Síndrome de Aicardi-Goutières: un caso familiar por alteración del gen RNASEH2B / Aicardi-Goutières syndrome: a family case due to alteration of the RNASEH2B gene

Introducción: El síndrome de Aicardi-Goutières es una encefalopatía progresiva de inicio en el primer año de vida que condiciona retraso psicomotor, microcefalia y disfunción piramidal. Tiene una prevalencia de 1-5 de cada 10.000 recién nacidos vivos. La mayoría de los casos tiene transmisión autosómica recesiva, por alteración en siete genes implicados en el metabolismo del interferón, lo cual condiciona un aumento de sus niveles en la sangre y el líquido cefalorraquídeo, y afecta al cerebro (leucodistrofia, atrofia corticosubcortical, calcificaciones en los núcleos basales…), la piel y el sistema inmunitario. Caso clínico: Se trata de dos hermanos que presentan la variante p.Ala177Thr en homocigosis en el gen RNASEH2B; ambos progenitores, consanguíneos, son portadores. El primer hermano comenzó a los 10 meses con hipotonía axial, hipertonía de las extremidades, regresión psicomotriz y movimientos distónicos. El segundo hermano presentó desde el nacimiento tono axial bajo con hipertonía de las extremidades, a los 4 meses se hallaron calcificaciones en los núcleos lenticuloestriados mediante ecografía transfontalar y a los 6 meses inició movimientos distónicos y nistagmo intermitente. Ambos han desarrollado tetraparesia espástica y permanecen estables con 8 y 10 años, pese a las complicaciones propias del síndrome. Conclusiones: El síndrome de Aicardi-Goutières es una entidad rara que debe tenerse presente ante situaciones que cursen con alteración del desarrollo psicomotor y calcificaciones intracraneales; destacamos la importancia del diagnóstico genético tanto para conocer el pronóstico de nuestros pacientes en función de su alteración genética como para ofrecer consejo genético a sus familias.(AU)

Introduction: Aicardi-Goutières syndrome is a progressive encephalopathy with onset in the first year of life that conditions psychomotor retardation, microcephaly and pyramidal dysfunction. It has a prevalence of 1-5 in 10,000 newly live births. Most cases have autosomal recessive transmission, due to alteration in seven genes involved in the metabolism of interferon, which causes an increase in its levels in the blood and cerebrospinal fluid, and affects the brain (leukodystrophy, corticosubcortical atrophy, calcifications in the basal ganglia…), the skin and the immune system. Clinical case: They are two brothers who present the homozygous p.Ala177Thr variant in the RNASEH2B gene; both of them parents, consanguineous, are carriers. The first sibling started at 10 months with axial hypotonia, hypertonia of the extremities, psychomotor regression and dystonic movements. The second brother presented from the birth low axial tone with hypertonia of the extremities, at 4 months calcifications were found in the nuclei lenticulostriated by transfontalar ultrasound and, at 6 months, she started dystonic movements and intermittent nystagmus. Both have developed spastic tetraparesis and remain stable at 8 and 10 years, despite complications typical of the syndrome. Conclusions: The Aicardi-Goutières syndrome is a rare entity that should be taken into account in situations that occur with altered psychomotor development and intracranial calcifications; we highlight the importance of diagnosis both to know the prognosis of our patients based on their genetic alteration and to offer genetic counseling to their families.(AU)

[Aicardi-Goutieres syndrome: a family case due to alteration of the RNASEH2B gene]. / Síndrome de Aicardi-Goutières: un caso familiar por alteración del gen RNASEH2B.

