RESUMEN
BACKGROUND: The construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations. METHODS: (13)C NMR spectroscopy and [1,2-(13)C2]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants. RESULTS: Rag8 showed a decrease of all (13)C glutamate fractional enrichments, except for [4-(13)C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4-(13)C]ketoglutarate pool not originating from mitochondria were suggested. (13)C lysine enrichments confirmed the presence of two compartmentalized α-ketoglutarate (α-KG) pools participating to glutamate and lysine synthesis. Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by (13)C NMR isotopomer analysis of alanine. CONCLUSIONS: (13)C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, (13)C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized α-KG pool. GENERAL SIGNIFICANCE: A compartmentalization of the α-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis. Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Ácido Glutámico/biosíntesis , Ácidos Cetoglutáricos/metabolismo , Kluyveromyces/metabolismo , Lisina/biosíntesisRESUMEN
BACKGROUND: In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag(-) phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing. METHODS: A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses. RESULTS: Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced. CONCLUSIONS: The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)(+)/NAD(P)H redox balance state. GENERAL SIGNIFICANCE: The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Kluyveromyces/metabolismo , Lípidos/química , Metabolómica , NADP/metabolismo , Quinasa de la Caseína I/genética , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Glucólisis , Kluyveromyces/genética , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
In a first study, we performed a cross-sectional analysis of urinary excretion of isoprostanes, IPF(2alpha-III) and (VI), and monocyte tissue factor (TF) antigen and activity between 11 antiphospholipid (APL) antibody-positive patients and 13 APL negative subjects. In a second study, 11 APL positive patients were randomly supplemented either with (n = 6) or without (n = 5) antioxidants (vitamin E at 900 IU day(-1), vitamin C at 2000 mg day(-1)) for 6 weeks. In a third study, TF and superoxide anion were measured in human monocytes incubated with anti-beta(2) glycoprotein 1 (beta(2)GP(1)) or control IgG, either with or without vitamin E. APL-positive patients had higher values of isoprostanes (P < 0.05) and monocyte TF antigen (P = 0.001) and activity (P = 0.0001) than APL-negative subjects. Only in APL positive patients did monocyte TF antigen correlate significantly with IPF(2alpha-III) (rho 0.79; P < 0.003) and IPF(2alpha-VI) (rho = 0.87; P < 0.0001). In patients who received antioxidant supplementation, we found a significant decrease of isoprostanes (P < 0.05) and monocyte TF antigen (P < 0.01) and activity (P < 0.007). In vitro experiments demonstrated that anti-beta(2)GP(1) antibodies dose-dependently enhanced the monocyte production of the superoxide anion and TF, which were significantly inhibited by vitamin E. This study demonstrates that in APL-positive patients, oxidative stress contributes to activate the clotting system via over-expression of monocyte TF. We suggest that anti-beta(2)GP(1) antibodies could play a pivotal role by enhancing the monocyte production of oxygen free radicals.
Asunto(s)
Anticuerpos Antifosfolípidos , Antioxidantes/farmacología , Monocitos/metabolismo , Estrés Oxidativo/fisiología , Tromboplastina/biosíntesis , Adulto , Síndrome Antifosfolípido/metabolismo , Estudios Transversales , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Lupus Eritematoso Sistémico/metabolismo , Masculino , Persona de Mediana Edad , SuperóxidosRESUMEN
The aerobic yeast Kluyveromyces lactis and the predominantly fermentative Saccharomyces cerevisiae share many of the genes encoding the enzymes of carbon and energy metabolism. The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis. We have isolated the KlCAT8 gene (a homologue of S. cerevisiae CAT8, encoding a DNA binding protein) as a multicopy suppressor of a fog1 mutation. The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of S. cerevisiae. While CAT8 controls the key enzymes of gluconeogenesis in S. cerevisiae, KlCAT8 of K. lactis does not (I. Georis, J. J. Krijger, K. D. Breunig, and J. Vandenhaute, Mol. Gen. Genet. 264:193-203, 2000). We therefore examined possible targets of KlCat8p. We found that the acetyl coenzyme A synthetase genes, KlACS1 and KlACS2, were specifically regulated by KlCAT8, but very differently from the S. cerevisiae counterparts. KlACS1 was induced by acetate and lactate, while KlACS2 was induced by ethanol, both under the control of KlCAT8. Also, KlJEN1, encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of KlCAT8.
