Luis López Rodríguez

Purification and characterization of an invertase from Candida utilis: comparison with natural and recombinant yeast invertases

A periplasmic invertase from the yeast Candida utilis was purified to homogeneity from cells fully derepressed for invertase synthesis. The enzyme was purified by successive Sephacryl S-300, and affinity chromatography and shown to be a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa. After EndoH treatment, the deglycosylated protein showed an apparent molecular weight of 60 kDa. The apparent K(m) values for sucrose and raffinose were 11 and 150 mM, respectively, similar to those reported in Saccharomyces cerevisiae. The range of optimum temperature was 60-75 degrees C. The optimum pH was 5.5 and the enzyme was stable over pH range 3-6.

Cloning and sequence analysis of the gene encoding invertase (INV1) from the yeast Candida utilis.

The gene INV1 encoding invertase from the yeast Candida utilis has been cloned using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed considering sequence comparisons between yeast invertases. The cloned gene was sequenced and found to encode a polypeptide of 533 amino acids that contain a 26 amino‐acid signal peptide and 12 potential N‐glycosylation sites. The nucleotide sequences of the 5′ and 3′ non‐coding regions were found to contain motifs probably involved in initiation, regulation and termination of gene transcription. The amino‐acid sequence shows significant identity with other yeast, bacterial and plant β‐fructofuranosidases. The INV1 gene from C. utilis was able to complement functionally the suc2 mutation of S. cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession Number Y12659.

Invertase secretion in Hansenula polymorpha under the AOX1 promoter from Pichia pastoris

A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha. We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H. polymorpha yeast.

Adecuación de las estancias hospitalarias en un servicio de neumología

Objetivo Establecer la tasa de inadecuacion de estancias en un servicio de neumologia a lo largo de un ano, asi como conocer las causas que motivan dicha inadecuacion y las variables predictoras de esta. Pacientes y metodos Se ha analizado una muestra representativa de las estancias hospitalarias generadas por los pacientes ingresados en el Servicio de Neumologia del Hospital de Valme durante el ano 2004, utilizando como instrumento de evaluacion el Appropriateness Evaluation Protocol (AEP) de forma retrospectiva. Realizaron la evaluacion 2 investigadores no neumologos. Se efectuo una regresion lineal multiple para determinar que variables eran predictoras de inadecuacion. Resultados Se analizo un total de 1.166 estancias, en las que se obtuvo una tasa de adecuacion del 89% (n = 1.038) y de inadecuacion del 11% (n = 128). La causa principal de inadecuacion fue la espera de pruebas diagnosticas o resultados (64%), mientras que los criterios que con mayor frecuencia justificaron la estancia fueron los tratamientos respiratorios (59,6%) y el tratamiento parenteral (46,1%). El modelo predictivo obtenido tras la regresion lineal multiple incluyo las siguientes variables: estancia en una planta diferente de la de neumologia, diagnostico de ingreso y estacion del ano. Conclusiones La tasa de inadecuacion de estancias fue baja en comparacion con otros estudios, y debida sobre todo a la espera de resultados o realizacion de pruebas diagnosticas. Las variables predictoras de inadecuacion fueron el diagnostico de ingreso, la estacion del ano y que el paciente estuviese ingresado en una planta diferente de la de neumologia.

Isolation and sequence analysis of the orotidine‐5'‐phosphate decarboxylase gene (URA3) of Candida utilis. Comparison with the OMP decarboxylase gene family

The URA3 gene of Candida utilis encoding orotidine‐5′‐phosphate decarboxylase enzyme was isolated by complementation in Escherichia coli pyrF mutation. The deduced amino‐acid sequence is highly similar to that of the Ura3 proteins from other yeast and fungal species. An extensive analysis of the family of orotidine‐5′‐phosphate decarboxylase is shown. The URA3 gene of C. utilis was able to complement functionally the ura3 mutation of Saccharomyces cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession Number Y12660.

Cloning and sequence analysis of the LEU2 homologue gene from Pichia anomala

The Pichia anomala LEU2 gene (PaLEU2) was isolated by complementation of a leu2 Saccharomyces cerevisiae mutant. The cloned gene also allowed growth of a Escherichia coli leuB mutant in leucine‐lacking medium, indicating that it encodes a product able to complement the β‐isopropylmalate dehydrogenase deficiency of the mutants. The sequenced DNA fragment contains a complete ORF of 1092 bp, and the deduced polypeptide shares significant homologies with the products of the LEU2 genes from S. cerevisiae (84% identity) and other yeast species. A sequence resembling the GC‐rich palindrome motif identified in the 5′ region of S. cerevisiae LEU2 gene as the binding site for the transcription activating factor encoded by the LEU3 gene was found at the promoter region. In addition, upstream of the PaLEU2 the 3′‐terminal half of a gene of the same orientation, encoding a homologue of the S. cerevisiae NFS1/SPL1 gene that encodes a mitochondrial cysteine desulphurase involved in both tRNA processing and mitochondrial metabolism, was found. The genomic organization of the PaNFS1–PaLEU2 gene pair is similar to that found in several other yeast species, including S. cerevisiae and Candida albicans, except that in some of them the LEU2 gene appears in the reverse orientation. The nucleotide sequence has been submitted to the EMBL database under Accession No. AJ294714. Copyright