INTRODUCTION: Aicardi-Goutieres syndrome is a progressive encephalopathy with onset in the first year of life that conditions psychomotor retardation, microcephaly and pyramidal dysfunction. It has a prevalence of 1-5 in 10,000 newly live births. Most cases have autosomal recessive transmission, due to alteration in seven genes involved in the metabolism of interferon, which causes an increase in its levels in the blood and cerebrospinal fluid, and affects the brain (leukodystrophy, corticosubcortical atrophy, calcifications in the basal ganglia…), the skin and the immune system. CLINICAL CASE: They are two brothers who present the homozygous p.Ala177Thr variant in the RNASEH2B gene; both of them parents, consanguineous, are carriers. The first sibling started at 10 months with axial hypotonia, hypertonia of the extremities, psychomotor regression and dystonic movements. The second brother presented from the birth low axial tone with hypertonia of the extremities, at 4 months calcifications were found in the nuclei lenticulostriated by transfontalar ultrasound and, at 6 months, she started dystonic movements and intermittent nystagmus. Both have developed spastic tetraparesis and remain stable at 8 and 10 years, despite complications typical of the syndrome. CONCLUSIONS: The Aicardi-Goutieres syndrome is a rare entity that should be taken into account in situations that occur with altered psychomotor development and intracranial calcifications; we highlight the importance of diagnosis both to know the prognosis of our patients based on their genetic alteration and to offer genetic counseling to their families.

TITLE: Síndrome de Aicardi-Goutières: un caso familiar por alteración del gen RNASEH2B.Introducción. El síndrome de Aicardi-Goutières es una encefalopatía progresiva de inicio en el primer año de vida que condiciona retraso psicomotor, microcefalia y disfunción piramidal. Tiene una prevalencia de 1-5 de cada 10.000 recién nacidos vivos. La mayoría de los casos tiene transmisión autosómica recesiva, por alteración en siete genes implicados en el metabolismo del interferón, lo cual condiciona un aumento de sus niveles en la sangre y el líquido cefalorraquídeo, y afecta al cerebro (leucodistrofia, atrofia corticosubcortical, calcificaciones en los núcleos basales…), la piel y el sistema inmunitario. Caso clínico. Se trata de dos hermanos que presentan la variante p.Ala177Thr en homocigosis en el gen RNASEH2B; ambos progenitores, consanguíneos, son portadores. El primer hermano comenzó a los 10 meses con hipotonía axial, hipertonía de las extremidades, regresión psicomotriz y movimientos distónicos. El segundo hermano presentó desde el nacimiento tono axial bajo con hipertonía de las extremidades, a los 4 meses se hallaron calcificaciones en los núcleos lenticuloestriados mediante ecografía transfontalar y a los 6 meses inició movimientos distónicos y nistagmo intermitente. Ambos han desarrollado tetraparesia espástica y permanecen estables con 8 y 10 años, pese a las complicaciones propias del síndrome. Conclusiones. El síndrome de Aicardi-Goutières es una entidad rara que debe tenerse presente ante situaciones que cursen con alteración del desarrollo psicomotor y calcificaciones intracraneales; destacamos la importancia del diagnóstico genético tanto para conocer el pronóstico de nuestros pacientes en función de su alteración genética como para ofrecer consejo genético a sus familias.

Evaluation of the metal phytoextraction potential of crop legumes. Regulation of the expression of O-acetylserine (thiol)lyase under metal stress.

The metal phytoextraction potential of three legumes belonging to different genera has been studied under greenhouse conditions. Legumes accumulate As and metals mainly in roots, although translocation to shoot is observed. Alfalfa did accumulate the highest concentrations of As and metals in shoots and aerial biomass was less affected by the toxic elements, indicating its good behaviour in phytoextraction. Clover accumulated less metal, but showed larger biomass. EDTA addition enhanced Pb phytoextraction up to levels similar to those described for plants proposed in phytoremediation. The regulation of O-acetylserine (thiol)lyase from legumes under metal stress has been analysed to test the possibility of establishing a possible correlation between the expression of OASTL in the presence of the metals and the metal accumulation in legume plant tissues. Cd and Pb(EDTA) produce the strongest increases of OASTL activity, with the higher enhancement seen in roots, in parallel with the higher metal accumulation. Arsenic produced an increase of root enzyme activity, whereas Cu produced a decrease, mainly in shoots. Western blots using antibodies against an A. THALIANA cytosolic OAS-TL recognised up to five protein bands in crude extracts from LOTUS and clover. A low molecular weight isoform of 32 kDa was induced in the presence of Cd and Pb. A partial RT-PCR sequence from clover has been obtained, showing 86 - 97 % identity with other described OASTLs. The PCR fragment has been used to analyse OASTL mRNA levels of legumes under metal stress. OASTL transcripts were increased by As, Cd, and Pb, especially in roots, where metal accumulation was maximal, while Cu produced a decrease in the transcript levels.