Asunto(s)
Acetato CoA Ligasa/genética , Proteínas Portadoras/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Kluyveromyces/metabolismo , Transportadores de Ácidos Monocarboxílicos , Proteínas de Saccharomyces cerevisiae , Simportadores , Transactivadores/metabolismo , Acetato CoA Ligasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/genética , Kluyveromyces/genética , Datos de Secuencia Molecular , Mutación , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo , Transactivadores/genética , Activación TranscripcionalRESUMEN
Inactivation, dissociation, and unfolding of tetrameric alcohol dehydrogenase I from Kluyveromyces lactis (KlADH I) were investigated using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence and gel filtration chromatography. At low denaturant concentrations (less than 0.3 M), reversible transformation of enzyme into tetrameric inactive form occurs. At denaturant concentrations between 0.3 and 0.5 M, the enzyme progressively dissociates into structured monomers through an irreversible reaction. At higher denaturant concentrations, the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only with the enzyme denatured between 0 and 0.3 M GdmCl concentrations. The enzyme denatured at GdmCl concentrations higher than 0.3 M refolds only partially. All together, our results indicate that unfolding of the KlADH I is a multistep process, i.e., inactivation of the structured tetramer, dissociation into partially structured monomers, followed by complete unfolding.
Asunto(s)
Alcohol Deshidrogenasa/efectos de los fármacos , Proteínas Fúngicas/efectos de los fármacos , Guanidina/farmacología , Kluyveromyces/enzimología , Alcohol Deshidrogenasa/química , Sitios de Unión , Cromatografía en Gel , Proteínas Fúngicas/química , Modelos Químicos , Concentración Osmolar , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica , Espectrometría de FluorescenciaRESUMEN
KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity, which is specifically induced by ethanol and insensitive to glucose repression. In this work, we report the molecular analysis of UAS(E), an element of the KlADH4 promoter which is essential for the induction of KlADH4 in the presence of ethanol. UAS(E) contains five stress response elements (STREs), which have been found in many genes of Saccharomyces cerevisiae involved in the response of cells to conditions of stress. Whereas KlADH4 is not responsive to stress conditions, the STREs present in UAS(E) seem to play a key role in the induction of the gene by ethanol, a situation that has not been observed in the related yeast S. cerevisiae. Gel retardation experiments showed that STREs in the KlADH4 promoter can bind factor(s) under non-inducing conditions. Moreover, we observed that the RAP1 binding site present in UAS(E) binds KlRap1p.
Asunto(s)
Alcohol Deshidrogenasa/genética , Etanol/farmacología , Kluyveromyces/genética , Elementos de Respuesta , Alcohol Deshidrogenasa/biosíntesis , Secuencia de Bases , Inducción Enzimática , Kluyveromyces/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
The yeast Kluyveromyces lactis was mutagenized with ethyl methane sulphonate and mutants unable to grow on respiratory carbon sources were isolated. Functional complementation of one of these mutants led to the isolation of KlCOX14, a gene encoding a 64 amino acid protein which is the functional homologue of Saccharomyces cerevisiae Cox14p, a protein necessary for the assembly of the cytochrome oxidase holoenzyme (Glerum et al., 1995). The disruption of KlCOX14 resulted in the absence of the absorption bands relative to cytochromes a and a(3) and in the complete loss of respiratory activity. Klcox14 mutants display the typical phenotype of pet mutants and have a reduced growth rate. In addition, unlike the wild-type, Klcox14 mutants are able to grow by fermentation also in the presence of low glucose. The nucleotide sequence of KlCOX14 has been deposited in the EMBL databank with Accession No. AJ238801.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Proteínas Fúngicas/genética , Kluyveromyces/genética , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Alelos , Secuencia de Aminoácidos , Antimicina A/análogos & derivados , Antimicina A/farmacología , Clonación Molecular , Complejo IV de Transporte de Electrones/genética , Fermentación , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Genes Fúngicos , Kluyveromyces/enzimología , Kluyveromyces/crecimiento & desarrollo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Consumo de Oxígeno , Análisis de Secuencia de ADN , Espectrofotometría/métodosRESUMEN
