RESUMEN
The formation of co-crystals by the assembly of molecules with complementary molecular recognition functionalities is a popular strategy to design or improve a range of solid-state properties, including those relevant for pharmaceuticals, photo- or thermoresponsive materials and organic electronics. Here, we report halogen-bonded co-crystals of a fluorinated azobenzene derivative with a volatile component-either dioxane or pyrazine-that can be cut, carved or engraved with low-power visible light. This cold photo-carving process is enabled by the co-crystallization of a light-absorbing azo dye with a volatile component, which gives rise to materials that can be selectively disassembled with micrometre precision using low-power, non-burning laser irradiation or a commercial confocal microscope. The ability to shape co-crystals in three dimensions using laser powers of 0.5-20 mW-substantially lower than those used for metals, ceramics or polymers-is rationalized by photo-carving that targets the disruption of weak supramolecular interactions, rather than the covalent bonds or ionic structures targeted by conventional laser beam or focused ion beam machining processes.
Asunto(s)
Halógenos , Luz , Cristalización , Electrónica , Halógenos/química , Polímeros/químicaRESUMEN
RATIONALE: The decline in estrogen concentrations in women after menopause can contribute to health related changes including impairments in cognition, especially memory. Because of the health concerns related to hormone replacement therapy (HRT), alternative approaches to treat menopausal symptoms, such as nutritional supplements and/or diet containing isoflavones, are of interest. OBJECTIVES: This study investigated whether soy isoflavones (soy milk and supplement) could improve cognitive functioning in healthy, postmenopausal women. PARTICIPANTS, INTERVENTION AND DESIGN: A total of 79 postmenopausal women, 48-65 years of age, completed a double-blind, placebo-controlled trial in which they were randomly assigned to one of three experimental groups: cow's milk and a placebo supplement (control); soy milk and placebo supplement (soy milk, 72 mg isoflavones/day); or cow's milk and isoflavone supplement (isoflavone supplement, 70 mg isoflavones/day). MEASUREMENTS: Cognitive functioning was assessed using various cognitive tasks before the intervention (baseline) and after the intervention (test). RESULTS: In contrast to predictions, soy isoflavones did not improve selective attention (Stroop task), visual long-term memory (pattern recognition), short-term visuospatial memory (Benton Visual Retention Test), or visuo-spatial working memory (color match task). Also, the soy milk group showed a decline in verbal working memory (Digit Ordering Task) compared to the soy supplement and control groups. CONCLUSION: Soy isoflavones consumed as a food or supplement over a 16-week period did not improve or appreciably affect cognitive functioning in healthy, postmenopausal women.
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Envejecimiento/psicología , Cognición/efectos de los fármacos , Cognición/fisiología , Isoflavonas/administración & dosificación , Recuerdo Mental/efectos de los fármacos , Leche de Soja , Anciano , Suplementos Dietéticos , Método Doble Ciego , Femenino , Humanos , Isoflavonas/farmacología , Memoria/efectos de los fármacos , Memoria/fisiología , Recuerdo Mental/fisiología , Persona de Mediana Edad , Posmenopausia , Escalas de Valoración PsiquiátricaRESUMEN
Trial 24 is one of three placebo-controlled trials within the ongoing bicalutamide ('Casodex') Early Prostate Cancer (EPC) programme evaluating bicalutamide 150 mg/day in addition to radical prostatectomy, radiotherapy or watchful waiting for T1b-4, any N, M0 prostate cancer. In Trial 24, at 5.1 y median follow-up, the addition of bicalutamide significantly (P < 0.0001) improved objective progression-free survival (PFS) and prostate-specific antigen PFS compared with standard care alone. There was no significant difference in overall survival (P = 0.746). In the context of the whole EPC programme, long-term bicalutamide is not appropriate for localised disease, yet provides advantages in delaying disease progression in patients with locally advanced prostate cancer.
