FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the ß-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.

Molecular basis of non-mutational derepression of ramA in Klebsiella pneumoniae

Objectives: The ram locus, consisting of the romA–ramA genes, is repressed by the tetracycline-type regulator RamR, where regulation is abolished due to loss-of-function mutations within the protein or ligand interactions. The aim of this study was to determine whether the phenothiazines (chlorpromazine and thioridazine) directly interact with RamR to derepress ramA expression.

Methods: Quantitative real-time PCR analyses were performed to determine expression levels of the romA–ramA genes after exposure to the phenothiazines. Electrophoretic mobility shift assays (EMSAs) and in vitro transcription experiments were performed to show direct binding to and repression by RamR. Direct binding of the RamR protein to the phenothiazines was measured by fluorescence spectroscopy experiments and molecular docking models were generated using the RamR crystal structure.

Results: Exposure to either chlorpromazine or thioridazine resulted in the up-regulation of the romA–ramA genes. EMSAs and in vitro transcription experiments demonstrated that both agents reduce/abolish binding and enhance transcription of the target PI promoter upstream of the ramR–romA genes in Klebsiella pneumoniae compared with RamR alone. Fluorescence spectroscopy measurements demonstrated that RamR directly binds both chlorpromazine and thioridazine with micromolar affinity. Molecular docking analyses using the RamR crystal structure demonstrated that the phenothiazines interact with RamR protein through contacts described for other ligands, in addition to forming unique strong polar interactions at positions D152 and K63.

Conclusions: These data demonstrate that phenothiazines can modulate loci linked to the microbe–drug response where RamR is an intracellular target for the phenothiazines, thus resulting in a transient non-mutational derepression of ramA concentrations.

Comparative biochemical analysis of three members of the Schistosoma mansoni TAL family: Differences in ion and drug binding properties

The tegumental allergen-like (TAL) proteins from Schistosoma mansoni are part of a family of calcium binding proteins found only in parasitic flatworms. These proteins have attracted interest as potential drug or vaccine targets, yet comparatively little is known about their biochemistry. Here, we compared the biochemical properties of three members of this family: SmTAL1 (Sm22.6), SmTAL2 (Sm21.7) and SmTAL3 (Sm20.8). Molecular modelling suggested that, despite similarities in domain organisation, there are differences in the three proteins’ structures. SmTAL1 was predicted to have two functional calcium binding sites and SmTAL2 was predicted to have one. Despite the presence of two EF-hand-like structures in SmTAL3, neither was predicted to be functional. These predictions were confirmed by native gel electrophoresis, intrinsic fluorescence and differential scanning fluorimetry: both SmTAL1 and SmTAL2 are able to bind calcium ions reversibly, but SmTAL3 is not. SmTAL1 is also able to interact with manganese, strontium, iron(II) and nickel ions. SmTAL2 has a different ion binding profile interacting with cadmium, manganese, magnesium, strontium and barium ions in addition to calcium. All three proteins form dimers and, in contrast to some Fasciola hepatica proteins from the same family; dimerization is not affected by calcium ions. SmTAL1 interacts with the anti-schistosomal drug praziquantel and the calmodulin antagonists trifluoperazine, chlorpromazine and W7. SmTAL2 interacts only with W7. SmTAL3 interacts with the aforementioned calmodulin antagonists and thiamylal, but not praziquantel. Overall, these data suggest that the proteins have different biochemical properties and thus, most likely, different in vivo functions.

Psychotropic prescribing patterns among adolescents in Northern Ireland presenting with psychotic symptoms during a 5-year period

Objective: To report new prescriptions of psychotropic medications among adolescents presenting with new onset psychotic symptoms during a 5-year period.
Methods: The Northern Ireland Early Onset Psychosis Study is a naturalistic longitudinal observational study of patients with an early onset first psychotic episode. All patients aged <18 years presenting to specialist mental health services across Northern Ireland with new onset psychotic symptoms between 2001 and 2006 were recruited (n = 113). Clinical case notes were analysed retrospectively for details of subsequent treatment with psychotropic medications.
Results: A total of 100 patients (88.5%) were prescribed some form of psychotropic medication. Over three-quarters of patients received an antipsychotic as their first medication. Risperidone (45.8%), olanzapine (24.0%) and chlorpromazine (12.5%) were the most commonly prescribed first-line antipsychotic medications. Of a total of 160 antipsychotic prescriptions,81 (50.6%) were off-label. Prescriptions were most likely to have been deemed off-label owing to medications not being licensed in under-18s (71.6% of off-label prescriptions) but other reasons were medications being used outsidelicensed age ranges (23.5%) and outside licensed indications (4.9%).
Conclusions: This is the first study examining psychotropic prescribing patterns in a complete sample of all children and adolescents presenting with early onset psychotic episodes in a single geographical area. The observation of risperidoneas the most commonly prescribed antipsychotic was in keeping with previous studies in child and adolescent populations. Rates of off-label prescribing were lower than previously observed although our study was the first to investigateoff-label prescribing solely in children and adolescents presenting with psychotic symptoms.