The effects of ethanol and 1-octanol on growth and fatty acid composition of different strains of Kluyveromyces lactis containing a mutation in the four different alcohol dehydrogenase (KlADH) genes were investigated. In the presence of ethanol and 1-octanol K. lactis reduced the fluidity of its lipids by decreasing the unsaturation index (UI) of its membrane fatty acids. In this way, a direct correlation between nonlethal ethanol concentrations and the decrease in the UI could be observed. At concentrations which totally inhibited cell growth no reaction occurred. These adaptive modifications of the fatty acid pattern of K. lactis to ethanol contrasted with those reported for Saccharomyces cerevisiae and Schizosaccharomyces pombe. Whereas these two yeasts increased the fluidity of their membrane lipids in the presence of ethanol, K. lactis reduced the fluidity (UI) of its lipids. Among the different isogenic adh negative strains tested, the strain containing no ADH (adh0) and that containing only KlADH1 were the most alcohol-sensitive. The strain with only KlADH2 showed nearly the same tolerance as reference strain CBS 2359/152 containing all four ADH genes. This suggests that the KlADH2 product could play an important role in the adaptation/detoxification reactions of K. lactis to high ethanol concentrations.
Asunto(s)
Alcohol Deshidrogenasa/genética , Membrana Celular/química , Etanol/farmacología , Ácidos Grasos/análisis , Kluyveromyces/enzimología , Mutación , 1-Octanol/farmacología , Adaptación Fisiológica , Alcohol Deshidrogenasa/metabolismo , Medios de Cultivo , Isoenzimas , Kluyveromyces/genética , Kluyveromyces/crecimiento & desarrolloRESUMEN
KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial beta-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UASE) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source.
Asunto(s)
Alcohol Deshidrogenasa/genética , Kluyveromyces/genética , Kluyveromyces/metabolismo , Regiones Promotoras Genéticas , Albúmina Sérica Bovina/biosíntesis , Albúmina Sérica Bovina/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Escherichia coli/enzimología , Escherichia coli/genética , Etanol/metabolismo , Fermentación , Expresión Génica , Genes Bacterianos , Genes Fúngicos , Genes Reporteros , Glucuronidasa/genética , Humanos , Cinética , Kluyveromyces/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Previous studies have shown that cirrhotic patients produce increased amounts of thrombin but the underlying mechanism is still unknown. AIMS: To analyse the relation between the rate of thrombin generation and monocyte expression of tissue factor (TF) in cirrhosis. PATIENTS: Thirty three cirrhotic patients classified as having low (n = 7), moderate (n = 17), or severe (n = 9) liver failure according to Child-Pugh criteria. METHODS: Prothrombin fragment F1 + 2, monocyte TF activity and antigen, and endotoxaemia were measured in all patients. Polymerase chain reaction (PCR) analysis of TF mRNA was performed in monocytes of five cirrhotic patients. RESULTS: Prothrombin fragment F1 + 2 was higher in cirrhotic patients than in controls (p < 0.0001). Monocytes from cirrhotic patients had higher TF activity and antigen than those from controls (p < 0.001) with a progressive increase from low to severe liver failure. Monocyte expression of TF was significantly correlated with plasma levels of F1 + 2 (TF activity: r = 0.98, p < 0.0001; TF antigen: r = 0.95, p < 0.0001) and with endotoxaemia (TF activity: r = 0.94, p < 0.0001; TF antigen: r = 0.91, p < 0.0001). PCR analysis of TF mRNA showed TF expression only in three patients with endotoxaemia (more than 15 pg/ml). CONCLUSIONS: Cirrhotic patients have enhanced expression of TF which could be responsible for clotting activation, suggesting that endotoxaemia might play a pivotal role.
