RESUMEN
BACKGROUND: The efficacy and safety of high versus medium doses of glucocorticoids for the treatment of patients with COVID-19 has shown mixed outcomes in controlled trials and observational studies. We aimed to evaluate the effectiveness of methylprednisolone 250 mg bolus versus dexamethasone 6 mg in patients with severe COVID-19. METHODS: A randomised, open-label, controlled trial was conducted between February and August 2021 at four hospitals in Spain. The trial was suspended after the first interim analysis since the investigators considered that continuing the trial would be futile. Patients were randomly assigned in a 1:1 ratio to receive dexamethasone 6 mg once daily for up to 10 days or methylprednisolone 250 mg once daily for 3 days. RESULTS: Of the 128 randomised patients, 125 were analysed (mean age 60 ± 17 years; 82 males [66%]). Mortality at 28 days was 4.8% in the 250 mg methylprednisolone group versus 4.8% in the 6 mg dexamethasone group (absolute risk difference, 0.1% [95% CI, -8.8 to 9.1%]; p = 0.98). None of the secondary outcomes (admission to the intensive care unit, non-invasive respiratory or high-flow oxygen support, additional immunosuppressive drugs, or length of stay), or prespecified sensitivity analyses were statistically significant. Hyperglycaemia was more frequent in the methylprednisolone group at 27.0 versus 8.1% (absolute risk difference, -18.9% [95% CI, -31.8 to - 5.6%]; p = 0.007). CONCLUSIONS: Among severe but not critical patients with COVID-19, 250 mg/d for 3 days of methylprednisolone compared with 6 mg/d for 10 days of dexamethasone did not result in a decrease in mortality or intubation.
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COVID-19 , Adulto , Masculino , Humanos , Persona de Mediana Edad , Anciano , Metilprednisolona , SARS-CoV-2 , Dexametasona , Resultado del TratamientoRESUMEN
BACKGROUND: COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. METHODS: A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. RESULTS: The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183-12.968], 0.025), viral RNA load (N1) (1.962 [1.244-3.096], 0.004); viral RNA load (N2) (2.229 [1.382-3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia). CONCLUSIONS: SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.
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COVID-19/complicaciones , ARN Viral/análisis , Carga Viral/inmunología , Adulto , Anciano , Biomarcadores/análisis , Biomarcadores/sangre , COVID-19/sangre , Distribución de Chi-Cuadrado , Enfermedad Crítica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/sangre , Estadísticas no ParamétricasRESUMEN
Flowering is a crucial process that demands substantial resources. Carbon metabolism must be coordinated with development through a control mechanism that optimizes fitness for any physiological need and growth stage of the plant. However, how sugar allocation is controlled during the floral transition is unknown. Recently, the role of a CONSTANS (CO) ortholog (Cr-CO) in the control of the photoperiod response in the green alga Chlamydomonas reinhardtii and its influence on starch metabolism was demonstrated. In this work, we show that transitory starch accumulation and glycan composition during the floral transition in Arabidopsis thaliana are regulated by photoperiod. Employing a multidisciplinary approach, we demonstrate a role for CO in regulating the level and timing of expression of the GRANULE BOUND STARCH SYNTHASE (GBSS) gene. Furthermore, we provide a detailed characterization of a GBSS mutant involved in transitory starch synthesis and analyze its flowering time phenotype in relation to its altered capacity to synthesize amylose and to modify the plant free sugar content. Photoperiod modification of starch homeostasis by CO may be crucial for increasing the sugar mobilization demanded by the floral transition. This finding contributes to our understanding of the flowering process.
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Arabidopsis/fisiología , Carbono/metabolismo , Flores/fisiología , Fotoperiodo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Modelos Biológicos , Mutación/genética , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad , Almidón/metabolismoRESUMEN
Inorganic polyphosphates (polyP) are linear polymers of tens to hundreds orthophosphate residues linked by phosphoanhydride bonds. These fairly abundant biopolymers occur in all extant forms of life, from prokaryotes to mammals, and could have played a relevant role in prebiotic evolution. Since the first identification of polyP deposits as metachromatic or volutin granules in yeasts in the nineteenth century, an increasing number of varied physiological functions have been reported. Due to their "high energy" bonds analogous to those in ATP and their properties as polyanions, polyP serve as microbial phosphagens for a variety of biochemical reactions, as a buffer against alkalis, as a storage of Ca(2+) and as a metal-chelating agent. In addition, recent studies have revealed polyP importance in signaling and regulatory processes, cell viability and proliferation, pathogen virulence, as a structural component and chemical chaperone, and as modulator of microbial stress response. This review summarizes the current status of knowledge and future perspectives of polyP functions and their related enzymes in the microbial world.
