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Candida auris is a multidrug-resistant fungus known to be a global public health problem. The skin-based transmission, together with the marked resistance to drugs, resulted in its rapid spread to all continents. The aim of this study was to identify an essential oil (EO) active in the fight against C. auris. A total of 15 EOs were tested against 10 clinical strains of C. auris. Cinnamomum zeylanicum EO (CZ-EO) was the most effective (MIC90 and MFC90 equal to 0.06% vol/vol). Three fractions obtained from CZ-EO, and the cinnamaldehyde (CIN), the major chemical compound, were tested to identify the principal compound effectives against C. auris. All CIN-containing samples showed anti-fungal activity. To study the synergy with fluconazole, CZ-EO, its active fraction (FR2), and CIN were tested in checkerboard tests. Results show that CZ-EO and FR2, but not CIN, synergize with fluconazole. Furthermore, only the copresence of CZ-EO or FR2 synergize with fluconazole at therapeutic concentrations of the drug (0.45 ± 0.32 µg/mL and 0.64 ± 0.67 µg/mL, respectively), while CIN only shows additive activity. In vivo studies conducted on Galleria mellonella larvae show the absence of toxicity of CZ-EO up to concentrations of 16% vol/vol, and the ability of CZ-EO to reactivate the efficacy of fluconazole when formulated at synergic concentrations. Finally, biochemical tests were made to study the mechanism of action of CZ-EO. These studies show that in the presence of both fluconazole and CZ-EO, the activity of fungal ATPases decreases and, at the same time, the amount of intracellular drug increases. IMPORTANCE This study highlights how small doses of CZ-EO are able to inhibit the secretion of fluconazole and promote its accumulation in the fungal cell. In this manner, the drug is able to exert its pharmacological effects bypassing the resistance of the yeast. If further studies will confirm this synergy, it will be possible to develop new therapeutic formulations active in the fight against C. auris resistances.
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When a batch of magma reaches Earth's surface, it forms a vent from which volcanic products are erupted. At many volcanoes, successive batches may open vents far away from previous ones, resulting in scattered, sometimes seemingly random spatial distributions. This exposes vast areas to volcanic hazards and makes forecasting difficult. Here, we show that magma pathways and thus future vent locations may be forecast by combining the physics of magma transport with a Monte Carlo inversion scheme for the volcano stress history. We validate our approach on a densely populated active volcanic field, Campi Flegrei (Italy), where we forecast future vents on an onshore semiannular belt located between 2.3 and 4.2 km from the caldera center. Our approach offers a mechanical explanation for the vent migration over time at Campi Flegrei and at many calderas worldwide and may be applicable to volcanoes of any type.
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Transient seismicity at active volcanoes poses a significant risk in addition to eruptive activity. This risk is powered by the common belief that volcanic seismicity cannot be forecast, even on a long term. Here we investigate the nature of volcanic seismicity to try to improve our forecasting capacity. To this aim, we consider Ischia volcano (Italy), which suffered similar earthquakes along its uplifted resurgent block. We show that this seismicity marks an acceleration of decades-long subsidence of the resurgent block, driven by degassing of magma that previously produced the uplift, a process not observed at other volcanoes. Degassing will continue for hundreds to thousands of years, causing protracted seismicity and will likely be accompanied by moderate and damaging earthquakes. The possibility to constrain the future duration of seismicity at Ischia indicates that our capacity to forecast earthquakes might be enhanced when seismic activity results from long-term magmatic processes, such as degassing.
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Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for serious hospital infections worldwide and represents a global public health problem. Curcumin, the major constituent of turmeric, is effective against MRSA but only at cytotoxic concentrations or in combination with antibiotics. The major issue in curcumin-based therapies is the poor solubility of this hydrophobic compound and the cytotoxicity at high doses. In this paper, we describe the efficacy of a composite nanoparticle made of curcumin (CU) and graphene oxide (GO), hereafter GOCU, in MRSA infection treatment. GO is a nanomaterial with a large surface area and high drug-loading capacity. GO has also antibacterial properties due mainly to a mechanical cutting of the bacterial membranes. For this physical mechanism of action, microorganisms are unlikely to develop resistance against this nanomaterial. In this work, we report the capacity of GO to support and stabilize curcumin molecules in a water environment and we demonstrate the efficacy of GOCU against MRSA at a concentration below 2 µg ml-1. Further, GOCU displays low toxicity on fibroblasts cells and avoids haemolysis of red blood cells. Our results indicate that GOCU is a promising nanomaterial against antibiotic-resistant MRSA.