[Ontogenic peculiarities of the human tympanic ossicular chain]. / Peculiaridades en la organización de la cadena osicular timpánica humana a lo largo de su ontogenia.

We have studied the development of the tympanic ossicles in 40 embryo-foetal human series aged between 32 days (6 mm) and newborn. Once performed the measurements to date chronologically embryos and foetuses, we did a meticulous dissection of temporal bones. After fix in 10% formol, decalcified with 2% nitric acid, embedded in Paraplast, sectioned in a sequence of 7 mm, and stained with Martin's trichrome. The tympanic ossicles are developed in the mesenchyme of the two first pharyngeal archs. The head of the malleus, the body and the short limb of the incus arise from the first arch while the handle of the malleus, the long limb of the incus and the mass of the stapes arise from the second arch. The vestibular side of the stapedial footplate develops in the otic capsule. The tympanic ossicles develop from endochondral ossification, while anterior process of the malleus has the membranous ossification. In their ontogenia 6 stages are observed. First stage, the formation of their sketch by mesenchimal condensation, in the second stage, "pre-cartilaginous", the cells of the primordia are differentiated into condroblasts, in the third stage "cartilaginous" the ossicles show a cartilaginous structure, in the forth stage the primary ossification centers are developed, in the fifth stage the ossicles arise in the periostic annulus and inside the endochondral bone, and in the last stage the osseous tissue grows until it acquires a compact osseous structure.

[Severe fulminant form of neonatal citrullinemia. Report of a case]. / Forma grave fulminante de citrulinemia neonatal. Comunicación de un caso.

INTRODUCTION: Citrullinemia is an autosomal recessive disease, which is caused by a deficiency of the argininosuccinate synthetase. The neonatal forms are serious and many times are associated with a high level of mortality. CASE REPORT: A newborn that came in again on her third day of life due to a apneic episodes which required mechanical ventilation. Previously, she rejected feeding, had poor suction, lethargy and remarkable hypoactivity. During the following hours, she showed serious neurologycal deterioration with multifocal convulsions and coma, passing away 20 hours after admission due to endocraneal hypertension. The metabolic evaluation confirmed very significant hyperammonemia, with important increase of citrullin and glutamin, and arginine in the low limits of normality. She was treated with sodium benzoate and arginine and she also needed exanguinotransfusion. It was not possible to put her on hemodyalisis. The findings of the autopsy confirmed massive cerebral edema and characteristic hystological changes in the liver. The determination of the enzymatical activity in liver tissue showed a partial deficiency, with a residual activity of 25% of the average control. CONCLUSIONS: This is a case of fulminant neonatal citrullinemia that we considered of interest in order to draw the attention of the clinical on this type of diseases. The prognosis depends on early diagnosis, witch is based on clinical suspicion and analytical determination of ammonia in every newborn with unexplained vomiting, lethargy or other symptoms of encephalopathy.

Forma grave fulminante de citrulinemia neonatal. Comunicación de un caso / Severe fulminant form of neonatal citrullinemia. Report of a case