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Anilidas/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anilidas/administración & dosificación , Anilidas/efectos adversos , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Método Doble Ciego , Humanos , Masculino , Persona de Mediana Edad , Nitrilos , Placebos , Prostatectomía , Neoplasias de la Próstata/radioterapia , Neoplasias de la Próstata/cirugía , Análisis de Supervivencia , Compuestos de Tosilo , Resultado del TratamientoRESUMEN
A 12-year-old boy presented with left shoulder pain during physical exercise and complained of uncommon sweating and fatigue. Diagnostic evaluation revealed a solitary pulmonary nodule in the left upper lobe. All laboratory values were within normal limits, except for an elevated level of antineutrophil cytoplasmic antibodies directed against myeloperoxidase (p-ANCA). Surgery was performed, and pathological examination showed a localized granulomatous vasculitis. Antineutrophil cytoplasmic antibodies directed against affinity purified proteinase 3 (p-ANCA) concentrations returned to baseline within 6 months, and the patient has done well during a follow-up period of 2 years. While nodular vasculitis is known to occur in Wegener's granulomatosis, to the best of our knowledge, this case represents the first c-ANCA negative primary pulmonary vasculitis in childhood.
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Enfermedades Pulmonares/diagnóstico , Nódulo Pulmonar Solitario/diagnóstico , Vasculitis/diagnóstico , Anticuerpos Anticitoplasma de Neutrófilos/metabolismo , Niño , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Humanos , Enfermedades Pulmonares/cirugía , Masculino , Nódulo Pulmonar Solitario/cirugía , Toracotomía , Tomografía Computarizada por Rayos XRESUMEN
Thrombembolic complications,which include the fat embolism syndrome, are well-known consequences of cementless and cemented femoral total hip replacement. Thrombembolic phenomena have been demonstrated in clinical and experimental situations with both these fixation techniques, but so far no exact quantification of the intravasated fat emboli has been performed. In a standardized animal model in 15 Merino sheep we investigated the intravasation of fat into the bloodstream during simultaneous bilateral prosthetic implantation (cemented versus cementless). After identical preparation of the intramedullary canal on both sides, a cement restrictor was additionally inserted on the cemented side and the canal was cleaned by 250 ml jet lavage. Catheters in the external iliac veins made it possible to collect the drained blood in two phases, after preparation of the intramedullary canal and during insertion of the prosthesis, and the fat content of these blood samples was measured. The amount of fat that passed into the venous draining system of the femur induced by cemented implantation (2.2749 g; S=+/-1.0079) was twice the amount seen with cementless implantation (1.1586 g; S=+/-0.4555) ( P=0.0002). An obvious effect of the canal preparation was recognizable with the cemented implantation, 8 of the 13 animals evaluated showing a peak in the fat intravasation caused by application of the cement restrictor. Our results emphasize the importance of a thorough preparation of the intramedullary canal, particularly when cemented fixation is performed. The jet lavage,which should be considered mandatory standard in cemented total hip arthroplasty, should be implemented before the insertion of the cement restrictor in order to further reduce the risk of fat embolism.
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Artroplastia de Reemplazo de Cadera/efectos adversos , Cementos para Huesos/uso terapéutico , Embolia Grasa/etiología , Animales , Cementos para Huesos/efectos adversos , Femenino , Factores de Riesgo , Ovinos , Irrigación TerapéuticaRESUMEN
An optical sensor system based on evanescent field excitation of fluorophore-labeled DNA-targets specifically binding to immobilized DNA probes has been developed, thus enabling for real-time analysis of hybridization events. Oligonucleotide probes are directly immobilized on the surface of the disposable sensor chip via biotin/neutravidin linkage and hybridize to complementary Cy5-labeled target DNA in the sample; this is recorded as an increase in the fluorescence signal. Under optimized conditions the hybridization rate was constant and directly proportional to the target concentration. When an 18mer oligonucleotide was used as a probe a linear calibration curve was obtained for a 56mer single-stranded DNA target derived from the neomycin phosphotransferase gene, a selection marker in a variety of genetically modified plants, with an estimated lower limit of detection of 0.21 nmol L(-1). No cross-hybridization to a 51mer actin DNA target was observed and even a single-nucleotide mismatch led to a negligible signal. A shutter in the readout device enabled separate detection of targets hybridizing to probes immobilized at the inlet and outlet sides, respectively, of the flow channel. This opens a route toward a real-time DNA array format with analysis times as short as 1-2 min. As a realistic sample a Cy5-labeled 56 bp PCR product was measured after separation of the double-stranded DNA by simple heat denaturation with a detection limit clearly lower than that of traditional gel electrophoresis.