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Polifosfatos/química , Polifosfatos/metabolismo , Células Procariotas/química , Células Procariotas/metabolismo , Células Eucariotas/química , Células Eucariotas/metabolismo , Polifosfatos/farmacologíaRESUMEN
The single-copy genes encoding putative polyphosphate-glucose phosphotransferases (PPGK, EC 2.7.1.63) from two nitrogen-fixing Cyanobacteria, Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102, were cloned and functionally characterized. In contrast to their actinobacterial counterparts, the cyanobacterial PPGKs have shown the ability to phosphorylate glucose using strictly inorganic polyphosphates (polyP) as phosphoryl donors. This has proven to be an economically attractive reagent in contrast to the more costly ATP. Cyanobacterial PPGKs had a higher affinity for medium-long-sized polyP (greater than ten phosphoryl residues). Thus, longer polyP resulted in higher catalytic efficiency. Also in contrast to most their homologs in Actinobacteria, both cyanobacterial PPGKs exhibited a modest but significant polyP-mannokinase activity as well. Specific activities were in the range of 180-230 and 2-3 µmol min(-1) mg(-1) with glucose and mannose as substrates, respectively. No polyP-fructokinase activity was detected. Cyanobacterial PPGKs required a divalent metal cofactor and exhibited alkaline pH optima (approx. 9.0) and a remarkable thermostability (optimum temperature, 45 °C). The preference for Mg(2+) was noted with an affinity constant of 1.3 mM. Both recombinant PPGKs are homodimers with a subunit molecular mass of ca. 27 kDa. Based on database searches and experimental data from Southern blots and activity assays, closely related PPGK homologs appear to be widespread among unicellular and filamentous mostly nitrogen-fixing Cyanobacteria. Overall, these findings indicate that polyP may be metabolized in these photosynthetic prokaryotes to yield glucose (or mannose) 6-phosphate. They also provide evidence for a novel group-specific subfamily of strictly polyP-dependent gluco(manno)kinases with ancestral features and high biotechnological potential, capable of efficiently using polyP as an alternative and cheap source of energy-rich phosphate instead of costly ATP. Finally, these results could shed new light on the evolutionary origin of sugar kinases.
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Glucoquinasa/aislamiento & purificación , Glucoquinasa/metabolismo , Hexosas/metabolismo , Nostoc/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Polifosfatos/metabolismo , Clonación Molecular , Coenzimas/análisis , Glucoquinasa/química , Glucoquinasa/genética , Concentración de Iones de Hidrógeno , Peso Molecular , Nostoc/genética , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Multimerización de Proteína , TemperaturaRESUMEN
The genome of the thermophilic green-sulfur bacterium Chlorobium tepidum TLS possesses two genes encoding putative exopolyphosphatases (PPX; EC 3.6.1.11), namely CT0099 (ppx1, 993 bp) and CT1713 (ppx2, 1557 bp). The predicted polypeptides of 330 and 518 aa residues are Ppx-GppA phosphatases of different domain architectures - the largest one has an extra C-terminal HD domain - which may represent ancient paralogues. Both ppx genes were cloned and overexpressed in Escherichia coli BL21(DE3). While CtPPX1 was validated as a monomeric enzyme, CtPPX2 was found to be a homodimer. Both PPX homologues were functional, K(+)-stimulated phosphohydrolases, with an absolute requirement for divalent metal cations and a marked preference for Mg(2+). Nevertheless, they exhibited remarkably different catalytic specificities with regard to substrate classes and chain lengths. Even though both enzymes were able to hydrolyse the medium-size polyphosphate (polyP) P13-18 (polyP mix with mean chain length of 13-18 phosphate residues), CtPPX1 clearly reached its highest catalytic efficiency with tripolyphosphate and showed substantial nucleoside triphosphatase (NTPase) activity, while CtPPX2 preferred long-chain polyPs (>300 Pi residues) and did not show any detectable NTPase activity. These catalytic features, taken together with the distinct domain architectures and molecular phylogenies, indicate that the two PPX homologues of Chl. tepidum belong to different Ppx-GppA phosphatase subfamilies that should play specific biochemical roles in nucleotide and polyP metabolisms. In addition, these results provide an example of the remarkable functional plasticity of the Ppx-GppA phosphatases, a family of proteins with relatively simple structures that are widely distributed in the microbial world.