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The aim of the study was to test 1) whether chronic and stable coronary artery disease (CAD) could downregulate epicardial fat adrenomedullin synthesis and secretion, and decrease intracoronary plasma adrenomedullin levels, and 2) whether intracoronary plasma adrenomedullin levels could be related to epicardial adipose tissue adrenomedullin gene and protein expression in subjects with CAD. We examined 12 patients with CAD who required coronary artery bypass graft (CABG) and 10 patients with non-CAD who underwent cardiac surgery for valve replacement. Plasma levels of adrenomedullin were measured in peripheral vein circulation, in left coronary artery (LCA) and coronary sinus (CS) during coronary angiography. Epicardial adipose tissue biopsy for Reverse Transcription and Real-Time PCR (RT-PCR) adrenomedullin mRNA analysis and Western Blotting (WB) protein expression was performed during cardiac surgery in all subjects. Peripheral, LCA, and CS plasma adrenomedullin levels were significantly lower in CAD patients than in those with non-CAD (3.0+/-0.9 vs. 4.4+/-0.9 pg/ml p<0.01; 2.9+/-1 vs. 4.05+/-0.8 pg/ml, p<0.01, 3.1+/-0.9 vs. 3.98+/-0.9 pg/ml p=0.04, respectively). However, CS adrenomedullin levels were not statistically different than those in LCA suggesting that adrenomedullin was not secreted from epicardial fat into the coronary artery lumen. Epicardial fat adrenomedullin mRNA levels and protein expression were lower in patients with CAD than in those with non-CAD (p<0.01 for both). We conclude that 1) epicardial fat adrenomedullin gene and protein expression can be downregulated in CAD subjects, and 2) intracoronary adrenomedullin levels are lower in CAD. No evidence that epicardial adipose tissue really contributes intracoronary adrenomedullin can be provided at this time.
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Tejido Adiposo/metabolismo , Adrenomedulina/análisis , Adrenomedulina/sangre , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/metabolismo , Pericardio/metabolismo , Tejido Adiposo/patología , Adrenomedulina/genética , Anciano , Enfermedad de la Arteria Coronaria/fisiopatología , Circulación Coronaria , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Pericardio/patología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Pratylenchus lentis n. sp. parasitizing roots of lentil in Sicily, Italy, is described and illustrated. The new species is characterized by a relatively high lip region with three annuli, mean stylet length of 16 mum, with anteriorly flattened knobs, cylindrical body with a relatively anterior vulva, large and ovoid spermatheca full of sperm, plump tail with truncate, irregularly annulated terminus, and by the presence of males. Molecular ITS-RFLP and sequencing analyses of the new species showed clear differences from other most morphologically similar species, such as P. thornei and P. mediterraneus. Preliminary host range tests revealed that chickpea, pea, faba bean and durum wheat are good hosts of P. lentis n. sp., whereas common bean, alfalfa and barley are less robust hosts and tomato, bell pepper, eggplant, melon and sunflower are poor hosts for the nematode.
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Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm(2) show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.
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Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos/genética , Hormona Paratiroidea/administración & dosificación , Hormona Paratiroidea/farmacología , Animales , Huesos/anatomía & histología , Huesos/citología , Cartílago/efectos de los fármacos , Recuento de Células , Humanos , Inmunohistoquímica , Masculino , Osteocitos/citología , Osteocitos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Meloidogyne mayaguensis is a damaging root-knot nematode able to reproduce on root-knot nematode-resistant tomato and other economically important crops. In a growth chamber experiment conducted at 22 and 33 degrees C, isolate 1 of M. mayaguensis reproduced at both temperatures on the Mi-1-carrying tomato lines BHN 543 and BHN 585, whereas M. incognita race 4 failed to reproduce at 22 degrees C, but reproduced well at 33 degrees C. These results were confirmed in another experiment at 26 +/- 1.8 degrees C, where minimal or no reproduction of M. incognita race 4 was observed on the Mi-1-carrying tomato genotypes BHN 543, BHN 585, BHN 586 and 'Sanibel', whereas heavy infection and reproduction of M. mayaguensis isolate 1 occurred on these four genotypes. Seven additional Florida M. mayaguensis isolates also reproduced on resistant 'Sanibel' tomato at 26 +/- 1.8 degrees C. Isolate 3 was the most virulent, with reproduction factor (Rf) equal to 8.4, and isolate 8 was the least virulent (Rf = 2.1). At 24 degrees C, isolate 1 of M. mayaguensis also reproduced well (Rf >/= 1) and induced numerous small galls and large egg masses on the roots of root-knot nematode-resistant bell pepper 'Charleston Belle' carrying the N gene and on three root-knot nematode-resistant sweet pepper lines (9913/2, SAIS 97.9001 and SAIS 97.9008) carrying the Tabasco gene. In contrast, M. incognita race 4 failed to reproduce or reproduced poorly on these resistant pepper genotypes. The ability of M. mayaguensis isolates to overcome the resistance of tomato and pepper genotypes carrying the Mi-1, N and Tabasco genes limits the use of resistant cultivars to manage this nematode species in infested tomato and pepper fields in Florida.