Adecuación de los ingresos hospitalarios en un servicio de neumología

Objetivos: Analizar la tasa de inadecuacion de ingresos en un servicio de neumologia a lo largo de un ano y conocer las causas que motivan dicha inadecuacion, asi como las variables predictoras de esta. Pacientes y metodos: Se analizaron todos los ingresos del Servicio de Neumologia del Hospital de Valme durante 2004 aplicando una version concurrente del Appropriateness Evaluation Protocol (AEP). Realizaron la evaluacion 2 facultativos no implicados en el ingreso de los pacientes. Se efectuo un analisis de regresion logistica para determinar las variables que predecian la inadecuacion de forma independiente. Resultados: Se analizaron 633 ingresos, de los que el 92,1% (n = 583) fueron adecuados y el 7,9% (n = 50) inadecuados. La causa principal de inadecuacion fue el ingreso de pacientes que podrian haber sido manejados ambulatoriamente (70%), mientras que los criterios que con mas frecuencia justificaron la adecuacion del ingreso fueron la necesidad de tratamiento parenteral (76,3%) y las terapias respiratorias (62%). Las variables que se relacionaron de forma independiente con la inadecuacion en la regresion logistica fueron el ingreso no urgente (odds ratio = 2,82; intervalo de confianza del 95%, 1,28-6,21; p = 0,01) y el diagnostico de neoplasia como motivo de ingreso (odds ratio = 8,57; intervalo de confianza del 95%, 2,69-27,24; p < 0,0005). Conclusiones: La tasa de inadecuacion de ingresos fue baja en comparacion con otros estudios y se debio sobre todo al ingreso de pacientes que podian haber sido manejados ambulatoriamente. La neoplasia como diagnostico de ingreso y los ingresos no urgentes fueron predictores independientes de inadecuacion.

Influencia del apoyo social en el control de las personas con diabetes

Introduccion: El apoyo social es un proceso interactivo en donde se obtiene ayuda emocional, instrumental y afectiva de la red social que nos rodea, teniendo un efecto protector sobre la salud y amortiguador del estres que supone una enfermedad. Las situaciones adversas que generan estres en el individuo y se acompanan de un cambio vital, alcanzan la categoria de acontecimientos vitales estresantes siendo un obstaculo para el cumplimiento del regimen terapeutico. Objetivo: Determinar si el apoyo social que perciben los diabeticos influye en el control metabolico. Metodologia: Estudio descriptivo transversal multicentrico del ambito de la Atencion Primaria, en 246 diabeticos tipo 2, donde se midio el control metabolico (cifra de HbA1c) y percepcion del apoyo social mediante el Cuestionario MOS. Para el analisis de datos se determinaron medias, desviaciones tipicas y porcentajes mediante SPSS y para estudiar la relacion de variables Chi-cuadrado, t-Student y regresion logistica multivariante. Resultados: El 88.6% de los sujetos estudiados percibian buen apoyo social. La comparacion de medias de HbA1c con Percepcion de Apoyo Social no mostro asociacion. Sin embargo en un primer corte con los primeros pacientes estudiados se apreciaba diferencias estadisticamente significativas de 1 punto en la cifra media de HbA1c con un IC 95% (0.2 - 1.75) p=0.01 y tras someter a regresion logistica multivariante a la percepcion de apoyo social se obtuvo un modelo que representa asociacion significativa de HbA1c (p=0.02 y OR=2.3) e IMC (p= 0.04 y OR= 1.15). Discusion: El cuestionario MOS requieren un clima favorecedor para que las respuestas del paciente sean fiables y este clima lo puede crear el tiempo de relacion profesional. Palabras clave: Apoyo social/ Diabetes Mellitus/ Atencion Primaria.

Isolation and characterization of mutants as an approach to a transformation system in Kluyveromyces marxianus.

Abstract A method to obtain K. marxianus mutants has been developed. Different auxotrophic mutants were isolated by nystatin and snail-enzyme enrichment procedures using an incubation time of 2 h before adding the antibiotic or the enzyme respectively. All his mutants analyzed by complementation tests turned out to belong to the same complementation group. Some of them were transformed and complemented by the S. cerevisiae HIS3 gene. These non-reverting his3 mutants contain no heterologous sequence, which is essential to make them acceptable for application in the food industry.

Characterization of Candida albicans orthologue of the Saccharomyces cerevisiae signal-peptidase-subunit encoding gene SPC3.

The Candida albicans orthologue of the SPC3 gene, which encodes one of the subunits essential for the activity of the signal peptidase complex in Saccharomyces cerevisiae, was isolated by complementation of a thermosensitive mutation in the S. cerevisiae SEC61 gene. The cloned gene (CaSPC3) encodes a putative protein of 192 amino acids that contains one potential membrane‐spanning region and shares significant homology with the corresponding products from mammalian (Spc22/23p) and yeast (Spc3p) cells. CaSPC3 is essential for cell viability, since a hemizygous strain containing a single copy of CaSPC3 under control of the methionine‐repressible MET3 promoter did not grow in the presence of methionine and cysteine. The cloned gene could rescue the phenotype associated with a spc3 mutation in S. cerevisiae, indicating that it is the true C. albicans orthologue of SPC3. However, in contrast with results previously described for its S. cerevisiae orthologue, CaSPC3 was not able to complement the thermosensitive growth associated with a mutation in the SEC11 gene. The heterologous complementation of the sec61 mutant suggests that Spc3p could play a role in the interaction that it is known to occur between the translocon (Sec61 complex) and the signal peptidase complex, at the endoplasmic reticulum membrane. Copyright