Introducción. La citrulinemia es una afección autosómica recesiva, debida a una deficiencia de la argininsuccinato sintetasa (ASS); las formas neonatales son graves y se asocian a un alto índice de mortalidad. Caso clínico. Recién nacida que reingresa al tercer día de vida por episodios apneicos, que requirieron intubación y ventilación mecánica. Previamente, presentó rechazo de las tomas, mala succión, letargia e hipoactividad marcada. En las horas siguientes presentó grave deterioro neurológico, con convulsiones multifocales y coma; falleció a las 20 horas de su ingreso por un cuadro de hipertensión endocraneal. La evaluación metabólica confirmó una hiperamonemia muy significativa, con importante aumento de citrulina y glutamina, adsí como arginina en los límites bajos de la normalidad. Se trató con benzoato sódico y arginina, y se le realizó una exanguinotransfusión de doble volumen; no fue posible practicarle hemodiálisis. Los hallazgos de la autopsia confirmaron un edema cerebral masivo y cambios histológicos característicos en el hígado. La medida de la actividad enzimática en el tejido hepático reveló una deficiencia parcial, con una actividad residual del 25 por ciento de la media control. Conclusión. Se trata de una observación de citrulinemia neonatal de curso fulminante que consideramos de interés, con la finalidad de alertar al clínico sobre este tipo de patología, ya que el pronóstico se va a relacionar con el diagnóstico precoz, basado en la sospecha clínica y determinación del amonio en todo recién nacido con vómitos inexplicables, letargia u otros signos de encefalopatía (AU)

Introduction. Citrullinemia is an autosomal recessive disease, which is caused by a deficiency of the argininosuccinate synthetase. The neonatal forms are serious and many times are associated with a high level of mortality. Case report. A newborn that came in again on her third day of life due to a apneic episodes which required mechanical ventilation. Previously, she rejected feeding, had poor suction, lethargy and remarkable hypoactivity. During the following hours, she showed serious neurologycal deterioration with multifocal convulsions and coma, passing away 20 hours after admission due to endocraneal hypertension. The metabolic evaluation confirmed very significant hyperammonemia, with important increase of citrullin and glutamin, and arginine in the low limits of normality. She was treated with sodium benzoate and arginine and she also needed exanguinotransfusion. It was not possible to put her on hemodyalisis. The findings of the autopsy confirmed massive cerebral edema and characteristic hystological changes in the liver. The determination of the enzymatical activity in liver tissue showed a partial deficiency, with a residual activity of 25% of the average control. Conclusions. This is a case of fulminant neonatal citrullinemia that we considered of interest in order to draw the attention of the clinical on this type of diseases. The prognosis depends on early diagnosis, witch is based on clinical suspicion and analytical determination of ammonia in every newborn with unexplained vomiting, lethargy or other symptoms of encephalopathy (AU)

Peculiaridades en la organización de la cadena osicular timpánica humana a lo largo de su ontogenia / Ontogenic peculiarities of the human tympanic ossicular chain

Hemos estudiado el desarrollo de los osículos timpánicos en 40 series embrio-fetales humanas de edades comprendidas entre los 32 días (6 mm) y el nacimiento. Una vez realizadas las mediciones para datar cronológicamente los embriones y fetos, se llevó a cabo una minuciosa y cuidada disección de los temporales en bloque; posteriormente fueron fijados en formol al 10 por ciento, descalcificados con ácido nítrico al 2 por ciento, deshidratados en alcoholes a concentraciones progresivamente crecientes, incluidos en parafina, cortados en serie a 7 mm, y teñidos en serie con la técnica del Tricrómico de Martins. Los osículos timpánicos se desarrollan en el mesénquima de los dos primeros arcos faríngeos. La cabeza del martillo, el cuerpo y la apófisis corta del yunque se originan del primer arco mientras que el mango del martillo, la apófisis larga del yunque y la masa del estribo lo hacen del segundo. La lámina vestibular de la platina se desarrolla en la cápsula ótica. Los osículos timpánicos se desarrollan por osificación endocondral con excepción de la apófisis anterior del martillo que lo hace por osificación membranosa. En su ontogenia se observan seis estadios. En el primero se produce la formación de sus esbozos por condensación del mesénquima, en el segundo "precartilaginoso" las células de los primordios se diferencian a condroblastos, en el tercero "cartilaginoso" los osículos presentan una estructura cartilaginosa, en el cuarto se forman los centros de osificación primarios, en el quinto aparecen en los osículos el collar perióstico y en su interior hueso endocondral, y en el último estadio el tejido óseo se va extendiendo progresivamente hasta adquirir una estructura ósea compacta (AU)