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Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/metabolismo , Chlorobium/enzimología , Chlorobium/genética , Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/aislamiento & purificación , Cationes Bivalentes/metabolismo , Clonación Molecular , Análisis por Conglomerados , Coenzimas/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Activadores de Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Hidrólisis , Cinética , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Polifosfatos/metabolismo , Potasio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia , Especificidad por SustratoRESUMEN
OBJECTIVES: To evaluate if the detection of N antigen of SARS-CoV-2 in plasma by a rapid lateral flow test predicts 90-day mortality in COVID-19 patients hospitalized at the wards. METHODS: The presence of N-antigenemia was evaluated in the first 36 hours after hospitalization in 600 unvaccinated COVID-19 patients, by using the Panbio COVID-19 Ag Rapid Test Device from Abbott (Abbott Laboratories Inc., Chicago, IL, USA). The impact of N-antigenemia on 90-day mortality was assessed by multivariable Cox regression analysis. RESULTS: Prevalence of N-antigenemia at hospitalization was higher in nonsurvivors (69% (82/118) vs. 52% (250/482); p < 0.001). The patients with N-antigenemia showed more frequently RNAemia (45.7% (148/324) vs. 19.8% (51/257); p < 0.001), absence of anti-SARS-CoV-2 N antibodies (80.7% (264/327) vs. 26.6% (69/259); p < 0.001) and absence of S1 antibodies (73.4% (240/327) vs. 23.6% (61/259); p < 0.001). The patients with antigenemia showed more frequently acute respiratory distress syndrome (30.1% (100/332) vs. 18.7% (50/268); p = 0.001) and nosocomial infections (13.6% (45/331) vs. 7.9% (21/267); p = 0.026). N-antigenemia was a risk factor for increased 90-day mortality in the multivariable analysis (HR, 1.99 (95% CI,1.09-3.61), whereas the presence of anti-SARS-CoV-2 N-antibodies represented a protective factor (HR, 0.47 (95% CI, 0.26-0.85). DISCUSSION: The presence of N-antigenemia or the absence of anti-SARS-CoV-2 N-antibodies after hospitalization is associated to increased 90-day mortality in unvaccinated COVID-19 patients. Detection of N-antigenemia by using lateral flow tests is a quick, widely available tool that could contribute to early identify those COVID-19 patients at risk of deterioration.
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COVID-19 , Anticuerpos Antivirales , COVID-19/diagnóstico , Prueba de COVID-19 , Humanos , Estudios Prospectivos , SARS-CoV-2RESUMEN
OBJECTIVES: To analyze the association between atmospheric levels of nitrogen dioxide (NO2) and the number of visits by adults to an emergency department (ED) for exacerbated asthma in an urban area with low levels of air pollution. MATERIAL AND METHODS: Retrospective ecological time-series study. We quantified ED visits for asthma by consecutive patients over the age of 14 years between 2010 and 2018 (3287 days). The association between the mean atmospheric concentration of NO2 and the number of daily visits to the ED for asthma was analyzed with generalized linear regression analysis (Poisson modeling). The impact of exposure on individual risk was assessed by crossover analysis of case periods. We adjusted for confounding meteorologic variables, potential variability due to seasonal changes was corrected by trend analysis, and 3 time lags were assessed (0, 1, and 3 days). RESULTS: We analyzed 2527 asthma emergencies in 1588 patients (70% female) with a mean (SD) age of 51 (21) years. A significant positive association (relative risk, 1.056, 95% CI, 1.006-1.108; P .05) between atmospheric NO2 concentration and greater risk of visiting an ED within 3 days was detected. An increase of 10 µg/m3 of NO2 accounted for 5.3% of the visits (attributable fraction, 5.30, 95% CI, 0.60-9.75; P .05). CONCLUSION: In an urban area with low pollution levels, an elevation in atmospheric NO2 is associated with more hospital ED visits for asthma attacks in adults.