Asunto(s)
Técnicas Biosensibles/instrumentación , ADN/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Actinas/genética , Técnicas Biosensibles/métodos , Calibración , Sondas de ADN , ADN de Cadena Simple/análisis , Colorantes Fluorescentes , Humanos , Kanamicina Quinasa/genética , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: To investigate the efficacy and tolerability of bicalutamide (Casodex) as immediate therapy, either alone or as adjuvant to treatment of curative intent, in patients with localized or locally advanced (T1b-T4, any nodal status, M0) prostate cancer. METHODS: This was a multicenter, prospective, randomized, double-blind, placebo-controlled trial in Europe, South Africa, Australia, and Mexico and is part of the Casodex Early Prostate Cancer program. RESULTS: A total of 3603 men were randomized to receive bicalutamide (n = 1798) or placebo (n = 1805). The patient demographics were well balanced between the two groups. Prior therapy of curative intent had been given to 64% of the patients (prostatectomy [44%], radiotherapy [18%], and prostatectomy and radiotherapy [2%]) and 36% had been monitored with watchful waiting. After a median follow-up of 2.6 years and a median exposure to the study drug of 2.2 years, a significant 43% reduction in the risk of objective progression was observed for the bicalutamide group compared with the placebo group (hazard ratio 0.57, 95% confidence interval 0.48 to 0.69, P << 0.0001). The time to prostate-specific antigen doubling was significantly delayed for the bicalutamide group compared with the placebo group (hazard ratio 0.37, 95% confidence interval 0.32 to 0.43, P << 0.001). The survival data were immature, with 7.2% overall mortality. The most frequently reported adverse events with bicalutamide were gynecomastia alone (17.4%), breast pain alone (17.6%), and gynecomastia with breast pain (47.5%). CONCLUSIONS: Bicalutamide 150 mg daily as immediate therapy, alone or as adjuvant to treatment of curative intent, significantly reduced the risk of disease progression in patients with localized or locally advanced prostate cancer. Longer follow-up is underway to assess any benefit in overall survival.
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Antagonistas de Andrógenos/uso terapéutico , Anilidas/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anilidas/efectos adversos , Biomarcadores de Tumor/sangre , Intervalos de Confianza , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Método Doble Ciego , Estudios de Seguimiento , Ginecomastia/inducido químicamente , Humanos , Masculino , Persona de Mediana Edad , Nitrilos , Dolor/inducido químicamente , Estudios Prospectivos , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/mortalidad , Tasa de Supervivencia , Compuestos de TosiloRESUMEN
Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.
Asunto(s)
Técnicas Biosensibles/métodos , Peróxido de Hidrógeno/análisis , Adsorción , Secuencia de Bases , Técnicas Biosensibles/instrumentación , Cristalización , Cartilla de ADN/genética , Enzimas Inmovilizadas/química , Oro , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Horseradish peroxidase is one of the most widely used marker enzymes in immunoassays. Several disadvantages are encountered upon chemical conjugation of peroxidase with antibodies or antigens, as are low reproducibility and undefined stoichiometry. We here describe for the first time the production of a recombinant fusion of a protein analyte with horseradish peroxidase in Escherichia coli, employing refolding of inclusion bodies and reconstitution with heme. The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and defined structure. As a protein analyte, the human heart fatty acid binding protein (H-FABP) was chosen, which is a new and sensitive marker for acute myocardial infarction. The recombinant conjugate was fully active [650 U/mg with 2,2-azino-bis(3-ethyl-thiazoline-6-sulfonate) as substrate] and obtained in a yield of 12 mg/L of E. coli culture, which is better than that for recombinant peroxidase alone. The competitive immunoassay that was developed with the recombinant conjugate requires fewer incubation steps than the traditional sandwich ELISA format. It permitted the detection of H-FABP directly in plasma in the range of 10-1500 ng/mL which is the relevant range for clinical decision-making.
