Development of an evaluation framework and assessment tools to assess the foot and mouth disease (FMD) control policies and their implementation in the proposed FMD-free zone in Thailand.

This paper proposes an evaluation framework and assessment tools for use in the evaluation of the current foot and mouth disease (FMD) control policies in Thailand and their implementation in the eastern region of the country (the proposed FMD-free zone). To develop the framework and assessment tools this study identified: a) the essential elements of a successful FMD control programme; b) stakeholders who are affected by the FMD control programme; and c) relevant Department of Livestock Development regulations and documents. These regulations and documents were used as the foundation for development of the framework and assessment tools. The proposed framework includes the essential characteristics of policy design and implementation that should be part of the FMD control programme in Thailand. The assessment tools include assessment matrices, three sets of questionnaires, and interview questions. When applied, the assessment matrices identify shortcomings of policy design, policy implementation, veterinary capacity and stakeholder engagement. Questionnaires and interview questions collect information that examines the consistency of elements of the FMD control programme against criteria in the assessment matrix. This framework and tools are currently being applied to assess the proposed FMD-free zone in Thailand.

Les auteurs proposent un cadre et des outils d'évaluation destinés à évaluer les mesures de lutte contre la fièvre aphteuse appliquées en Thaïlande et décrivent leur mise en oeuvre dans la région orientale du pays (correspondant à la zone dont la Thaïlande propose la reconnaissance en tant qu'indemne de fièvre aphteuse). Afin d'élaborer ce cadre et ces outils d'évaluation, l'étude a pris en compte : a) les composantes essentielles de la réussite d'un programme de contrôle de la fièvre aphteuse ; b) les parties prenantes concernées par l'application du programme de contrôle de la fièvre aphteuse ; c) les réglementations et documents pertinents du Département thaïlandais de développement de la production animale (DLD). Ces textes ont servi de base pour élaborer le cadre et les outils d'évaluation. Le cadre proposé recouvre les caractéristiques essentielles des mesures intégrant le programme de lutte contre la fièvre aphteuse en Thaïlande, en termes de conception et de mise en oeuvre. Les outils d'évaluation regroupent plusieurs matrices, trois séries de questionnaires et les questions à aborder lors d'entretiens. Les matrices d'évaluation permettent de détecter les failles dans la conception des mesures et d'évaluer la mise en oeuvre de celles-ci, ainsi que les capacités vétérinaires et la participation des parties prenantes. Les questionnaires et les questions posées lors d'entretiens permettent de recueillir des informations visant à éprouver la solidité du programme de lutte contre la fièvre aphteuse au regard des critères mis en exergue dans la matrice d'évaluation. Ce cadre et ces outils sont actuellement appliqués par la Thaïlande pour évaluer la zone proposée en tant qu'indemne de fièvre aphteuse.

Los autores proponen un sistema de referencia y varias herramientas que puedan emplearse para evaluar las políticas vigentes de lucha contra la fiebre aftosa en Tailandia, así como su aplicación en la región oriental del país (zona propuesta como «libre de la enfermedad¼). Con el estudio efectuado para elaborar dicho marco de referencia y dichas herramientas se pudieron determinar: a) los elementos esenciales para que un programa de lucha contra la fiebre aftosa tenga éxito; b) las partes interesadas que se ven afectadas por tal programa de lucha; y c) los reglamentos y documentos aplicables del Departamento de Desarrollo Ganadero, reglamentos y documentos que sirvieron de base para definir el marco y las herramientas de evaluación en cuestión. El marco de referencia propuesto integra las características esenciales del proceso de concepción y aplicación de políticas que deberían formar parte del programa de lucha contra la fiebre aftosa en Tailandia. Las herramientas de evaluación son: matrices de evaluación, tres conjuntos de cuestionarios y una serie de preguntas de entrevista. El uso de las matrices de evaluación sirve para detectar deficiencias en la concepción y aplicación de políticas, la capacidad veterinaria y la participación de las partes interesadas. Las preguntas de cuestionario y de entrevista permiten reunir información para valorar en qué medida los elementos del programa de lucha contra la fiebre aftosa se ajustan a los criterios de la matriz de evaluación. Este marco de referencia y estas herramientas se están aplicando actualmente en la zona de Tailandia propuesta como zona libre de fiebre aftosa.

Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris.

Fusion proteins composed of a cellulose-binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B were constructed using different linker peptides. The aim was to create proteolytically stable linkers that were able to join the functional modules without disrupting their function. Six fusion variants containing linkers of 4-44 residues were expressed in Pichia pastoris and analysed. Three variants were found to be stable throughout 7-day cultivations. The cellulose-binding capacities of fusion proteins containing short linkers were slightly lower compared with those containing long linkers. The lipase-specific activities of all variants, in solution or immobilized on to cellulose, were equal to that of the wild-type lipase.

Levels of ochratoxin A in blood from Norwegian and Swedish blood donors and their possible correlation with food consumption.

Blood levels of ochratoxin A were determined in 406 Scandinavian blood donors (206 from Oslo, Norway, and 200 from Visby on the island of Gotland, Sweden), using an HPLC method. In connection with the blood collection, the subjects were asked to fill in a food questionnaire to obtain individual dietary information relevant to ochratoxin A exposure. The mean plasma level of ochratoxin A was 0.18 ng/ml in Oslo and slightly higher, 0.21 ng/ml (P=0.046) in Visby. There was no correlation between plasma levels of ochratoxin A and the estimated total dietary intake of ochratoxin A based on consumption data and levels in food (retrieved from the literature), neither was the plasma level of ochratoxin A correlated with the total amount of food consumed. However, consumption of several foods, including cereal products, wine, beer and pork, were to some minor degree related to high plasma levels of ochratoxin A. The strongest correlations (correlation coefficient r>0.4; P<0.001) were observed for women in relation to the consumption of beer or medium brown bread. Correlation analysis of combinations of two or more food categories did not result in any statistically significant correlation.

Rational design of enantioselective enzymes requires considerations of entropy.

Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (Delta(R-S)DeltaH(++)) and entropy (Delta(R-S)DeltaS(++)) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. Delta(R-S)DeltaS(++) was in all cases equally significant as Delta(R-S)DeltaH(++) to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++). Although the changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++) were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger Delta(R-S)DeltaH(++) than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in Delta(R-S)DeltaS(++).

Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain.

Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.

Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study.

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.

Improved enantioselectivity of a lipase by rational protein engineering.

A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity. 1-Halo-2-octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast-reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutations gave rise to one variant with doubled enantioselectivity as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity. The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.

Mass transport limitations reduce the effective stereospecificity in enzyme-catalyzed kinetic resolution.

[reaction-see text] The kinetic resolution of seudenol catalyzed by Candida antarctica lipase B in hexane was investigated. Large differences in reaction rate and stereospecificity were observed when different enzyme preparations were used. These differences were ascribed to mass transport limitations which reduced both reaction rate and stereospecificity. Lyophilized enzyme preparations were more apt to give this problem than immobilized preparations. Further, low substrate concentrations enhanced the effect. Thus, high alcohol concentrations and enzyme immobilization can be recommended.

An active-site titration method for lipases.

A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.

Placental and lactational transfer of ochratoxin A in rats.