OBJETIVO: Analizar la asociación entre los niveles ambientales de dióxido de nitrógeno (NO2) y el número de consultas a urgencias por un episodio de agudización de asma bronquial en la población adulta de un entorno urbano con bajos niveles de contaminación. METODO: Estudio ecológico retrospectivo de series temporales. Se consideraron las visitas por asma de pacientes mayores de 14 años que acudieron a un servicio de urgencias de forma consecutiva entre 2010 y 2018 (3.287 días). La asociación entre la concentración media de NO2 y el número diario de visitas a urgencias por asma se estudió mediante un modelo lineal generalizado con regresión de Poisson. Se evaluó el impacto en el riesgo individual mediante un análisis de casos cruzados. Se ajustó por las variables confusoras meteorológicas, se corrigió la estacionalidad mediante análisis de tendencias y se evaluaron tres lags temporales (0, 1 y 3 días). RESULTADOS: Se analizaron 2.527 urgencias por asma correspondientes a 1.588 pacientes (edad media 51 ± 21 años, 70% mujeres). Hubo una asociación positiva significativa (riesgo relativo: RR = 1,056, IC 95%: 1,006-1,108; p 0,05) entre la concentración de NO2 y un mayor riesgo de consulta a urgencias por asma a los 3 días. Un incremento de 10 µgr/m3 de NO2 explicó el 5,3% de las consultas (fracción atribuible: FA = 5,30, IC 95%: 0,60-9,75; p 0,05). CONCLUSIONES: El incremento de los niveles ambientales de NO2 se asocia con un mayor número de urgencias hospitalarias por exacerbación de asma en adultos en un entorno con baja contaminación.
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Contaminantes Atmosféricos , Contaminación del Aire , Asma , Adolescente , Adulto , Contaminantes Atmosféricos/efectos adversos , Contaminación del Aire/efectos adversos , Contaminación del Aire/análisis , Asma/epidemiología , Estudios Cruzados , Servicio de Urgencia en Hospital , Femenino , Humanos , Masculino , Dióxido de Nitrógeno/efectos adversos , Dióxido de Nitrógeno/análisis , Estudios Retrospectivos , Factores de TiempoRESUMEN
OBJECTIVES: To compare the prognostic value of 3 severity scales: the Pneumonia Severity Index (PSI), the CURB-65 pneumonia severity score, and the Severity Community-Acquired Pneumonia (SCAP) score. To build a new predictive model for in-hospital mortality in patients over the age of 75 years admitted with pneumonia due to the coronavirus disease 2019 (COVID-19). MATERIAL AND METHODS: Retrospective study of patients older than 75 years admitted from the emergency department for COVID-19 pneumonia between March 12 and April 27, 2020. We recorded demographic (age, sex, living in a care facility or not), clinical (symptoms, comorbidities, Charlson Comorbidity Index [CCI]), and analytical (serum biochemistry, blood gases, blood count, and coagulation factors) variables. A risk model was constructed, and the ability of the 3 scales to predict all-cause in-hospital mortality was compared. RESULTS: We included 186 patients with a median age of 85 years (interquartile range, 80-89 years); 44.1% were men. Mortality was 47.3%. The areas under the receiver operating characteristic curves (AUCs) were as follows for each tool: PSI, 0.74 (95% CI, 0.64-0.82); CURB-65 score, 0.71 (95% CI, 0.62-0.79); and SCAP score, 0.72 (95% CI, 0.63-0.81). Risk factors included in the model were the presence or absence of symptoms (cough, dyspnea), the CCI, and analytical findings (aspartate aminotransferase, potassium, urea, and lactate dehydrogenase. The AUC for the model was 0.81 (95% CI, 0.73-0.88). CONCLUSION: This study shows that the predictive power of the PSI for mortality is moderate and perceptibly higher than the CURB-65 and SCAP scores. We propose a new predictive model for mortality that offers significantly better performance than any of the 3 scales compared. However, our model must undergo external validation.