Asunto(s)
Cirrosis Hepática/metabolismo , Monocitos/metabolismo , Tromboplastina/metabolismo , Adulto , Anciano , Antígenos/análisis , Coagulación Sanguínea , Estudios de Casos y Controles , Estudios Transversales , Endotoxemia/complicaciones , Endotoxemia/inmunología , Endotoxemia/metabolismo , Femenino , Humanos , Cirrosis Hepática/complicaciones , Cirrosis Hepática/inmunología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Fragmentos de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , Protrombina/metabolismo , ARN Mensajero/análisis , Análisis de Regresión , Tromboplastina/genéticaRESUMEN
The cytosolic and mitochondrial alcohol dehydrogenases from Kluyveromyces lactis (KlADHs) were purified and characterised. Both the N-terminally blocked cytosolic isozymes, KlADH I and KlADH II, were strictly NAD-dependent and exhibited catalytic properties similar to those previously reported for other yeast ADHs. Conversely, the mitochondrial isozymes, KlADH III and KlADH IV, displayed Ala and Asn, respectively, as N-termini and were able to oxidise at an increased rate primary alcohols with aliphatic chains longer than ethanol, such as propanol, butanol, pentanol and hexanol. Interestingly, the mitochondrial KlADHs, at variance with cytosolic isozymes and the majority of ADHs from other sources, were capable of accepting as a cofactor, and in some case almost equally well, either NAD or NADP. Since Asp-223 of horse liver ADH, thought to be responsible for the selection of NAD as coenzyme, is strictly conserved in all the KlADH isozymes, this amino-acid residue should not be considered critical for the coenzyme discrimination with respect to the other residues lining the coenzyme binding pocket of the mitochondrial isozymes. The relatively low specificity of the mitochondrial KlADHs both toward the alcohols and the cofactor could be explained on the basis of an enhanced flexibility of the corresponding catalytic pockets. An involvement of the mitochondrial KlADH isozymes in the physiological reoxidation of the cytosolic NADPH was also hypothesized. Moreover, both cytosolic and KlADH IV isozymes have an additional cysteine, not involved in zinc binding, that could be responsible for the increased activity in the presence of 2-mercaptoethanol.
Asunto(s)
Alcohol Deshidrogenasa/aislamiento & purificación , Isoenzimas/aislamiento & purificación , Kluyveromyces/enzimología , Alcohol Deshidrogenasa/química , Secuencia de Aminoácidos , Citosol/enzimología , Isoenzimas/química , Cinética , Mercaptoetanol/farmacología , Mitocondrias/enzimología , Datos de Secuencia Molecular , Peso Molecular , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Especificidad por SustratoRESUMEN
BACKGROUND: Anti-phospholipid antibodies (aPLs) were associated with an ongoing prothrombotic state in patients with systemic lupus erythematosus (SLE). Because aPLs are able to shift endothelial function toward procoagulant activity in vitro, we investigated the relationship among aPLs, ongoing prothrombotic state, and endothelial perturbation in SLE patients. METHODS AND RESULTS: We measured aPLs, anti-EC antibodies, circulating levels of prothrombin fragment F1 + 2 (F1 + 2), tumor necrosis factor-alpha (TNF-alpha), tissue-type plasminogen activator (TPA), and von Willebrand factor (vWF) in 43 SLE patients and 25 healthy subjects. Patients positive for aPLs (n = 23) had a higher prevalence of anti-EC antibodies (P = .02) and higher levels of F1 + 2 (P = .003) than aPL(-) patients. Endothelial perturbation, defined by elevated plasma levels of both TPA and vWF, was significantly associated with aPL positivity (P = .001). F1 + 2 > 1 nmol/L (mean +/- 2 SD of controls) was detected in all but one patient in whom aPL positivity and endothelial perturbation coexisted and in no aPL(+) patient without endothelial perturbation (P = .0039). F1 + 2 was significantly correlated with vWF (rho = .6, P = .004) and TPA (114 = .70, P = .0006) only in aPL(+) patients. Endothelial perturbation was closely associated with high values of TNF-alpha (P = .0001), anti-phospholipid (P = .001), and anti-EC antibodies (P = .012). In 31 patients without a clinical history of thrombosis followed up for 3 years, aPL(+) patients with endothelial perturbation showed higher F1 + 2 and TNF-alpha values than aPL(+) patients without endothelial dysfunction. CONCLUSIONS: This study shows that in SLE patients, aPL positivity is associated with an ongoing prothrombotic state only in the presence of endothelial perturbation. Our findings also suggest that aPLs and TNF-alpha might cooperate in inducing endothelial perturbation.
