Identification of NF-AT-like transcription factor in Schistosoma mansoni: its possible involvement in the antiparasitic action of cyclosporin A.

Cyclosporin A (CsA) has been found to exert potent anti-parasite activity against a wide range of protozoan and helminth parasites. In schistosomes, evidence has been accumulated to propose that the drug damages parasites by mechanisms independent of its immunosuppressive properties. Moreover, the absence of correlation between anti-schistosomal properties and inhibition of peptidyl-prolyl cis-trans isomerase activity of cyclophilins (CsA receptors) for various drug analogs, argued against a direct implication of cyclophilins in the lethal effect of CsA. We describe, in S. mansoni, the existence of NF-AT-like transcription factors, a protein family already characterized by its sensitivity to CsA. The observation that CsA treatment of S. mansoni larvae inhibited the expression of the Sm28GST protein and the characterization of a functional NF-AT-like site in the gene encoding this protein, provide new insights in the understanding of the antischistosomal effect of CsA. Our results also support the hypothesis that the regulatory function of NF-AT-like proteins might be responsible for parasite development and survival in the host and open new perspectives in studies of helminth biology.

Conformational requirements of a recombinant ferredoxin-NADP+ reductase precursor for efficient binding to and import into isolated chloroplasts.

The cytosolic precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase was expressed in Escherichia coli rendering a soluble protein that contained bound FAD and could be imported by isolated chloroplasts. The mechanism of plastid translocation was studied under defined conditions using this recombinant precursor holoprotein and intact pea chloroplasts. The first step in the import pathway, namely, binding of the reductase precursor to isolated chloroplasts, was saturable at about 2000 molecules/plastid, and showed a high-affinity interaction with a dissociation constant Kd of approximately 5 nM. Binding was not affected by the addition of soluble leaf extracts or by prior denaturation of the precursor with urea. Analysis of the initial import rates at different precursor concentrations indicated the existence of a single translocation system for this protein. Inclusion of leaf extracts in the assay resulted in a three-fold increase of the maximal import rates to 14,000 molecules . min-(1).chloroplast-(1), with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 microM to 100-150 nM. Comparison of Km and Kd values under various conditions indicated that the binding step of the translocation process is largely irreversible, favouring import and processing. In the absence of extract, a denatured precursor obtained by incubation with urea was a better substrate for plastid import than the holoprotein. Treatment of the precursor with either extract or urea resulted in similar increases in import efficiency (V/Km), suggesting that stimulation by leaf extracts is probably related to unfolding of the precursor prior to translocation.

The precursor of pea ferredoxin-NADP+ reductase synthesized in Escherichia coli contains bound FAD and is transported into chloroplasts.

The precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.

One-step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase.

Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988). A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase. When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences. The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. It showed both transferase and reductase activities. Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous. The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host. The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells. The system is potentially useful for the purification of activity-impaired forms of the flavoprotein.

DNA polymerase activity of tomato fruit chromoplasts.

DNA polymerase activity was measured in chromoplasts of ripening tomato fruits. Plastids isolated from young leaves or mature red fruits showed similar DNA polymerase activities. The same enzyme species was present in either chloroplasts or chromoplasts as judged by pH and temperature profiles, sensitivities towards different inhibitors and relative molecular mass (Mr 88 kDa). The activities analyzed showed the typical behaviour of plastid-type polymerases. The results presented here suggest that chromoplast maintain their DNA synthesis potential in fruit tissue at chloroplast levels. Consequently, the sharp decrease of the plastid chromosome transcription observed at the onset of fruit ripening could not be due to limitations in the availability of template molecules. Other mechanisms must be involved in the inhibition of chromoplast RNA synthesis.

[Semantic scales for noise evaluation. Third stage: subjective characterization of sound stimuli]. / Escalas semánticas para la evaluación del ruido. Tercera etapa: caracterización subjectiva de estímulos sonoros.