Asunto(s)
Peroxidasa de Rábano Silvestre/química , Inmunoensayo/métodos , Proteínas de Neoplasias , Proteínas Recombinantes de Fusión/química , Proteínas Supresoras de Tumor , Biomarcadores/análisis , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Peroxidasa de Rábano Silvestre/genética , Humanos , Infarto del Miocardio/diagnóstico , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Peroxisome proliferator-activated receptor alpha (PPARalpha) is a key regulator of lipid homeostasis in hepatocytes and target for fatty acids and hypolipidemic drugs. How these signaling molecules reach the nuclear receptor is not known; however, similarities in ligand specificity suggest the liver fatty acid binding protein (L-FABP) as a possible candidate. In localization studies using laser-scanning microscopy, we show that L-FABP and PPARalpha colocalize in the nucleus of mouse primary hepatocytes. Furthermore, we demonstrate by pull-down assay and immunocoprecipitation that L-FABP interacts directly with PPARalpha. In a cell biological approach with the aid of a mammalian two-hybrid system, we provide evidence that L-FABP interacts with PPARalpha and PPARgamma but not with PPARbeta and retinoid X receptor-alpha by protein-protein contacts. In addition, we demonstrate that the observed interaction of both proteins is independent of ligand binding. Final and quantitative proof for L-FABP mediation was obtained in transactivation assays upon incubation of transiently and stably transfected HepG2 cells with saturated, monounsaturated, and polyunsaturated fatty acids as well as with hypolipidemic drugs. With all ligands applied, we observed strict correlation of PPARalpha and PPARgamma transactivation with intracellular concentrations of L-FABP. This correlation constitutes a nucleus-directed signaling by fatty acids and hypolipidemic drugs where L-FABP acts as a cytosolic gateway for these PPARalpha and PPARgamma agonists. Thus, L-FABP and the respective PPARs could serve as targets for nutrients and drugs to affect expression of PPAR-sensitive genes.
Asunto(s)
Proteínas Portadoras/fisiología , Núcleo Celular/metabolismo , Ácidos Grasos/fisiología , Regulación de la Expresión Génica , Hipolipemiantes/farmacología , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor , Animales , Línea Celular , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Humanos , Hígado/metabolismo , Ratones , Unión Proteica , Transducción de Señal , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Expression of brain fatty acid-binding protein (B-FABP) is spatially and temporally correlated with neuronal differentiation during brain development. Isothermal titration calorimetry demonstrates that recombinant human B-FABP clearly exhibits high affinity for the polyunsaturated n-3 fatty acids alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, and for monounsaturated n-9 oleic acid (K(d) from 28 to 53 nm) over polyunsaturated n-6 fatty acids, linoleic acid, and arachidonic acid (K(d) from 115 to 206 nm). B-FABP has low binding affinity for saturated long chain fatty acids. The three-dimensional structure of recombinant human B-FABP in complex with oleic acid shows that the oleic acid hydrocarbon tail assumes a "U-shaped" conformation, whereas in the complex with docosahexaenoic acid the hydrocarbon tail adopts a helical conformation. A comparison of the three-dimensional structures and binding properties of human B-FABP with other homologous FABPs, indicates that the binding specificity is in part the result of nonconserved amino acid Phe(104), which interacts with double bonds present in the lipid hydrocarbon tail. In this context, analysis of the primary and tertiary structures of human B-FABP provides a rationale for its high affinity and specificity for polyunsaturated fatty acids. The expression of B-FABP in glial cells and its high affinity for docosahexaenoic acid, which is known to be an important component of neuronal membranes, points toward a role for B-FABP in supplying brain abundant fatty acids to the developing neuron.