Asunto(s)
Anticuerpos Antifosfolípidos/análisis , Endotelio Vascular/fisiopatología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/fisiopatología , Trombosis/etiología , Adulto , Femenino , Humanos , Lupus Eritematoso Sistémico/complicaciones , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Trombina/análisisRESUMEN
We report the sequence of a 8,061 bp fragment of Saccharomyces cerevisiae chromosome VII. Five open reading frames (ORFs) of at least 100 amino acids were identified. Three show similarities to the amino-acid sequence of known gene products. ORF G9374 corresponds to the gene coding for the yTAFII145 protein: a TBP-associated factor whose amino-acid sequence was previously reported (Reese et al., 1994). The remaining ORF does not display similarities to known sequences.
Asunto(s)
Cromosomas Fúngicos/genética , Proteínas de Unión al ADN/genética , Genes Fúngicos/genética , Sistemas de Lectura Abierta/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
We have determined the nucleotide sequence of a segment of VII of the yeast Saccharomyces cerevisiae contained in the cosmid clone pEGH101 for a total of 7 kbp. This sequence contains a large open reading frame (ORF) called G9365, coding for a protein of 1967 amino acids that shows a significant homology with the product of the SKI2 gene of S. cerevisiae and contains domains characteristics of RNA-helicases. The ORF is transcribed in vegetative cells but it is not essential for viability as demonstrated by gene disruption.
Asunto(s)
Cromosomas Fúngicos/genética , Proteínas Fúngicas/genética , Genes Fúngicos/genética , ARN Nucleotidiltransferasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Fenotipo , ARN Helicasas , ARN Bicatenario/análisis , ARN de Hongos/análisis , Mapeo Restrictivo , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Esporas Fúngicas , Transcripción GenéticaRESUMEN
The lactose-utilizing yeast Kluyveromyces lactis is an essentially aerobic organism in which both respiration and fermentation can coexist depending on the sugar concentration. Despite a low fermentative capacity as compared to Saccharomyces cerevisiae, four structural genes encoding alcohol dehydrogenase (ADH) activities are present in this yeast. Two of these activities, namely K1ADH III and K1ADH IV, are located within mitochondria and their presence is dependent on the carbon sources in the medium. In this paper we demonstrate by transcription and activity analysis that KlADH3 is expressed in the presence of low glucose concentrations and in the presence of respiratory carbon sources other than ethanol. Indeed ethanol acts as a strong repressor of this gene. On the other hand, KlADH4 is induced by the presence of ethanol and not by other respiratory carbon sources. We also demonstrate that the presence of KLADH III and KLADH IV in K. lactis cells is dependent on glucose concentration, glucose uptake and the amount of ethanol produced. As a consequence, these activities can be used as markers for the onset of respiratory and fermentative metabolism in this yeast.
Asunto(s)
Alcohol Deshidrogenasa/biosíntesis , Regulación Enzimológica de la Expresión Génica , Isoenzimas/biosíntesis , Kluyveromyces/enzimología , Mitocondrias/enzimología , Alcohol Deshidrogenasa/genética , Aldehído Oxidorreductasas/biosíntesis , Aldehído Oxidorreductasas/genética , Etanol/farmacología , Fermentación , Glucosa/farmacología , Isoenzimas/genética , Kluyveromyces/efectos de los fármacos , Kluyveromyces/genética , Kluyveromyces/metabolismo , Mitocondrias/genética , Mutagénesis , Consumo de Oxígeno , Factores de Tiempo , Transcripción Genética , Transformación GenéticaRESUMEN
BACKGROUND & AIMS: Hyperfibrinolysis may complicate the clinical course of liver cirrhosis. The aim of this study was to evaluate if, in cirrhosis, hyperfibrinolysis is primary or secondary to intravascular clotting activation and if endotoxemia is associated with activation of clotting and/or the fibrinolytic system. METHODS: Clotting, fibrinolytic indexes, and endotoxemia were studied in 41 cirrhotic patients and 20 healthy subjects. RESULTS: Twenty-seven cirrhotic patients (66%) had high plasma levels of prothrombin fragment F1 + 2, a marker of thrombin generation. Nineteen patients had elevated values of D-dimer, a marker of fibrinolysis in vivo. All patients with high values of D-dimer also had high values of prothrombin fragment F1 + 2. Endotoxemia was elevated in patients with severe liver failure and significantly correlated to prothrombin fragment F1 + 2. Thirty patients were treated for 7 days either with standard therapy (n = 15) or with standard therapy plus nonabsorbable antibiotics (n = 15). Although standard therapy did not significantly change laboratory indexes, a significant reduction of endotoxemia, prothrombin fragment F1 + 2, and D-dimer was found in those patients who received the combined treatment. CONCLUSIONS: This study shows that, in cirrhotic patients, hyperfibrinolysis is not a primary phenomenon but occurs as a consequence of clotting activation and that endotoxemia might play a pathophysiological role.
