A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferro... more A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferrooxidans ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using twodimensional polyacrylamide gel electrophoresis. N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A. ferrooxidans ATCC 23270. The genomic context around several of these genes suggests their involvement in the energetic metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function. The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome c 552 , a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others. The results suggest in general a separation of the iron and sulfur utilization pathways. Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides. During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available.
By proteomic analysis we found a 21-kDa protein (P21) from Acidithiobacillus ferrooxidans ATCC 19... more By proteomic analysis we found a 21-kDa protein (P21) from Acidithiobacillus ferrooxidans ATCC 19859 whose synthesis was greatly increased by growth of the bacteria in pyrite, thiosulfate, elemental sulfur, CuS, and ZnS and was almost completely repressed by growth in ferrous iron. After we determined the N-terminal amino acid sequence of P21, we used the available preliminary genomic sequence of A. ferrooxidans ATCC 23270 to isolate the DNA region containing the p21 gene. The nucleotide sequence of this DNA fragment contained a putative open reading frame (ORF) coding for a 23-kDa protein. This difference in size was due to the presence of a putative signal peptide in the ORF coding for P21. When p21 was cloned and overexpressed in Escherichia coli, the signal peptide was removed, resulting in a mature protein with a molecular mass of 21 kDa and a calculated isoelectric point of 9.18. P21 exhibited 27% identity and 42% similarity to the Deinococcus radiodurans thiosulfate-sulfur transferase (rhodanese; EC 2.8.1.1) and similar values in relation to other rhodaneses, conserving structural domains and an active site with a cysteine, both characteristic of this family of proteins. However, the purified recombinant P21 protein did not show rhodanese activity. Unlike cytoplasmic rhodaneses, P21 was located in the periphery of A. ferrooxidans cells, as determined by immunocytochemical analysis, and was regulated depending on the oxidizable substrate. The genomic context around gene p21 contained other ORFs corresponding to proteins such as thioredoxins and sulfate-thiosulfate binding proteins, clearly suggesting the involvement of P21 in inorganic sulfur metabolism in A. ferrooxidans.
EFE, ISSN 1575-5533, XVII, 2008 RESUMEN Para estudiar las distintas alteraciones de la voz, el la... more EFE, ISSN 1575-5533, XVII, 2008 RESUMEN Para estudiar las distintas alteraciones de la voz, el laringólogo se ha fundamentado en la exploración indirecta de las cuerdas vocales. De entre las distintas posibilidades exploratorias, la estroboscopia laríngea es hoy por hoy la principal herramienta clínica para el diagnóstico etiológico de los trastornos de la voz, porque permite analizar imágenes de la laringe en alta resolución y simultáneamente aporta datos sobre el patrón vibratorio de la mucosa vocal. La videoestroboscopia no es más que una técnica especial de iluminación de las cuerdas vocales por emisión flashes cortos de luz de forma sincronizada o asincronizada con la frecuencia fundamental, lo que permite visualizar el movimiento de ondulación de la mucosa vocal a cámara lenta. La exploración estroboscópica permite examinar la laringe a diferente tono, intensidad y calidad de voz. Los parámetros que se evalúan en el estudio estroboscópico son los siguientes: frecuencia fundamental, simetría de los movimientos de cierre/apertura de las cuerdas vocales, regularidad o periodicidad de las vibraciones, cierre glótico, amplitud del desplazamiento horizontal de las cuerdas vocales, y las características y grado de ondulación de la mucosa vocal A lo largo del presente taller de trabajo se detallará la metodología exploratoria que se realiza en el marco clínico de la consulta otorrinolaringológica, presentándose ejemplos de un amplio número de exploraciones normales y patológicas.
A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferro... more A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferrooxidans ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using twodimensional polyacrylamide gel electrophoresis. N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A. ferrooxidans ATCC 23270. The genomic context around several of these genes suggests their involvement in the energetic metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function. The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome c 552 , a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others. The results suggest in general a separation of the iron and sulfur utilization pathways. Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides. During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available.
By proteomic analysis we found a 21-kDa protein (P21) from Acidithiobacillus ferrooxidans ATCC 19... more By proteomic analysis we found a 21-kDa protein (P21) from Acidithiobacillus ferrooxidans ATCC 19859 whose synthesis was greatly increased by growth of the bacteria in pyrite, thiosulfate, elemental sulfur, CuS, and ZnS and was almost completely repressed by growth in ferrous iron. After we determined the N-terminal amino acid sequence of P21, we used the available preliminary genomic sequence of A. ferrooxidans ATCC 23270 to isolate the DNA region containing the p21 gene. The nucleotide sequence of this DNA fragment contained a putative open reading frame (ORF) coding for a 23-kDa protein. This difference in size was due to the presence of a putative signal peptide in the ORF coding for P21. When p21 was cloned and overexpressed in Escherichia coli, the signal peptide was removed, resulting in a mature protein with a molecular mass of 21 kDa and a calculated isoelectric point of 9.18. P21 exhibited 27% identity and 42% similarity to the Deinococcus radiodurans thiosulfate-sulfur transferase (rhodanese; EC 2.8.1.1) and similar values in relation to other rhodaneses, conserving structural domains and an active site with a cysteine, both characteristic of this family of proteins. However, the purified recombinant P21 protein did not show rhodanese activity. Unlike cytoplasmic rhodaneses, P21 was located in the periphery of A. ferrooxidans cells, as determined by immunocytochemical analysis, and was regulated depending on the oxidizable substrate. The genomic context around gene p21 contained other ORFs corresponding to proteins such as thioredoxins and sulfate-thiosulfate binding proteins, clearly suggesting the involvement of P21 in inorganic sulfur metabolism in A. ferrooxidans.
EFE, ISSN 1575-5533, XVII, 2008 RESUMEN Para estudiar las distintas alteraciones de la voz, el la... more EFE, ISSN 1575-5533, XVII, 2008 RESUMEN Para estudiar las distintas alteraciones de la voz, el laringólogo se ha fundamentado en la exploración indirecta de las cuerdas vocales. De entre las distintas posibilidades exploratorias, la estroboscopia laríngea es hoy por hoy la principal herramienta clínica para el diagnóstico etiológico de los trastornos de la voz, porque permite analizar imágenes de la laringe en alta resolución y simultáneamente aporta datos sobre el patrón vibratorio de la mucosa vocal. La videoestroboscopia no es más que una técnica especial de iluminación de las cuerdas vocales por emisión flashes cortos de luz de forma sincronizada o asincronizada con la frecuencia fundamental, lo que permite visualizar el movimiento de ondulación de la mucosa vocal a cámara lenta. La exploración estroboscópica permite examinar la laringe a diferente tono, intensidad y calidad de voz. Los parámetros que se evalúan en el estudio estroboscópico son los siguientes: frecuencia fundamental, simetría de los movimientos de cierre/apertura de las cuerdas vocales, regularidad o periodicidad de las vibraciones, cierre glótico, amplitud del desplazamiento horizontal de las cuerdas vocales, y las características y grado de ondulación de la mucosa vocal A lo largo del presente taller de trabajo se detallará la metodología exploratoria que se realiza en el marco clínico de la consulta otorrinolaringológica, presentándose ejemplos de un amplio número de exploraciones normales y patológicas.
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