1,2-octanediol. A tandem hydrolysis was carried out using lipases from Candida antarctica (Novozym® 435), Rhizomucor miehei (Lipozyme IM), and “resting cells” from a Rhizopus oryzae strain that was not mycotoxigenic. The influence of the enzyme and the reaction medium on the selective hydrolysis of isomeric mixtures of halohydrin esters is described. Novozym® 435 allowed preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol after 1–3 h ofreaction at 40 °C in [BMIM][PF6].

Esterification of oleic acid catalayzed by an Aspergillus niger strain. Influence of water activity and alcohol concentration

Effects of water activity and 1-propanol concentration on synthesis of propyl oleate from oleic acid using Aspergillus niger cell-bound lipases in isooctane are described. A. niger produces lipases (EC 3.1.1.3) which partly bind to the mycelium during growth. Ester production was monitored for 72 hours at different 1-propanol concentrations and water activities. Aliquots were sequentially withdrawn and propyl esters were quantified using GC and methyl palmitate as an internal standard. In all assayed conditions A. niger cell-bound lipases catalysed propyl oleate synthesis, but at different degrees.

Lipase Improvement: Goals and strategies

Lipases have received great attention as industrial biocatalysts in areas like oils and fats processing, detergents, baking, cheese making, surface cleaning, or fine chemistry . They can catalyse reactions of insoluble substrates at the lipid-water interface, preserving their catalytic activity in organic solvents. This makes of lipases powerful tools for catalysing not only hydrolysis, but also various reverse reactions such as esterification, transesterification, aminolysis, or thiotransesterifications in anhydrous organic solvents. Moreover, lipases catalyse reactions with high specificity, regio and enantioselectivity, becoming the most used enzymes in synthetic organic chemistry. Therefore, they display important advantages over classical catalysts, as they can catalyse reactions with reduced side products, lowered waste treatment costs, and under mild temperature and pressure conditions. Accordingly, the use of lipases holds a great promise for green and economical process chemistry.

Organo-gel: um novo sistema para a imobilização de lipases e sua aplicação em síntese orgânica

Lipases have been immobilized in microemulsion-based organogels (MBG's) and successfully utilized for the enantioselective esterification, diesterification and transesterification reactions, in organic solvents at 25ºC. This methodology is described as a new alternative for the use of enzymes in organic solvents. High enzymic stability has been observed. We have also used this methodology for the successful resolution of chiral secondary alcohols. This is a convenient way of using this catalyst in organic solvents which employs small amounts of the enzyme (250mg/mL).