We have studied the development of the tympanic ossicles in 40 embryo-foetal human series aged between 32 days (6 mm) and newborn. Once performed the measurements to date chronologically embryos and foetuses, we did a meticulous dissection of temporal bones. After fix in 10% formol, decalcified with 2% nitric acid, embedded in Paraplast, sectioned in a sequence of 7 mm, and stained with Martin's trichrome. The tympanic ossicles are developed in the mesenchyme of the two first pharyngeal archs. The head of the malleus, the body and the short limb of the incus arise from the first arch while the handle of the malleus, the long limb of the incus and the mass of the stapes arise from the second arch. The vestibular side of the stapedial footplate develops in the otic capsule. The tympanic ossicles develop from endochondral ossification, while anterior process of the malleus has the membranous ossification. In their ontogenia 6 stages are observed. First stage, the formation of their sketch by mesenchimal condensation, in the second stage, "pre-cartilaginous", the cells of the primordia are differentiated into condroblasts, in the third stage "cartilaginous" the ossicles show a cartilaginous structure, in the forth stage the primary ossification centers are developed, in the fifth stage the ossicles arise in the periostic annulus and inside the endochondral bone, and in the last stage the osseous tissue grows until it acquires a compact osseous structure (AU)

[Contribution to the development of the union between the manubrium of the malleus and the tympanic membrane in human fetus]. / Contribución al desarrollo de la unión del mango del martillo y la membrana del tímpano en fetos humanos.

The development of the union between the manubrium of the malleus and the tympanic membrane was studied in human embryo. For that purpose 25 temporal bones of human foetus, aged from 36 days (14 mm) to 29 weeks (270 mm) were analyzed. Samples were fixed in a 10% formaldehyde solution, decalcified with 2% nitric acid, embedded in Paraplast, cut in sections of 7 microns thick and stained with Martin's trichrome method. During the development a pseudojoint between the malleus and the tympanic membrane several stages were seen. In the first stage, the manubrium was adhered to membrane mesenchyme of primitive tympanum, in the second one this mesenchyme was loose and there appeared capillars in it, in the third one there were collagen fibers in a radial disposition, and in the forth stage, the hollow in the meatal plug gave independence to the tympanic membrane for the external acoustic meatus. The distal portion of the manubrium, included in tympanic membrane, has remained with a cartilaginous structure during end of the time it has been studied by us. The collagen fibers at the level of the umbo surround the manubrium, while the rest of it remains in its anterior edge.

Contribución al desarrollo de la unión del mango del martillo y la membrana del tímpano en fetos humanos / Contribution to the development of the union between the manubrium of the malleus and the tympanic membrane in human fetus

Se ha estudiado el desarrollo de la unión entre el mango del martillo y la membrana del tímpano en embriones y fetos humanos.Para ello se han utilizado 25 huesos temporales de fetos humanos, cuyas edades cronológicas van desde los 36 días (14 mm) hasta las 29 semanas (270 mm). Las muestras fueron fijadas en formol al 10 por ciento, descalcificadas con ácido nítrico al 2 por ciento, incluidas en parafina, cortadas en serie a 7 µm, y teñidas con el método Tricrómico de Martins. Se han identificado varias etapas en el desarrollo de la pseudoarticulación entre el martillo y la membrana del tímpano. En la primera el mango se adhiere íntimamente al mesénquima de la membrana del tímpano primitiva, en la segunda dicho mesénquima se hace laxo y en él aparecen capilares, en la tercera aparecen fibras de colágeno de disposición radial, y en la cuarta el ahuecamiento del tapón meatal da independencia a la membrana del tímpano del conducto auditivo externo. La porción distal del mango, incluida en la membrana del tímpano, ha permanecido con una estructura cartilaginosa durante todo el tiempo estudiado. Las fibras de colágeno a nivel del umbo rodean al extremo terminal del mango mientras que en el resto de él lo hacen solamente en su arista anterior (AU)