This is the third step of a research for which Osgood's Differential Semantic Technique was resorted to in order to get suitable scales on a subjective evaluation of sound stimuli. Thus, a three-stage work was fulfilled. The first one dealt with the construction of seven-point bipolar scales starting from experimentally selected adjectives. The second one succeeded in finding the subjacent dimensions of the said scales by means of Factorial Analysis Method. The present stage aims at characterizing 40 sound stimuli through the discovered dimensions most representative scales. A special Semantic Differential Program was used to getting the following Index: Scalar Scores, Polarity, Familiarity and Interconcept Distances. The interpretation of the Index showed the best characterized stimuli altogether with their affective meanings and their similarities and differences as well.

Escalas semánticas para la evaluación del ruido tercera etapa: caracterización subjetiva de estímulos sonoros / Semantic scales for noise evaluation third part: subjective characterization of sound stimuli

Tercera y última etapa de una investigación (5, 6) cuya finalidad fue la construcción de escalas adecuadas para la evaluación subjetiva de estímulos sonoros, utilizando la Técnica del diferencial semántico. El objetivo de esta etapa fue caracterizar 40 estímulos sonoros a través de un grupo de las escalas antes mencionadas y teniendo en cuenta las dimensiones psicológicas subyacentes obtenidas por el análisis factorial de las mismas. Para ello se aplicó un programa especial de diferencial semántico obteniéndose los siguientes índices: puntaje escalar, polaridad, familiaridad y distancia inter-conceptos. Se determinaron los estímulos sonoros mejor caracterizados y las escalas más adecuadas para evaluar cada concepto

Escalas semánticas para la evaluación del ruido tercera etapa: caracterización subjetiva de estímulos sonoros / Semantic scales for noise evaluation third part: subjective characterization of sound stimuli

Tercera y última etapa de una investigación (5, 6) cuya finalidad fue la construcción de escalas adecuadas para la evaluación subjetiva de estímulos sonoros, utilizando la Técnica del diferencial semántico. El objetivo de esta etapa fue caracterizar 40 estímulos sonoros a través de un grupo de las escalas antes mencionadas y teniendo en cuenta las dimensiones psicológicas subyacentes obtenidas por el análisis factorial de las mismas. Para ello se aplicó un programa especial de diferencial semántico obteniéndose los siguientes índices: puntaje escalar, polaridad, familiaridad y distancia inter-conceptos. Se determinaron los estímulos sonoros mejor caracterizados y las escalas más adecuadas para evaluar cada concepto (AU)

Escalas semánticas para la evaluación del ruido. Tercera etapa: caracterización subjectiva de estímulos sonoros. / [Semantic scales for noise evaluation. Third stage: subjective characterization of sound stimuli]

This is the third step of a research for which Osgoods Differential Semantic Technique was resorted to in order to get suitable scales on a subjective evaluation of sound stimuli. Thus, a three-stage work was fulfilled. The first one dealt with the construction of seven-point bipolar scales starting from experimentally selected adjectives. The second one succeeded in finding the subjacent dimensions of the said scales by means of Factorial Analysis Method. The present stage aims at characterizing 40 sound stimuli through the discovered dimensions most representative scales. A special Semantic Differential Program was used to getting the following Index: Scalar Scores, Polarity, Familiarity and Interconcept Distances. The interpretation of the Index showed the best characterized stimuli altogether with their affective meanings and their similarities and differences as well.

[Semantic scales for the evaluation of noise (II). Subjacent dimensions in subjective judgments of sound stimuli]. / Escalas semánticas para la evaluación del ruido (II). Dimensiones subyacentes en los juicios subjetivos de estímulos sonoros.