Asunto(s)
Química Encefálica , Proteínas Portadoras/química , Proteína P2 de Mielina/química , Proteínas de Neoplasias , Proteínas Supresoras de Tumor , Secuencia de Bases , Calorimetría , Proteínas Portadoras/genética , Clonación Molecular , Cristalografía por Rayos X , Cartilla de ADN , Proteína 3 de Unión a Ácidos Grasos , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Humanos , Modelos Moleculares , Proteína P2 de Mielina/genética , Conformación Proteica , TermodinámicaRESUMEN
Liver-type fatty acid binding protein (L-FABP) has been proposed to be involved in the transport of fatty acids and peroxisome proliferators from the cytosol into the nucleus for interaction with the peroxisome proliferator-activated receptors (PPARs). On the basis of this premise, we investigated by isothermal titration calorimetry the binding of myristic, stearic, oleic, and docosahexaenoic acids to three orthologous L-FABPs and compared these results to those obtained for several xenobiotics [Wy14,643, bezafibrate, 5,8,11,14-eicosatetraynoic acid (ETYA), and BRL48,482] known for their peroxisome proliferating activity in rodents. Recombinant human, murine, and bovine L-FABPs were analyzed and the thermodynamic data were obtained. Our studies showed that fatty acids bound with a stoichiometry of 2:1, fatty acid to protein, with dissociation constants for the first binding site in the nanomolar range. With dissociation constants above 1 microM the drug peroxisome proliferators showed weaker binding, with the exception of arachidonate analogue ETYA, which bound with a similar affinity as the natural fatty acid. Some of the thermodynamic data obtained for fatty acid binding could be explained by differences in protein structure. Moreover, our results revealed that binding affinities were not determined by ligand solubility in the aqueous phase.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Hígado/metabolismo , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Proliferadores de Peroxisomas/metabolismo , Proteínas Supresoras de Tumor , Ácido 5,8,11,14-Eicosatetrainoico/metabolismo , Secuencia de Aminoácidos , Animales , Bezafibrato/química , Bezafibrato/metabolismo , Calorimetría , Proteínas Portadoras/química , Bovinos , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Ácidos Grasos/química , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Proteína P2 de Mielina/química , Proliferadores de Peroxisomas/química , Unión Proteica , Pirimidinas/química , Pirimidinas/metabolismoRESUMEN
Clean polycrystalline gold electrodes were modified with native glycosylated horseradish peroxidases (HRP) or two different recombinant (carbohydrate free) HRPs; recombinant wild-type HRP (rec-HRP) and recombinant HRP containing a six histidine-tag at the C-terminus of the polypeptide chain (rec-HRP-His), respectively. Only the electrodes modified with the recombinant HRPs exhibited high current responses to H2O2 due to relatively rapid direct electron transfer (ET) between recombinant HRP and gold. The absence of a carbohydrate shell on rec-HRP and the additionally existing histidine-tag on rec-HRP-His improved the electrode sensitivity to H2O2 by more than 100 times if compared with the response observed at gold modified with native HRP. Rotating disk electrode experiments indicated that the heterogeneous electron transfer rates are equal to 4.7 and 7.5 s-1 for direct electron transfer between the gold electrode and rec-HRP or rec-HRP-His, respectively.
Asunto(s)
Transporte de Electrón , Oro/química , Peroxidasa de Rábano Silvestre/química , Electroquímica , Enzimas Inmovilizadas , Proteínas Recombinantes/químicaRESUMEN
The human breast cancer cell line MCF7 does not express heart-type fatty acid binding protein (H-FABP), a marker protein for differentiated mammary gland. MCF7 cells transfected with the bovine H-FABP cDNA expressed the corresponding protein and were characterized by growth inhibition and lower tumorgenicity in nude mice [22]. By enzyme linked immunoassay we now determined the amount of bovine H-FABP in these cells as 638 +/- 80 ng/mg protein and used the transfected cells to study the role of H-FABP in fatty acid metabolism. Compared to control cells the uptake of radioactively labelled palmitic acid and oleic acid into MCF7 cells after 30 or 60 min was increased by 67% in H-FABP expressing transfectants, demonstrating a stimulatory role for this FABP-type in fatty acid metabolism. However, preferential targeting of [14C]oleic acid into neutral or phospholipid classes was not observed by the criterion of high performance thin layer chromatography followed by autoradiography. A reason for the modest increase of fatty acid uptake in H-FABP transfected MCF7 cells may be the basal expression of epidermal-type FABP, which was detected for the first time in these cells. It appears that the small amount of E-FABP expressed in MCF7 cells fulfils the need of the cells for a cytosolic fatty acid carrier under culture conditions and that even high concentrations of another FABP do only slightly increase the uptake due to limitations of fatty acid transport through the plasma membrane or of metabolism.