The development of the union between the manubrium of the malleus and the tympanic membrane was studied in human embryo. For that purpose 25 temporal bones of human foetus, aged from 36 days (14 mm) to 29 weeks (270 mm) were analyzed. Samples were fixed in a 10% formaldehyde solution, decalcified with 2% nitric acid, embedded in Paraplast, cut in sections of 7 microns thick and stained with Martin's trichrome method. During the development a pseudojoint between the malleus and the tympanic membrane several stages were seen. In the first stage, the manubrium was adhered to membrane mesenchyme of primitive tympanum, in the second one this mesenchyme was loose and there appeared capillars in it, in the third one there were collagen fibers in a radial disposition, and in the forth stage, the hollow in the meatal plug gave independence to the tympanic membrane for the external acoustic meatus. The distal portion of the manubrium, included in tympanic membrane, has remained with a cartilaginous structure during end of the time it has been studied by us. The collagen fibers at the level of the umbo surround the manubrium, while the rest of it remains in its anterior edge (AU)

[Parotid metastases of a neuroendocrine cutaneous carcinoma]. / Metástasis parotídea de un carcinoma neuroendocrino cutáneo.

A case of metastatic Merkel-cell carcinoma to lymph nodes on the left side of the neck nad left parotid is described. Neuroendocrine cutaneous carcinoma, also called Merkel cell carcinoma (MCC), is an uncommon primary skin tumor most often seen in elderly. Literature is reviewed and comment about more important features of these lesions, like differential diagnosis, prognosis and treatment.

Metástasis parotídea de un carcinoma neuroendocrino cutáneo / Parotideal metastasis of a cutaneous neuroendocrine carcinoma

Presentamos un caso de carcinoma neuroendocrino cutáneo con metástasis cervicales y en glándula parótida izquierda. El carcinoma neuroendocrino cutáneo, también llamado carcinoma de células de Merkel (MCC), es un raro tumor de la piel que aparece en edades avanzadas de la vida. Una vez revisada la literatura, discutimos sobre las características más importantes de estas lesiones, como sonel diagnóstico diferencial, pronóstico y tratamiento (AU)

A case of metastatic Merkel-cell carcinoma to lymph nodes on the left side of the neck nad left parotid is described. Neuroendocrine cutaneous carcinoma, also called Merkel cell carcinoma (MCC), is an uncommon primary skin tumor most often seen in elderly. Literature is reviewed and comment about more important features of these lesions, like differential diagnosis, prognosis and treatment (AU)

The cytosolic O-acetylserine(thiol)lyase gene is regulated by heavy metals and can function in cadmium tolerance.

Regulation of the expression of the cytosolic O-acetylserine(thiol)lyase gene (Atcys-3A) from Arabidopsis thaliana under heavy metal stress conditions has been investigated. Northern blot analysis of Atcys-3A expression shows a 7-fold induction after 18 h of cadmium treatment. Addition of 50 microm CdCl(2) to the irrigation medium of mature Arabidopsis plants induces a rapid accumulation of the mRNA throughout the leaf lamina, the root and stem cortex, and stem vascular tissues when compared with untreated plants, as observed by in situ hybridization. High pressure liquid chromatography analysis of GSH content shows a transient increase after 18 h of metal treatment. Our results are compatible with a high cysteine biosynthesis rate under heavy metal stress required for the synthesis of GSH and phytochelatins, which are involved in the plant detoxification mechanism. Arabidopsis-transformed plants overexpressing the Atcys-3A gene by up to 9-fold show increased tolerance to cadmium when grown in medium containing 250 microm CdCl(2), suggesting that increased cysteine availability is responsible for cadmium tolerance. In agreement with these results, exogenous addition of cystine can, to some extent, also favor the growth of wild-type plants in cadmium-containing medium. Cadmium accumulates to higher levels in leaves of tolerant transformed lines than in wild-type plants.

