RESUMEN
In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Δpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDH(R46E,Q47E). Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Δpgi E. coli strains were replaced by DNA encoding LmG6PDH(R46E,Q47E). Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59â% in the Δpgi strain, while falling 44â% in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden-Meyerhof pathway. Interestingly, a genomic database search suggested that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.
Asunto(s)
Escherichia coli/enzimología , Escherichia coli/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , NAD/metabolismo , Simulación por Computador , Escherichia coli/crecimiento & desarrollo , Eliminación de Gen , Perfilación de la Expresión Génica , Glucosafosfato Deshidrogenasa/genética , Leuconostoc/enzimología , Leuconostoc/genética , Ingeniería Metabólica , Análisis de Flujos Metabólicos , Redes y Vías Metabólicas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinación Genética , Biología de SistemasRESUMEN
Snyder-Robinson syndrome (SRS, OMIM 309583) is a rare X-linked syndrome characterized by mental retardation, marfanoid habitus, skeletal defects, osteoporosis, and facial asymmetry. Linkage analysis localized the related gene to Xp21.3-p22.12, and a G-to-A transition at point +5 of intron 4 of the spermine synthase gene, which caused truncation of the SMS protein and loss of enzyme activity, was identified in the original family. Here we describe another family with Snyder-Robinson syndrome in two Mexican brothers and a novel mutation (c.496T>G) in the exon 5 of the SMS gene confirming its involvement in this rare X-linked mental retardation syndrome.
Asunto(s)
Cromosomas Humanos X , Genes Ligados a X , Discapacidad Intelectual Ligada al Cromosoma X/genética , Mutación Missense , Espermina Sintasa/genética , Adulto , Análisis Mutacional de ADN , Exones , Ligamiento Genético , Humanos , Discapacidad Intelectual/genética , Masculino , Síndrome de Marfan/genética , Osteoporosis/genética , Linaje , Escoliosis/genética , Hermanos , Adulto JovenRESUMEN
High levels of sHLA-I (soluble HLA--class I) have been correlated with rejection episodes in solid organ transplant recipients and with graft versus host disease in bone marrow recipients. Studies of human infection with parasitic worms of the gut have suggested that certain individuals may be genetically predisposed to intense infection. In this study, the influence of parasitic helminth infection on levels of sHLA-I in plasma was investigated in 155 HLA typed individuals from St. Lucia, exposed to the gut parasite Trichuris trichiura. The results confirmed previous findings showing increased levels of sHLA-I in HLA-A9, and in this case HLA-A23 positive individuals. However, HLA-A9 positive individuals with high worm burden had significantly lower levels of sHLA-I in their plasma compared with HLA-A9 positive subjects with low worm burden. These results suggest that the intensity of T. trichiura infection influences the ability of HLA-A9 positive subjects to maintain high levels of sHLA-I.
Asunto(s)
Antígenos HLA/sangre , Antígenos de Histocompatibilidad Clase I/sangre , Tricuriasis/inmunología , Animales , Niño , Preescolar , Antígenos HLA-A/sangre , Humanos , Santa Lucia , Solubilidad , Tricuriasis/parasitología , Trichuris/aislamiento & purificaciónRESUMEN
High levels of sHLA-I(soluble HLA class 1) have been correlated with rejection episodes in solid organ transport recipients and with graft versus host disease in bone marrow recipients. Studies of human infection with parasitic worms of the gut have suggested that certain individuals may be genetically predisposed to intense infection. In this study, the influence of parasitic helminth infection on levels of sHLA-I in plasma was investigated in 155 HLA typed individuals from St. Lucia exposed to the gut parasite Trichuris trichiura. The results confirmed previous findings showing increased levels of sHLA-I in HLA-A9, and in this case HLA-A23 postive individuals. However, HLA-A9 positive individuals with high worm burden had significantly lower levels of sHLA-I in their plasma compared with HLA-A9 positive subjects with low worm burden. These results suggest that the intensity of T. trichiuria infection infection influences the ability of HLA-A9 positive subjects to maintain high levels of sHLA-I. (AU)