The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Self-assembling of phenothiazine compounds investigated by small-angle X-ray scattering and electron paramagnetic resonance spectroscopy

Small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) have been carried out to investigate the structure of the self-aggregates of two phenothiazine drugs, chlorpromazine (CPZ) and trifluoperazine (TFP), in aqueous solution. In the SAXS studies, drug solutions of 20 and 60 mM, at pH 4.0 and 7.0, were investigated and the best data fittings were achieved assuming several different particle form factors with a homogeneous electron density distribution in respect to the water environment. Because of the limitation of scattering intensity in the q range above 0.15 angstrom(-1), precise determination of the aggregate shape was not possible and all of the tested models for ellipsoids, cylinders, or parallelepipeds fitted the experimental data equally well. The SAXS data allows inferring, however, that CPZ molecules might self-assemble in a basis set of an orthorhombic cell, remaining as nanocrystallites in solution. Such nanocrystals are composed of a small number of unit cells (up to 10, in c-direction), with CPZ aggregation numbers of 60-80. EPR spectra of 5- and 16-doxyl stearic acids bound to the aggregates were analyzed through simulation, and the dynamic and magnetic parameters were obtained. The phenothiazine concentration in EPR experiments was in the range of 5-60 mM. Critical aggregation concentration of TFP is lower than that for CPZ, consistent with a higher hydrophobicity of TFP. At acidic pH 4.0 a significant residual motion of the nitroxide relative to the aggregate is observed, and the EPR spectra and corresponding parameters are similar to those reported for aqueous surfactant micelles. However, at pH 6.5 a significant motional restriction is observed, and the nitroxide rotational correlation times correlate very well with those estimated for the whole aggregated particle from SAXS data. This implies that the aggregate is densely packed at this pH and that the nitroxide is tightly bound to it producing a strongly immobilized EPR spectrum. Besides that, at pH 6.5 the differences in motional restriction observed between 5- and 16-DSA are small, which is different from that observed for aqueous surfactant micelles.

Electroanalytical determination of promethazine hydrochloride in pharmaceutical formulations on highly boron-doped diamond electrodes using square-wave adsorptive voltammetry

The electrochemical oxidation of promethazine hydrochloride was made on highly boron-doped diamond electrodes. Cyclic voltammetry experiments showed that the oxidation mechanisms involved the formation of an adsorbed product that is more readily oxidized, producing a new peak with lower potential values whose intensity can be increased by applying the accumulation potential for given times. The parameters were optimized and the highest current intensities were obtained by applying +0.78 V for 30 seconds. The square-wave adsorptive voltammetry results obtained in BR buffer showed two well-defined peaks, dependent on the pH and on the voltammetric parameters. The best responses were obtained at pH 4.0, frequency of 50 s(-1), step of 2 mV, and amplitude of 50 mV. Under these conditions, linear responses were obtained for concentrations from 5.96 x 10(-7) to 4.76 x 10(-6) mol L-1, and calculated detection limits of 2.66 x 10(-8) mol L-1 (8.51 mu g L-1) for peak 1 and of 4.61 x 10(-8) mol L-1 (14.77 mu g L-1) for peak 2. The precision and accuracy were evaluated by repeatability and reproducibility experiments, which yielded values of less than 5.00% for both voltammetric peaks. ne applicability of this procedure was tested on commercial formulations of promethazine hydrochloride by observing the stability, specificity, recovery and precision of the procedure in complex samples. All results obtained were compared to recommended procedure by British Pharmacopeia. The voltammetric results indicate that the proposed procedure is stable and sensitive, with good reproducibility even when the accumulation steps involve short times. It is therefore very suitable for the development of the electroanalytical procedure, providing adequate sensitivity and a reliable method.