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas Portadoras/genética , Ácidos Grasos/metabolismo , Proteína P2 de Mielina/genética , Proteínas de Neoplasias , Proteínas Supresoras de Tumor , Animales , Radioisótopos de Carbono , Proteínas Portadoras/metabolismo , Bovinos , Ensayo de Inmunoadsorción Enzimática , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Femenino , Humanos , Proteína P2 de Mielina/metabolismo , Miocardio/metabolismo , Ácido Oléico/metabolismo , Ácido Palmítico/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
We describe the crystal structure of human epidermal-type fatty acid binding protein (E-FABP) that was recently found to be highly upregulated in human psoriatic keratinocytes. To characterize E-FABP with respect to ligand-binding properties and tertiary structure, we cloned the respective cDNA, overexpressed the protein in Escherichia coli and purified it to homogeneity by a combination of ion-exchange and size-exclusion chromatographic steps with a yield of 30 mg/L broth. The purified protein revealed a 5-fold higher affinity for stearic acid than for oleic and arachidonic acids. The crystal structure of recombinant human E-FABP was determined to 2.05 A and refined to an R(factor) of 20.7%. The initial residual electron density maps clearly showed the presence of a ligand, which was identified as endogenous bacterial fatty acid. Within a central cavity of 252 A(3), this ligand is bound in a U-shaped conformation, its carboxyl group interacting with tyrosine 131 and arginines 129 and 109, the latter via an ordered water molecule. The E-FABP crystal structure is unique in the FABP family because of the presence of a disulfide bridge between cysteines 120 and 127 that may be physiologically as well as pathophysiologically relevant. Cysteines 67 and 87 are also in close vicinity but in contrast do not form a disulfide bridge. We postulate that this protein belongs to a particular FABP subfamily whose members share common structural as well as functional features.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Ácidos Grasos/metabolismo , Proteína P2 de Mielina/biosíntesis , Proteína P2 de Mielina/química , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Supresoras de Tumor , Tejido Adiposo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Bovinos , Cristalización , Cristalografía por Rayos X , Epidermis , Escherichia coli/genética , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteína P2 de Mielina/genética , Proteína P2 de Mielina/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Piel , Relación Estructura-ActividadRESUMEN
This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.
Asunto(s)
Análisis de los Alimentos , Ingeniería Genética , Glycine max/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Zea mays/genética , Agrobacterium tumefaciens/genética , Caulimovirus/genética , Electroforesis en Gel de Poliacrilamida , Reacciones Falso Negativas , Reacciones Falso Positivas , Regiones Promotoras Genéticas , Regiones Terminadoras GenéticasRESUMEN
Branched-chain phytanic acid is metabolized in liver peroxisomes. Sterol carrier protein 2/sterol carrier protein x (SCP2/SCPx) knockout mice, which develop a phenotype with a deficiency in phytanic acid degradation, accumulate dramatically high concentrations of this fatty acid in serum (Seedorf at al. 1998. Genes Dev. 12: 1189-1201) and liver. Concomitantly, a 6.9-fold induction of liver fatty acid binding protein (L-FABP) expression is observed in comparison to wild-type animals fed standard chow, possibly mediated by the peroxisome proliferator-activated receptor alpha (PPARalpha). Cytosolic transport of phytanic acid to either peroxisomal membranes or to the nucleus for activation of PPARalpha may be mediated by L-FABP, which gives rise to the question whether phytanic acid is a transactivator of this protein. Here we show first that phytanic acid binds to recombinant L-FABP with high affinity. Then the increase of the in vivo phytanic acid concentration by phytol feeding to mice results in a 4-fold induction of L-FABP expression in liver, which is in the order of that attained with bezafibrate, a known peroxisome proliferator. Finally to test in vitro whether this induction is conferred by phytanic acid, we cotransfected HepG2 cells with an expression plasmid for murine PPARalpha and a CAT-reporter gene with 176 bp of the murine L-FABP promoter, containing the peroxisome proliferator responsive element (PPRE). After incubation with phytanic acid, we observed a 3.2-fold induction of CAT expression. These findings, both in vivo and in vitro, demonstrate that phytanic acid is a transcriptional activator of L-FABP expression and that this effect is mediated via PPARalpha.