Second stage of a research in which Osgood's Diferential Semantic Technique was used in two experiments with the following aims: a) to find out the subyacent dimensions of subjective evaluation of sound stimuli; b) to know if Osgood's Evaluation, Potency and Activity dimensions appear when sound stimuli are judged; c) to obtain a group of semantic scales that represent the found dimensions, which can be used in other researches in the same field. In the first experiment 37 scales were used by 98 Ss for judging 10 sound stimuli. In the second, 120 Ss performed the same task through 46 scales and 40 sound stimuli. Data of both experiments were processed by factor analysis method and a Varimax rotation was done. Having obtained 12 similar factors in both experiments the results were analized in a comparative way. Eight dimensions that explain the subject-sound stimuli relation were found. The other dimensions deal with the stimulis control, cognitive experience and structural caracteristics of sound stimuli, being the affective component also present in some of their scales. In the third stage the sound stimuli used will be analized through the found dimensions.

Escalas semanticas para la evaluacion del ruido (II). Dimensiones subyacentes en los juicios subjetivos de estimulos sonoros. / Semantic scales for the noise (II). Subjacent dimensions in subjective judgement of sound stimuli

Segunda etapa de una investigacion en la cual utilizando la tecnica del diferencial semantico de Osgood se realizaron dos experiencias con el objeto de investigar: a) las dimensiones que subyacen en la evaluacion de estimulos sonoros; b) si las dimensiones EPA del mismo autor aparecen en el dominio estudiado; c) si existe un grupo de escalas semanticas representativas de las dimensiones encontradas. Dos muestras de sujetos juzgaron diferentes grupos de estimulos sonoros mediante escalas de adjetivos bipolares.Los datos obtenidos en cada experiencia fueron procesados por el metodo de analisis factorial, interpretandose los resultados en forma comparativa.Obtuvieron 8 dimensiones que explican la relacion sujeto-juicio-estimulo sonoro, comprobandose la importancia de las dimensiones afectivas en esta relacion

Escalas semánticas para la evaluación del ruido (II). Dimensiones subyacentes en los juicios subjetivos de estímulos sonoros. / [Semantic scales for the evaluation of noise (II). Subjacent dimensions in subjective judgments of sound stimuli]

Second stage of a research in which Osgoods Diferential Semantic Technique was used in two experiments with the following aims: a) to find out the subyacent dimensions of subjective evaluation of sound stimuli; b) to know if Osgoods Evaluation, Potency and Activity dimensions appear when sound stimuli are judged; c) to obtain a group of semantic scales that represent the found dimensions, which can be used in other researches in the same field. In the first experiment 37 scales were used by 98 Ss for judging 10 sound stimuli. In the second, 120 Ss performed the same task through 46 scales and 40 sound stimuli. Data of both experiments were processed by factor analysis method and a Varimax rotation was done. Having obtained 12 similar factors in both experiments the results were analized in a comparative way. Eight dimensions that explain the subject-sound stimuli relation were found. The other dimensions deal with the stimulis control, cognitive experience and structural caracteristics of sound stimuli, being the affective component also present in some of their scales. In the third stage the sound stimuli used will be analized through the found dimensions.

Escalas semanticas para la evaluacion del ruido (II). Dimensiones subyacentes en los juicios subjetivos de estimulos sonoros. / Semantic scales for the noise (II). Subjacent dimensions in subjective judgement of sound stimuli

Segunda etapa de una investigacion en la cual utilizando la tecnica del diferencial semantico de Osgood se realizaron dos experiencias con el objeto de investigar: a) las dimensiones que subyacen en la evaluacion de estimulos sonoros; b) si las dimensiones EPA del mismo autor aparecen en el dominio estudiado; c) si existe un grupo de escalas semanticas representativas de las dimensiones encontradas. Dos muestras de sujetos juzgaron diferentes grupos de estimulos sonoros mediante escalas de adjetivos bipolares.Los datos obtenidos en cada experiencia fueron procesados por el metodo de analisis factorial, interpretandose los resultados en forma comparativa.Obtuvieron 8 dimensiones que explican la relacion sujeto-juicio-estimulo sonoro, comprobandose la importancia de las dimensiones afectivas en esta relacion

[Construction of semantic scales for the subjective evaluation of noise]. / Construcción de escalas semánticas para la evaluación subjetiva del ruido.