Self-assembling of phenothiazine compounds investigated by small-angle X-ray scattering and electron paramagnetic resonance spectroscopy

Small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) have been carried out to investigate the structure of the self-aggregates of two phenothiazine drugs, chlorpromazine (CPZ) and trifluoperazine (TFP), in aqueous solution. In the SAXS studies, drug solutions of 20 and 60 mM, at pH 4.0 and 7.0, were investigated and the best data fittings were achieved assuming several different particle form factors with a homogeneous electron density distribution in respect to the water environment. Because of the limitation of scattering intensity in the q range above 0.15 angstrom(-1), precise determination of the aggregate shape was not possible and all of the tested models for ellipsoids, cylinders, or parallelepipeds fitted the experimental data equally well. The SAXS data allows inferring, however, that CPZ molecules might self-assemble in a basis set of an orthorhombic cell, remaining as nanocrystallites in solution. Such nanocrystals are composed of a small number of unit cells (up to 10, in c-direction), with CPZ aggregation numbers of 60-80. EPR spectra of 5- and 16-doxyl stearic acids bound to the aggregates were analyzed through simulation, and the dynamic and magnetic parameters were obtained. The phenothiazine concentration in EPR experiments was in the range of 5-60 mM. Critical aggregation concentration of TFP is lower than that for CPZ, consistent with a higher hydrophobicity of TFP. At acidic pH 4.0 a significant residual motion of the nitroxide relative to the aggregate is observed, and the EPR spectra and corresponding parameters are similar to those reported for aqueous surfactant micelles. However, at pH 6.5 a significant motional restriction is observed, and the nitroxide rotational correlation times correlate very well with those estimated for the whole aggregated particle from SAXS data. This implies that the aggregate is densely packed at this pH and that the nitroxide is tightly bound to it producing a strongly immobilized EPR spectrum. Besides that, at pH 6.5 the differences in motional restriction observed between 5- and 16-DSA are small, which is different from that observed for aqueous surfactant micelles.

Optimized synthesis of the melatonin metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo comparativo entre a acepromazina, clorpromazina e levomepromazina em diferentes doses, através do exame bispectral, termo e pressoalgimetria, em cães

The study's objective was to realize comparisons among different acepromazine, chlorpromazine and methotrimeprazine doses, evaluate parametric changes, test analgesia using press and term algimetry, and evaluate bispectral condition. 90 mongrel dogs were used, male and female, adult, weighting 10 to 15 Kg as a rule, distributed in 9 groups with 10 animals each. At first, second and third groups acepromazine was used at 0,1; 0,05 e 0,025 mg/Kg, respectively. At forth, fifth and sixth groups, chlorpromazine was used at 1,0; 0,5 and 0,25 mg/Kg, respectively. At seventh, eighth and ninth groups, methotrimeprazine at 1,0; 0,5 and 0,25 mg/Kg was used, respectively. All drugs were administered intravenously. objects of study: heart rate (HR), non invasive blood pressure (SAP, MAP, DAP), respiratory rate (f), capnography (ETCO(2)), pulse oxymetry (SatO(2)), mouth and rectal temperature, bispectral index (BIS), electromyography (EMG%), press and term algimetry. Somatic analgesia was evaluated by animal's response to nociceptives stimulus. We concluded that chlorpromazine had more bypotension. Dogs showed higher hypnosis level at: chlorpromazine group, with evident myorelaxation. All groups showed analgesia to thermic and mechanic stimulus. Acepromazine group showed high duration to both pain stimuli. Bispectral index was shorten at chlorpromazine group at 1,0 mg/kg doses, showing higher hypnosis index, and acepromazine was the less depressing considering the bispectral index.

Bloqueio do nervo alveolar mandibular com ropivacaína a 0,5 % em gatos

Objetivou-se, com este experimento, avaliar a ação da ropivacaína a 0,5% no bloqueio do nervo alveolar mandibular de gatos. Vinte gatos adultos, sem raça definida, machos ou fêmeas, receberam clorpromazina (1,0mg kg-1, VO) e propofol (3,0mg kg-1, IV). Ropivacaína a 0,5% foi administrada com uma agulha 13x3,8 em forma de L, inserida no ângulo da mandíbula direita, aproximadamente 1,0cm rostral ao processo angular e 0,5cm dorsal à superfície medial do ramo da mandíbula, a fim de depositá-la próximo ao nervo alveolar mandibular, no forame mandibular. As freqüências cardíaca e respiratória foram mensuradas antes da administração da clorpromazina, 20 minutos após administração desta (T0), 20 minutos após o bloqueio do nervo alveolar mandibular com ropivacaína (T20) e, em intervalos de 20 minutos, até a volta da sensibilidade na região anestesiada. Observou-se o período de latência e a duração da anestesia por meio do pinçamento da pele e gengiva da região lateral direita da mandíbula. Encontrou-se início da anestesia após 22 minutos, com duração de 164,25 minutos. Os parâmetros de freqüência cardíaca e freqüência respiratória tiveram alterações, porém sem significado clínico para a espécie. A ropivacaína a 0,5% anestesia a região dos dentes pré-molares, molares, caninos, incisivos, pele e mucosa oral e lábio inferior, sem causar efeitos colaterais.