Homology predicted structure and functional interaction of ferredoxin from the eukaryotic alga Chlamydomonas reinhardtii with nitrite reductase and glutamate synthase.

Ferredoxin (Fd) from Chlamydomonas reinhardtii is composed of 94 amino-acid residues and a [2Fe-2S] cluster. The homology modelling technique has been used to predict the tertiary structure of C. reinhardtii Fd. The overall structure shows the typical fifth-stranded beta-grasp plus two additional beta-sheets and three alpha-helices. Site-directed mutagenesis of recombinant Fd has allowed us to obtain four point mutants and one double mutant--all mutations being located in the short alpha-helix at the carboxy-terminal segment as well as a triple mutant affected on helix alpha1. Crosslinking studies and measurement of enzymatic activities reveal that the residues changed are critical for the interaction of Fd with glutamate synthase (GOGAT) and nitrite reductase (NiR). Potentiometric analyses of the Fd mutants show that the replacement of glutamate in position 91 drastically changes the redox potential value (70 mV), thereby suggesting that such a glutamate can modulate the reactivity of Fd towards its reaction partners. According to results herein presented, the reported mutations modify the electrostatic interactions within the complex formed between Fd and GOGAT or NiR.

Glutathione biosynthesis in Arabidopsis trichome cells.

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode gamma-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238+/-25, 80+/-2, and 144+/-19 microM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH](cyt) is reduced to 47+/-5 microM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.

Salt-specific regulation of the cytosolic O-acetylserine(thiol)lyase gene from Arabidopsis thaliana is dependent on abscisic acid.

The expression of Atcys-3A gene coding for cytosolic O-acetylserine(thiol)lyase, a key enzyme in cysteine biosynthesis, from Arabidopsis thaliana is significantly induced by exposure to salt and heavy-metal stresses. Addition of NaCl to mature plants induced a rapid accumulation of the mRNA throughout the leaf lamina and roots, and later on in stems, being mainly restricted to vascular tissues. The salt-specific regulation of Atcys-3A was also mediated by abscisic acid (ABA) since: (1) exogenous addition of ABA to the culture medium mimicked the salt-induced plant response by raising the level of Atcys-3A transcript, and (2) Arabidopsis mutants aba-1 and abi2-1 were not able to respond to NaCl. Our results suggest that a high rate of cysteine biosynthesis is required in Arabidopsis under salt stress necessary for a plant protection or adaptation mechanism. This hypothesis was supported by the observation that intracellular levels of cysteine and glutathione increased up to 3-fold after salt treatment.

Cysteine biosynthesis in Chlamydomonas reinhardtii. Molecular cloning and regulation of O-acetylserine(thiol)lyase.

A cDNA, Cys1ACr, encoding an isoform of O-acetylserine(thiol) lyase has been isolated from Chlamydomonas reinhardtii, using a PCR-based approach. The inclusion of dimethylsulfoxide in the PCR reaction has been demonstrated to be essential for the correct amplification of C. reinhardtii templates with complex secondary structures caused by a high G + C content. The deduced amino acid sequence exhibited highest similarity with plant O-acetylserine(thiol)lyase isoforms, indicating that the C. reinhardtii enzyme was structurally more similar to higher plant O-acetylserine(thiol)lyase than to the corresponding prokaryotic enzymes. The N-terminal extension present in Cys1ACr showed several characteristics of an organellar transit peptide, with a length typical for C. reinhardtii. Southern blot analysis suggested that the C. reinhardtii genome may contain a single copy of the organellar O-acetylserine(thiol)lyase gene. O-acetylserine(thiol)lyase activity was strongly induced by sulfur-deficient conditions (up to sevenfold the level observed in a sulfur-repleted cell culture) and required the presence of a nitrogen source. Northern blot analysis showed a different pattern of regulation of Cys1ACr to that observed at the activity level. To obtain an increase of transcript abundance a longer period of sulfur limitation was required, reaching a maximum level of approximately threefold Cys1ACr mRNA when compared with the level of a sulfate-grown culture.