The placental and lactational transfer of ochratoxin A (OA) was investigated in a cross-fostering study in rats. Dams were given 50 microg OA(-1) kg body weight by gastric intubations 5 times a week for 2 weeks before mating, during gestation and then 7 days a week during lactation. Neonates from OA-treated dams were cross-fostered at birth to control dams treated with only vehicle. In the same way, neonates from control dams were cross-fostered to OA-treated dams. Treatment with OA did not result in any effects on birth weight or growth development of the pups during the first 21 days of life. There were no effects on milk quality as measured by milk lipids, protein or lactose concentrations, or on milk production, assessed by the mammary gland content of RNA and DNA. A mean milk:blood ratio of approximately 0.6 was found. The dose of OA from milk to the suckling pup at 14 days of age can be calculated to about 50 microg kg(-1) body weight(-1) day, which is similar to the dose given to the dams. Pups exposed to OA only via milk had blood and kidney levels of OA approximately 3 times higher than their dams, indicating a high absorption and/or a low excretion of OA in the sucklings. At 14 days of age the highest blood and kidney levels of OA were found in offspring exposed both via placenta and milk, with the highest contribution from milk. Offspring exposed only via milk had about 4-5 times higher levels of OA in blood and kidney compared to offspring exposed only via placenta. As milk could be a significant source of OA exposure in newborns, adverse health effects resulting from postnatal exposure should be studied and evaluated in the risk assessment of OA.

Molecular modeling of the enantioselectivity in lipase-catalyzed transesterification reactions.

Two strategies based on the use of subsets for calculating the enantioselectivity in lipase-catalyzed transesterifications using the CHARMM force field were investigated. Molecular dynamics was used in our search for low energy conformations. Molecular mechanics was used for refining these low energy conformations. A tetrahedral intermediate with a rigid central part was used for mimicking the transition state. The energy differences between the transition states of the diastereomeric enzyme-substrate complexes were calculated. The way of defining the subsets was based on two fundamentally different strategies. The first strategy used predefined parts of the enzyme and the substrate as subsets. The second approach formed energy-based subsets, varying in size with the substrates studied. The selection of residues to be included in these energy-based subsets was based on the energy of the interaction between the specific residue or water molecule and the transition state. The reaction studied was the kinetic resolution of secondary alcohols in transesterifications using the Candida antarctica lipase B as chiral biocatalyst. The secondary alcohols used in the study were 2-butanol, 3-methyl-2-butanol, and 3,3-dimethyl-2-butanol.

Ochratoxin A in rice cultivars after inoculation of Penicillium verrucosum.

The natural occurrence of ochratoxin A in grain samples of 23 rice cultivars was in the range 0.01-1.0 ng g(-1) rice. Samples from the same cultivars were surface sterilized with NaClO, dried to 19% water content and equilibrated at water activity (a(w)) 0.75 and 20 degrees C for 8 days. Varietal differences in equilibrium w/w water content (p < 0.0001) were found, reflected by differences in amylose and protein contents. Samples were then inoculated with an isolate of Penicillium verrucosum with 1 ml spore suspension to each 50 g rice sample; and incubated at a(w) 0.75 and 20 degrees C for 23 weeks. During incubation, ochratoxin A was accumulated in all cultivars. Significant varietal differences in ochratoxin A accumulation were observed (p < 0.0001). Grain samples with less than 19.5% equilibrium water content accumulated less ochratoxin A (p < 0.005). In a multiple regression analysis accumulated ochratoxin A content was expressed as a function of natural occurrence of ochratoxin A (p < 0.05), equilibrium water content at time of inoculation (p < 0.005), 1000-grain weight (p < 0.1), and chalkiness of endosperm (p < 0.05), with p < 0.0001 for the full function. Naturally occurring ochratoxin A was the strongest independent variable with p < 0.0005 for the slope coefficient in single regression. Rice cultivars IR8, IR24, IR620030-18-2-2 and R91-1081-1 had exceptionally low accumulation of ochratoxin A.

The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase.