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PROBLEM: Nitric oxide (NO), an important mediator of the inflammatory response, is involved in several reproductive processes including pregnancy and labor. Uterus, placenta and fetal membranes are significant sources of NO. Presently, there is no information on factors regulating NO production by fetal membranes. METHOD OF STUDY: Human fetal membranes at term gestation were cultured for 24 hr in the presence of oxytocin. The concentrations of NO metabolites nitrites in culture medium were determined by the Griess reaction. The presence of inducible nitric oxide synthase (iNOS) was determined by reverse transcriptase-polymerase chain reaction and Western blot. RESULTS: Oxytocin increased nitrite release by fetal membranes. Messenger ribonucleic acid iNOS expression was also enhanced by oxytocin. These effects were more marked in tissues obtained after labor than before labor. CONCLUSIONS: Oxytocin exerts an overall stimulatory effect on NO release by fetal membranes. This action might be of relevance in the biomolecular processes leading to parturition.
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Membranas Extraembrionarias/efectos de los fármacos , Membranas Extraembrionarias/metabolismo , Óxido Nítrico/metabolismo , Oxitocina/farmacología , Parto/metabolismo , Dimerización , Femenino , Humanos , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas de Cultivo de TejidosRESUMEN
Analysis of a genomic fragment from the plant parasitic nematode Meloidogyne artiellia revealed the presence of a gene which, in bacteria, is involved in the formation of polyglutamate capsule. Searching of various databases, including the Caenorhabditis elegans genome sequence and the large EST datasets from a variety of parasitic nematodes, showed that no similar genes have been identified in other nematodes or in any other eukaryotic organisms. The M. artiellia gene has a typical eukaryotic structure and its mRNA is present in the intestine. The gene is expressed in all life cycle stages tested. These findings demonstrate horizontal gene transfer may be important in catalyzing the diversification of nematode lineages.
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Transferencia de Gen Horizontal , Genes Bacterianos , Genes de Helminto , Ácido Poliglutámico/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Tylenchoidea/genética , Secuencia de Aminoácidos , Animales , Exones , Genoma , Hibridación in Situ , Mucosa Intestinal/metabolismo , Intestinos/enzimología , Intrones , Estadios del Ciclo de Vida , Datos de Secuencia Molecular , ARN de Helminto/genética , ARN de Helminto/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Tylenchoidea/enzimología , Tylenchoidea/crecimiento & desarrolloRESUMEN
Although the presence of chitin in nematodes is well documented little is known about its synthesis in this phyletic group. The recently completed genome sequence of Caenorhabditis elegans predicts two sequences with homology to chitin synthases (chitin-UDP acetyl-glucosaminyl transferase; EC 2.4.1.16). We show that these genes are differentially expressed in a pattern that may reflect different functional roles. One gene is expressed predominantly in the adult hermaphrodite (the main egg-producing stage in the nematode) and later larval stages, which is consistent with a role in production of chitin for the eggshell. The other gene, however, is expressed in the cells that form the pharynx, and only in the period directly preceding a moult. These data suggest that the product of this gene is involved in synthesis of the feeding apparatus, which is replaced during each moult. We have also isolated a full-length genomic sequence of a chitin synthase orthologue from the plant parasitic nematode Meloidogyne artiellia. The single gene present in M. artiellia shows an expression pattern that is consistent with a role for the protein in production of the eggshell.
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Quitina Sintasa/genética , Nematodos/genética , Plantas/parasitología , Animales , Southern Blotting , ADN de Helmintos , Regulación Enzimológica de la Expresión Génica , Nematodos/enzimología , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r = 0.993, P < 0.001). Similar ML levels were measured in drug-induced apoptotic cells (A approximately 30-40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter < or = 1.0 microm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, 'biochemically active' phase of programmed cell death.