Asunto(s)
Coagulación Intravascular Diseminada/complicaciones , Fibrinólisis , Cirrosis Hepática/sangre , Toxemia/complicaciones , Adulto , Anciano , Estudios Transversales , Femenino , Humanos , Cirrosis Hepática/complicaciones , Masculino , Persona de Mediana EdadRESUMEN
One hundred sixty-five patients with cirrhosis diagnosed by needle liver biopsy were followed for 2 years to evaluate the relation between clotting factors and survival. Patients with spontaneous bacterial peritonitis, hepatic carcinoma, and cholestatic liver diseases were excluded. Patients were classified as A (n = 34), B (n = 75), or C (n = 56) according to Child-Pugh criteria. During the follow-up 45 patients died of liver failure or gastrointestinal hemorrhage. Nonsurvivor patients had significantly higher values of bilirubin and D-dimer, a marker of fibrinolysis in vivo, lower values of albumin, prothrombin activity, fibrinogen, prekallikrein, factor VII, and a more prolonged activated partial thromboplastin time than survivors. All these variables and Child-Pugh classification were significantly associated with survival in a univariate analysis. Multivariate analysis (Cox's model) showed that only prekallikrein and factor VII were independently predictors of survival. Ninety-three percent of patients with prekallikrein values < 32% died within 32 months of follow-up, whereas factor VII < 34% identified 93% of patients who died within 10 months of follow-up. This study suggests that factor VII is an early predictor of survival and may be a useful test to better identify cirrhotic patients who should be candidates for liver transplantation.
Asunto(s)
Coagulación Sanguínea , Fibrinólisis , Cirrosis Hepática/sangre , Anciano , Factor VII/análisis , Femenino , Estudios de Seguimiento , Humanos , Cirrosis Hepática/mortalidad , Masculino , Persona de Mediana Edad , Precalicreína/análisis , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de SupervivenciaRESUMEN
Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.
Asunto(s)
Alcohol Deshidrogenasa/genética , Etanol/metabolismo , Glucosa/metabolismo , Kluyveromyces/genética , Alcohol Deshidrogenasa/fisiología , Genes Fúngicos , Isoenzimas/genética , Isoenzimas/fisiología , Kluyveromyces/fisiología , MutaciónRESUMEN
The relation between coagulation indexes and survival rate was studied and analyzed in 46 patients with advanced liver cirrhosis (grade B and C Child-Pugh Classification), during a follow-up of 1 year. Twenty-four patients (52%) died of liver failure or fatal haemorrhage within 12 months of follow-up. Prothrombin activity, fibrinogen, fibrin(ogen) degradation products, prekallikrein and factor VII, serum bilirubin, and the degree of liver insufficiency, scored by Child-Pugh classification, proved to be significantly correlated with survival by univariate analysis. A multivariate survival analysis (Cox regression model) disclosed two variables, prekallikrein and factor VII, that predicted survival. The rate ratios of death increased to 2.8 and 7.6 with values of prekallikrein < 26% and factor VII < 39%, respectively. This study shows that some simple laboratory tests exploring the clotting system may identify patients with poor prognosis in severe liver failure.