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteína P2 de Mielina/genética , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Ácido Fitánico/metabolismo , Ácido Fitánico/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Bezafibrato/farmacología , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , ADN Complementario , Escherichia coli/genética , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Expresión Génica/efectos de los fármacos , Hipolipemiantes/farmacología , Hígado/metabolismo , Ratones , Fitol/farmacología , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Activación Transcripcional , TransfecciónRESUMEN
The liver-type fatty acid binding protein (L-FABP), a member of a family of mostly cytosolic 14-15 kDa proteins known to bind fatty acids in vitro and in vivo, is discussed to play a role in fatty acid uptake. Cells of the hepatoma HepG2 cell line endogenously express this protein to approximately 0.2% of cytosolic proteins and served as a model to study the effect of L-FABP on fatty acid uptake, by manipulating L-FABP expression in two approaches. First, L-FABP content was more than doubled upon treating the cells with the potent peroxisome proliferators bezafibrate and Wy14,643 and incubation of these cells with [1-14C]oleic acid led to an increase in fatty acid uptake rate from 0.55 to 0.74 and 0.98 nmol/min per mg protein, respectively. In the second approach L-FABP expression was reduced by stable transfection with antisense L-FABP mRNA yielding seven clones with L-FABP contents ranging from 0.03% to 0.14% of cytosolic proteins. This reduction to one sixth of normal L-FABP content reduced the rate of [1-14C]oleic acid uptake from 0.55 to 0. 19 nmol/min per mg protein, i.e., by 66%. The analysis of peroxisome proliferator-treated cells and L-FABP mRNA antisense clones revealed a direct correlation between L-FABP content and fatty acid uptake.
Asunto(s)
Proteínas Portadoras/biosíntesis , Ácidos Grasos/metabolismo , Proteína P2 de Mielina/biosíntesis , Proteínas de Neoplasias , Proliferadores de Peroxisomas/farmacología , Proteínas Supresoras de Tumor , Bezafibrato/farmacología , Carcinoma Hepatocelular , Proteínas Portadoras/genética , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Humanos , Proteína P2 de Mielina/genética , Pirimidinas/farmacología , ARN sin Sentido , Transfección , Células Tumorales CultivadasRESUMEN
Due to their hydrophobic nature, free fatty acids require carriers for transport across and within the cells. The endothelial layer is the first barrier to be traversed by the fatty acids, from the plasma to the underlying cells and tissues. We tried to find out whether cytosolic fatty acid-binding proteins (FABPs) are present in the endothelium of large vessels (aortic endothelial cells) and small vessels (myocardial capillaries) using the following experimental approaches: (i) loading the delipidated aortic endothelial cell (EC) homogenate and the heart cytosolic proteins and membrane proteins with [14C]palmitate or [14C]oleate, respectively, followed by autoradiographic detection of electrophoretically separated bands; (ii) detection by immunoprecipitation of heart-type FABP (H-FABP) using an affinity-purified antibody raised against bovine H-FABP (anti-H-FABP), and (iii) localization of FABP by indirect immunofluorescence and gold-immunocytochemistry applied to cultured EC and to thick and thin frozen sections of mouse heart. The results showed that: (i) within the EC homogenate proteins that express affinity for [14C]palmitate have an apparent Mr of 15000, and 40000-45000, that correspond as molecular mass to cytosolic and membrane FABPs, respectively. Similar affinity was found by incubation with [14C]oleate, that binds to a protein of Mr 15000 in the heart cytosol, and to a 40-45 kDa protein in the membrane fraction; (ii) anti-H-FABP immunoprecipitated specifically a cytosolic 15 kDa peptide (H-FABP); (iii) by indirect immunofluorescence, cytosolic H-FABP was localized on heart microvessels and myocytes and also in cultured aortic EC where intense spotted fluorescence characteristic for cytosolic antigens was present; (iv) by immunocytochemistry, H-FABP was detected in the EC cytoplasm, and in close proximity to the cytoplasmic aspect of plasmalemma and vesicle membranes. Together the data attest the presence of the 15 kDa, heart-type FABP in the endothelium of aorta and heart microvessels.