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Citoplasma/metabolismo , Lípidos/análisis , Linfocitos T/metabolismo , Apoptosis , Colorantes Fluorescentes , Técnica de Fractura por Congelación , Humanos , Ionomicina , Células Jurkat , Activación de Linfocitos , Espectroscopía de Resonancia Magnética , Microscopía Electrónica , Oxazinas , Linfocitos T/ultraestructura , Acetato de TetradecanoilforbolRESUMEN
Severe infections of white mulberry feeder roots and heavy soil infestations by Meloidogyne arenaria race 2 were found in southern Spain. This is the first record of M. arenaria on white mulberry in Europe. Morphometric observations, analysis of the esterase electrophoretic pattern, and artificial inoculations of race differentials were used to characterize nematodes. Nematode-induced mature galls were spherical and usually contained one or more females, males, and egg masses with eggs. Feeding sites were characterized by the development of giant cells that contained granular cytoplasm and many hypertrophied nuclei. Giant cell cytoplasm was aggregated along a thickened cell wall. Vascular tissues within galls appeared disorganized. The relationship between the initial nematode population density (Pi) in a series from 0 to 1,024 eggs and juveniles/cm3 soil and growth of white mulberry seedlings was tested in the greenhouse. A Seinhorst model was fitted to plant height and top fresh weight. Tolerance limits of white mulberry to M. arenaria race 2 for plant height and top fresh weight were, respectively, 1.1 and 1.38 eggs and juveniles/cm3 soil. The minimum relative values for plant height and top fresh weight were 0 at Pi ≥ 64 and Pi ≥ 128 eggs and juveniles/cm3 soil, respectively. Maximum nematode reproduction rate was 435-fold at the lowest Pi.
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This paper reports the results of 31P and 1H nuclear magnetic resonance (NMR) studies on the uptake and phosphorylation of 3'-azido-3'-deoxythymidine (AZT) in the human CD4+ T-lymphoblastoid cell line CCRF-CEM (CEM-1.3) and in its AZT-resistant cell variant MT-500, isolated by prolonged culturing of CEM cells in the presence of increasing AZT concentrations. After 3 hr of incubation in the presence of 0.5 mM AZT, both AZT and its monophosphorylated form (AZT-MP) could be detected in the sensitive cell line in concentrations above the NMR detection levels. In another cell line, MOLT-4, which is less sensitive to AZT effects, the intracellular level of AZT-MP was much lower and was only slightly raised by increasing the concentration of AZT in the extracellular and intracellular compartments. In the AZT-resistant clone MT-500, characterized by a very low thymidine kinase (TK, EC 2.7.1.21) activity with respect to the parental clone, the intracellular AZT-MP concentration was below detection (<0.02 nmol/10(6) cells). Since, however, not only AZT-MP but also AZT signals failed to be detected in MT-500 extracts following cell incubation with AZT, it was concluded that a TK deficiency cannot be the exclusive mechanism of AZT resistance in these cells. The possible effects of additional mechanisms of drug resistance, such as specific AZT cell extrusion and limited permeation, are discussed, together with the new prospects offered by NMR spectroscopy to further evaluate the limiting steps for the utilization of antiretroviral nucleoside analogues.
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Fármacos Anti-VIH/metabolismo , Linfocitos T/metabolismo , Zidovudina/metabolismo , Línea Celular , Humanos , Espectroscopía de Resonancia Magnética , Fosforilación , Timidina Quinasa/genéticaRESUMEN
The cut-1 gene coding for cuticlin-1 has been isolated from the plant parasitic nematode Meloidogyne artiellia. The sequence of the cut-1 gene was compared with the corresponding sequence from the free-living nematode Caenorhabditis elegans. The high degree of similarity between the amino acid sequences, together with the occurrence of characteristic sequence motifs, indicates that the cuticlin-1 is a non-collagenous component of the cuticle also in plant parasitic nematodes. Studies on the expression pattern during the development of M. artiellia indicate that there is a burst of expression of this gene during moulting. Then, the expression rate is reduced in the infective juveniles, which migrate in the soil. In the sedentary females, in contrast, no expression is detected, while in the males which move freely through the soil, the gene is expressed and the transcript fully processed. These data strongly suggest that the gene is developmentally regulated. It is proposed that the production of cuticlin plays an important role in determining the mechanical properties of the cuticle. Furthermore, evidence is provided to indicate that the modulation of cut-1 expression is achieved by regulation of the cis-splicing mechanism in the infective second-stage juvenile.
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Proteínas de Caenorhabditis elegans , Regulación de la Expresión Génica , Proteínas del Helminto/genética , Empalme del ARN/genética , Tylenchoidea/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Clonación Molecular , ADN de Helmintos , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes de Helminto , Masculino , Datos de Secuencia Molecular , ARN de Helminto/genética , Triticum/parasitologíaRESUMEN
Some of the main experimental requirements limiting, till now, the clinical development of nuclear magnetic resonance (NMR) spectroscopy are discussed, with particular attention to the scarce sensitivity, low resolution and problems of spatial localization. A concise description is then given of the principal biological compounds which can be studied by this peculiar type of spectroscopy, together with the main categories of biochemical information that can be extracted from their detection, i.e. especially at the level of intermediate and energetic metabolism. In conclusion, some of the more outstanding examples selected from human clinical studies are depicted, particularly emphasizing those focused on the skeletal muscle system.