This is the first stage of a research the aim of which is to construct proper scales for the subjective evaluation of noise, using Osgood's Differential Semantic Technique. 137 adjectives were selected using two criteria. For the first criterium a method based on Gougenheim's and Hogenraad's researches was used. 200 subjects were asked to write 30 adjectives that could be used for qualifying sonorous stimuli. The frequencies of the adjectives given by the subjects were calculated and 25 of them were selected for being under the 10% limit of the probability, arbitrarily chosen as a criterium for the selection. For the second criterium 10 acoustic researchers were asked to write all those adjectives they considered important for noise evaluation. Thus, 112 adjectives were selected. The 137 adjectives got by the two criteria were judged by 15 university professors of different careers, for getting their opposites. A level of agreement of at least 6 of 15 judges was considered. 62 adjectives with their opposites were got and using other two criteria they were reduced to 37. A hypothesis was formulated grouping the adjectives into 8 factors taking into account the three universal factors: Evaluation (E), Potency (P) and Activity (A) according to the Differential Semantic Technique. Seven point scales were constructed with the adjectives. The construction of the scales, the selection of concepts, the application of them to a sample of subjects and data analysis will be the second stage of this research.

Construccion de escalas semanticas para la evaluacion subjetiva del ruido. / Construction of semantic scales for the subjective evaluation of noise

Primera etapa de un trabajo cuyo objetivo es construir mediante la tecnica del Diferencial Semantico, escalas adecuadas para evaluar subjetivamente estimulos sonoros.Se seleccionaron en primer lugar adjetivos utilizando 2 criterios experimentales y se obtuvieron sus opuestos mediante el Sistema de Jueces. Se seleccionaron los mas relevantes, teniendo en cuenta todos los aspectos de la relacion sujeto-estimulo sonoro. Se formularon hipotesis sobre los factores subyacentes, quedando la construccion y seleccion de escalas asi como la comprobacion de los factores para la segunda etapa de esta investigacion

Construcción de escalas semánticas para la evaluación subjetiva del ruido. / [Construction of semantic scales for the subjective evaluation of noise]

This is the first stage of a research the aim of which is to construct proper scales for the subjective evaluation of noise, using Osgoods Differential Semantic Technique. 137 adjectives were selected using two criteria. For the first criterium a method based on Gougenheims and Hogenraads researches was used. 200 subjects were asked to write 30 adjectives that could be used for qualifying sonorous stimuli. The frequencies of the adjectives given by the subjects were calculated and 25 of them were selected for being under the 10

limit of the probability, arbitrarily chosen as a criterium for the selection. For the second criterium 10 acoustic researchers were asked to write all those adjectives they considered important for noise evaluation. Thus, 112 adjectives were selected. The 137 adjectives got by the two criteria were judged by 15 university professors of different careers, for getting their opposites. A level of agreement of at least 6 of 15 judges was considered. 62 adjectives with their opposites were got and using other two criteria they were reduced to 37. A hypothesis was formulated grouping the adjectives into 8 factors taking into account the three universal factors: Evaluation (E), Potency (P) and Activity (A) according to the Differential Semantic Technique. Seven point scales were constructed with the adjectives. The construction of the scales, the selection of concepts, the application of them to a sample of subjects and data analysis will be the second stage of this research.

Construccion de escalas semanticas para la evaluacion subjetiva del ruido. / Construction of semantic scales for the subjective evaluation of noise

Primera etapa de un trabajo cuyo objetivo es construir mediante la tecnica del Diferencial Semantico, escalas adecuadas para evaluar subjetivamente estimulos sonoros.Se seleccionaron en primer lugar adjetivos utilizando 2 criterios experimentales y se obtuvieron sus opuestos mediante el Sistema de Jueces. Se seleccionaron los mas relevantes, teniendo en cuenta todos los aspectos de la relacion sujeto-estimulo sonoro. Se formularon hipotesis sobre los factores subyacentes, quedando la construccion y seleccion de escalas asi como la comprobacion de los factores para la segunda etapa de esta investigacion