OBJETIVO: Los objetivos son comparar la utilidad pronóstica de tres escalas de gravedad (Pneumonia Severity Index: PSI; CURB-65 scale; Severity Community Acquired Pneumonia Score: SCAP) y diseñar un nuevo modelo predictivo de mortalidad hospitalaria en pacientes mayores de 75 años ingresados por neumonía por COVID-19. METODO: Estudio retrospectivo de pacientes mayores de 75 años ingresados por neumonía por COVID-19 desde el servicio de urgencias entre el 12 de marzo y el 27 de abril de 2020. Se recogieron variables demográficas (edad, sexo, institucionalización), clínicas (síntomas, comorbilidades, índice de Charlson) y analíticas (bioquímica en suero, gasometría, hematimetría, hemostasia). Se derivó un modelo de riesgo y se compararon las escalas de gravedad PSI, CURB-65 y SCAP para predecir la mortalidad intrahospitalaria por cualquier causa. RESULTADOS: Se incluyeron 186 pacientes, con una mediana de edad de 85 años (RIC 80-89), un 44,1% varones. La mortalidad fue del 47,3%. Las escalas PSI, CURB-65 y SCAP tuvieron un área bajo la curva (ABC) de 0,74 (IC 95% 0,64-0,82), 0,71 (IC 95% 0,62-0,79) y 0,72 (IC 95% 0,63-0,81), respectivamente. El modelo predictivo compuesto por la ausencia o presencia de síntomas (tos y disnea), comorbilidad (índice de Charlson) y datos analíticos (aspartato- aminotransferasa, potasio, urea y lactato-deshidrogenasa) tuvo un ABC de 0,81 (IC 95% 0,73-0,88). CONCLUSIONES: Este estudio muestra que la escala PSI tiene una capacidad predictiva de mortalidad moderada, notablemente mejor que las escalas CURB-65 y SCAP. Se propone un nuevo modelo predictivo de mortalidad que mejora significativamente el rendimiento de estas escalas, siendo necesario verificar su validez externa.
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COVID-19/mortalidad , Mortalidad Hospitalaria , Modelos Teóricos , Índice de Severidad de la Enfermedad , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Femenino , Humanos , Masculino , Curva ROC , Estudios RetrospectivosRESUMEN
Infection with SARS-CoV-2 is becoming the leading cause of death in most countries during the 2020 pandemic. The objective of this study is to assess the association between COVID-19 and cause-specific death. The design is retrospective cohort study. We included data from inpatients diagnosed with COVID-19 between March 18 and April 21, 2020, who died during their hospital stay. Demographic, clinical and management data were collected. Causes of death were ascertained by review of medical records. The sample included 128 individuals. The median age was 84 (IQR 75-89), 57% were men. In 109 patients, the death was caused by SARS-CoV-2 infection, whereas in 19 (14.8%, 95 CI 10-22%), the infection acted only as a precipitating factor to decompensate other pathologies. This second group of patients was older (88y vs 82, p < 0.001). In age-adjusted analysis, they had a greater likelihood of heart failure (OR 3.61 95% CI 1.15-11.32), dependency in activities of daily living (OR 12.07 95% CI 1.40-103.86), frailty (OR 8.73 95% CI 1.37-55.46). The presence of X-ray infiltrates was uncommon (OR 0.07, 95% CI 0.02-0.25). A higher percentage of patient deaths from causes unrelated to COVID-19 complications occurred during the two first weeks of the pandemic. Fifteen percent of patients with COVID-19 infection died from decompensation of other pathologies and the cause of death was unrelated to COVID-19 severe complications. Most of these patients had more comorbidities and were frail and elderly. These findings can partially explain the excess mortality in older people.
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Causas de Muerte/tendencias , Infecciones por Coronavirus/mortalidad , Pandemias/estadística & datos numéricos , Neumonía Viral/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , COVID-19 , Estudios de Cohortes , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/epidemiología , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Neumonía Viral/complicaciones , Neumonía Viral/epidemiología , Insuficiencia Respiratoria/complicaciones , Insuficiencia Respiratoria/epidemiología , Estudios Retrospectivos , España/epidemiología , Estadísticas no Paramétricas , Tromboembolia/complicaciones , Tromboembolia/epidemiologíaRESUMEN
A study was carried out to determine the changes in phenolic composition induced by tuberculosis infection in olive trees. Four ethanolic extracts were compared: olive leaf from shoots affected by Pseudomonas savastanoi pv. Savastanoi, nodules induced by this bacteria, leaf from healthy (asymptomatic) shoots, and shoots. Among the differences found, the presence of a phenolic compound in nodules was significant in much larger quantities than in leaf or shoots. Mass spectrometric analysis showed this compound to be verbascoside. The enhancement of its biosynthesis could be related to the defense mechanisms of the tree in the nodules induced by P. savastanoi and suggests the possibility of exploration of natural and biotechnological sources of this compound.