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Espectroscopía de Resonancia Magnética/métodos , HumanosRESUMEN
The first part of this paper is devoted to discuss the necessity of using time-varying magnetic fields, with frequencies up to a few kHz, during NMR tomographic and spectroscopic clinical examinations. It is then shown that these magnetic fields induce in the biological systems an electric field, with associated current densities, whose intensities span from physiological currents to currents able to depolarize the axonal membrane. Therefore particular emphasis is given to the involvement of excitable tissues, which can produce biological responses of variable intensity, from the less hazardous, such as magnetophosphenes, to muscle twitches, to more serious phenomena, such as extrasistoles and ventricular fibrillation. The thresholds above which these effects become physiologically relevant depend upon the different conditions and modalities of stimulus administration. Regarding weak electric currents, with their associated biological effects, the more important mechanisms of action at present hypothesized, along with some better studied experimental models, have been shortly outlined.
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Imagen por Resonancia Magnética , Campos Electromagnéticos , Humanos , Factores de TiempoRESUMEN
31P NMR studies were carried out on the parental drug-sensitive human T-lymphoblastoid cell line CCRI-CEM (CEM) and its multi-drug-resistant (MDR) CEM-VBL100 variants, to assess the role of the pentose phosphate (PP) in MDR expression. CEM and CEM-VBL100 were incubated in the presence of 2-deoxyglucose, as recently proposed by our group (Clin. Chim. Acta 208: 39, 1992). Accumulation of 2-deoxyglucose 6-phosphate was much lower in the drug-resistant than in sensitive cells, indicating PP shunt activation in the MDR variants. This result was confirmed by enzymatic analyses, which demonstrated that, with respect to the parental line, the MDR variant was characterized by a) unaltered hexokinase activity; b) higher glucose 6-phosphate dehydrogenase activity; c) increased levels of reduced glutathione and marked increase of glutathione peroxidase activity after cell exposure to an oxidizing agent (tert-butylhydroperoxide). These results support the view that cell detoxification mechanisms mediated by the pentose phosphate pathway may contribute to the expression of MDR in tumours.
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Resistencia a Medicamentos/fisiología , Glucosa-6-Fosfato/análogos & derivados , Vía de Pentosa Fosfato , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Northern Blotting , Proteínas Portadoras/análisis , Proteínas Portadoras/biosíntesis , Dactinomicina/toxicidad , Doxorrubicina/toxicidad , Resistencia a Medicamentos/genética , Variación Genética , Glucofosfatos/metabolismo , Humanos , Cinética , Espectroscopía de Resonancia Magnética/métodos , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/biosíntesis , Fósforo , Leucemia-Linfoma Linfoblástico de Células Precursoras , Linfocitos T , Células Tumorales Cultivadas , Vinblastina/toxicidadRESUMEN
The flux of 13C-labeled glucose through the Embden-Meyerhof and pentose phosphate pathways was studied by 13C NMR in intact erythrocytes isolated from normal subjects or from patients suffering of glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) deficiency. Similar rates of glucose catabolism and similar fluxes of the 13C-label into 2,3-bisphosphoglycerate and lactate were found, under basal conditions, in normal and in G6PD-deficient erythrocytes incubated in the presence of either [1-13C]- or D[6-13C]glucose. Exposure to oxidative stress by preincubation with tert-butylhydroperoxide induced in normal, but not in G6PD-deficient erythrocytes, a significant enhancement of glucose consumption, as well as a substantial reduction in 13C-label transfer from C1-glucose into lactate. It was also possible, by 31P NMR, to evaluate the conversion of 2-deoxyglucose to its phosphate-containing metabolites. The oxidation and subsequent decarboxylation of 2-deoxyglucose-6-phosphate was assessed in reconstituted systems and could subsequently be evidenced also in ethanolic extracts from normal (but not from G6PD-deficient) erythrocytes which had been exposed to oxidative stress. The results indicate that, in terms of glucose flux through the glycolytic pathway, there is little or no difference between normal and G6PD-deficient erythrocytes, regardless of previous exposure to oxidative stress. Faster consumption of either glucose or 2-deoxyglucose is induced, only in normal cells, by treatment with tert-butylhydroperoxide, essentially as a consequence of the activation of the pentose-phosphate pathway.