The importance of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase for the hydrolytic activity at the water/lipid interface was investigated by site-directed mutagenesis. It was found that the effect on the hydrolytic activity upon the replacement of Trp89 with Phe, Leu, Gly or Glu was substrate dependent. The Trp89 mutants displayed an altered chain length specificity towards triglycerides, with a higher relative activity towards triacetin and trioctanoin compared with tributyrin. Trp89 was shown to be less important in the hydrolysis of vinyl esters compared with ethyl esters and triglycerides. An exclusive effect on the acylation reaction rate by the mutation of Trp89 was consistent with the data. It is suggested that Trp89 is important in the process of binding the acyl chain of the substrate into the active site for optimal acylation reaction rate. The Trp89Phe mutation resulted in an increased hydrolytic activity towards 2-alkylalkanoic acid esters. This is suggested to be due to reduction of unfavourable van der Waals contacts between Trp89 and the 2-substituent of the substrate. Thus, in contrast to natural substrates, Trp89 has a negative impact on the catalytic efficiency when substrates with bulky acyl chains are used. In contrast to the Trp89 mutations, the effect on the hydrolytic activity of the Glu87Ala mutation was almost substrate independent, 35-70% activity of wild-type lipase. A reduction of both the acylation and deacylation reaction was consistent with the data.

A structural basis for enantioselective inhibition of Candida rugosa lipase by long-chain aliphatic alcohols.

Molecular modeling showed that the enantiomers of heptyl 2-methyldecanoate are productively bound to the active site of Candida rugosa lipase in quite different conformations. The fast-reacting S-enantiomer may well occupy the previously identified acyl-binding tunnel in the active site of the lipase. By contrast, the slow-reacting R-enantiomer must be bound to the active site, leaving the tunnel empty to allow the formation of two catalytically essential hydrogen bonds between His 449 of the catalytic triad and the transition state of the catalyzed reaction. This information enables us to propose a molecular mechanism explaining how long-chain aliphatic alcohols act as enantioselective inhibitors of this lipase in the resolution of 2-methyldecanoic acid. Long-chain aliphatic alcohols may coordinate to the acyl-binding tunnel of the C. rugosa lipase, thereby selectively inhibiting the turnover of the fast-reacting S-enantiomer, thus resulting in a lowered enantioselectivity in the resolution.

Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols.

Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols.

On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase.

The interfacial activation of Candida antarctica lipase A (CALA) and B (CALB) has been investigated and compared with that of Humicola lanuginosa lipase (HLL). CALB displayed no interfacial activation towards p-nitrophenyl butyrate (PNPB) when exceeding the solubility limit of the substrate. No activation was observed towards p-nitrophenyl acetate (PNPA) at the addition of sodium dodecyl sulfate (SDS) nor in the presence of a solid polystyrene surface. The catalytic action of CALB was very different from that of Humicola lanuginosa lipase, which showed a pronounced interfacial activation with the same substrates. The basis for the anomalous behaviour of CALB is proposed to be due to the absence of a lid that regulates the access to the active site. In contrast to CALB, CALA expressed interfacial activation, but the activation was not as prominent as for Humicola lanuginosa lipase (HLL). The structural basis for the activation of CALA is unknown.

Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.

Probing a functional role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase through transesterification reactions in organic solvent.

To reveal the functional role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased the Vmax,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. The Km,app for tributyrin was not affected by the Glu87Ala mutation, but the Km,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreased Vmax,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on the Km,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased the Km,app for tributyrin and vinyl butyrate by a factor of > 5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0-680 mM) studied here. With vinyl butyrate as a substrate the Vmax,app was only 6% of that obtained with wild-type enzyme.

Toxicokinetics of ochratoxin A in rat following intratracheal administration.

The toxicokinetic parameters of ochratoxin A in rat following intratracheal administration of 50 ng ochratoxin A/g body weight were studied. The absorption of ochratoxin A from the lungs was very efficient. The elimination pattern of the toxin was studied by the analysis of blood samples. The biological half-life of ochratoxin A was 127 h and the calculated apparent volume of distribution equalled 168 ml/kg. The bioavailability of the toxin was very high, 98%. The plasma clearance of the toxin was 0.92 ml/kg.h.