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Glucósidos/análisis , Olea/microbiología , Fenoles/análisis , Enfermedades de las Plantas/microbiología , Pseudomonas , Antiinfecciosos/análisis , Extractos Vegetales/análisis , Hojas de la Planta/químicaRESUMEN
Sucrose-phosphate phosphatase (SPP) catalyses the final step in the sucrose biosynthesis pathway. Arabidopsis thaliana genome codifies four SPP isoforms. In this study, the four Arabidopsis thaliana genes coding for SPP isoforms have been cloned, expressed in Escherichia coli and the kinetic and regulatory properties of the purified enzymes analysed. SPP2 is the isoform showing the highest activity, with SPP3b and SPP3a showing lower activity levels. No activity was detected for SPP1. We propose that this lack of activity is probably due to the absence of an essential amino acid participating in catalysis and/or in the binding of the substrate, sucrose-6-phosphate (Suc6P). The expression patterns of Arabidopsis SPP genes indicate that SPP2 and SPP3b are the main isoforms expressed in different tissues and organs, although the non-catalytic SPP1 is the main isoform expressed in roots. Thus, SPP1 could have acquired new unknown functions. We also show that the three catalytically active SPPs from Arabidopsis are dimers. By generating a chimeric SPP composed of the monomeric cyanobacterial SPP fused to the higher plant non-catalytic S6PPc domain (from SPP2), we show that the S6PPc domain is responsible for SPP dimerization. This is the first experimental study on the functionality and gene expression pattern of all the SPPs from a single plant species.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Multimerización de Proteína , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Escherichia coli/metabolismo , Fluorescencia , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Peso Molecular , Mutagénesis Sitio-Dirigida , Fotosíntesis/efectos de los fármacos , Filogenia , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Sacarosa/farmacologíaRESUMEN
Amine fungicides are widely used as crop protectants. Their success is believed to be related to their ability to inhibit postlanosterol sterol biosynthesis in fungi, in particular sterol-Δ(8),Δ(7)-isomerases and sterol-Δ(14)-reductases, with a concomitant accumulation of toxic abnormal sterols. However, their actual cellular effects and mechanisms of death induction are still poorly understood. Paradoxically, plants exhibit a natural resistance to amine fungicides although they have similar enzymes in postcicloartenol sterol biosynthesis that are also susceptible to fungicide inhibition. A major difference in vacuolar ion homeostasis between plants and fungi is the presence of a dual set of primary proton pumps in the former (V-ATPase and H(+)-pyrophosphatase), but only the V-ATPase in the latter. Abnormal sterols affect the proton-pumping capacity of V-ATPases in fungi and this has been proposed as a major determinant in fungicide action. Using Saccharomyces cerevisiae as a model fungus, we provide evidence that amine fungicide treatment induced cell death by apoptosis. Cell death was concomitant with impaired H(+)-pumping capacity in vacuole vesicles and dependent on vacuolar proteases. Also, the heterologous expression of the Arabidopsis thaliana main H(+)-pyrophosphatase (AVP1) at the fungal vacuolar membrane reduced apoptosis levels in yeast and increased resistance to amine fungicides. Consistently, A. thaliana avp1 mutant seedlings showed increased susceptibility to this amine fungicide, particularly at the level of root development. This is in agreement with AVP1 being nearly the sole H(+)-pyrophosphatase gene expressed at the root elongation zones. All in all, the present data suggest that H(+)-pyrophosphatases are major determinants of plant tolerance to amine fungicides.
RESUMEN
Countercurrent supercritical fluid extraction (CC-SFE) at a pilot scale plant was used for fractionation of high-added-value products from a raw extract of olive leaves in hexane. Compounds found in the raw extract were waxes, hydrocarbons, squalene, beta-carotene, triglycerides, alpha-tocopherol, beta-sitosterol, and alcohols. The CC-SFE extraction process was investigated according to a 2(3) full factorial experimental design using the following variables and ranges: extraction pressure, 75-200 bar; extraction temperature, 35-50 degrees C; and ethanol as modifier, 0-10%. Data were analyzed in terms of extraction yield, enrichment, recovery, and selectivity. Higher extraction yields were attained at 200 bar. For most of the compounds analyzed enrichment was attained at the same conditions, that is, 75 bar, 35 degrees C, and 10% ethanol. Hydrocarbons were usually recovered in the separators, whereas waxes and alpha-tocopherol remain in the raffinate. Selectivity data reveal that alpha-tocopherol is the most easily separable compound. The influence of the experimental factors on the recovery of all the compounds was studied by means of regression models. The best fitted model was attained for beta-sitosterol, with R2 = 99.25%.