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The initial stages of the nucleation of cubic (c-) GaN in heterophase epitaxy on a Si v-groove are investigated. Growth of GaN on a nanoscale {111}-faceted v-groove fabricated into a Si(001) substrate proceeds in the hexagonal (h-) phase that induces a secondary v-groove replicating the substrate topography with two opposing {0001} facets. The secondary v-groove is then orientationally mismatched at the junction of the h-GaN facets (h -h junction) resulting in structural instability. This instability is relieved either by the formation of voids that reduce the actual junction area or by the transition to c-phase (h-c transition) suppressing further extension of the h-h junction. The distribution of voids that is locally affected by the island growth mode of h-GaN on Si(111) and the imperfection in the groove geometry impacts the initial stage of heterophase epitaxy. Primarily, The h-c transition is observed as a non-local phenomenon; it occurs homogeneously and simultaneously along the bottom of the entire secondary groove and forms a one-dimensional (1D) seed layer except for some interruptions where the h-h junction is defected by gaps or incomplete voids. Between these interruptions, epitaxy retains a single crystal but results in a series of c-GaN nanodots on the seed layer with large fluctuation in size and spacing. The adatom incorporation observed in this heterophase epitaxy is a 1D analog to the wetting of a substrate followed by the self-assembly in conventional quantum dot epitaxy. The surface morphology of the c-GaN nanodots is governed by the faceting mostly composed of (001)- and (11n)-orientations and the roughening between these facets that ultimately affect the morphology of the final top surface of the c-III-N. The interruptions interfere with the homogeneity of the h-c transition and can cause antiphase defects and mosaicity. Based on experimental results, a solution to improve these issues is proposed.
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BACKGROUND: PET represents a valuable tool for glioma imaging. In addition to amino acid tracers such as 18F-FET, PET targeting the 18-kDa mitochondrial translocator-protein (TSPO) is of high interest for high-grade glioma (HGG) imaging due to its upregulation in HGG cells. 18F-GE-180, a novel TSPO ligand, has shown a high target-to-background contrast in HGG. Therefore, we intra-individually compared its uptake characteristics to dynamic 18F-FET PET and contrast-enhanced MRI in patients with HGG. METHODS: Twenty HGG patients (nine IDH-wildtype, 11 IDH-mutant) at initial diagnosis (n = 8) or recurrence (n = 12) were consecutively included and underwent 18F-GE-180 PET, dynamic 18F-FET PET, and MRI. The maximal tumour-to-background ratios (TBRmax) and biological tumour volumes (BTV) were evaluated in 18F-GE-180 and 18F-FET PET. Dynamic 18F-FET PET analysis included the evaluation of minimal time-to-peak (TTPmin). In MRI, the volume of contrast-enhancement was delineated (VOLCE). Volumes were spatially correlated using the Sørensen-Dice coefficient. RESULTS: The median TBRmax tended to be higher in 18F-GE-180 PET compared to 18F-FET PET [4.58 (2.33-8.95) vs 3.89 (1.56-7.15); p = 0.062] in the overall group. In subgroup analyses, IDH-wildtype gliomas showed a significantly higher median TBRmax in 18F-GE-180 PET compared to 18F-FET PET [5.45 (2.56-8.95) vs 4.06 (1.56-4.48); p = 0.008]; by contrast, no significant difference was observed in IDH-mutant gliomas [3.97 (2.33-6.81) vs 3.79 (2.01-7.15) p = 1.000]. Only 5/20 cases showed higher TBRmax in 18F-FET PET compared to 18F-GE-180 PET, all of them being IDH-mutant gliomas. No parameter in 18F-GE-180 PET correlated with TTPmin (p > 0.05 each). There was a tendency towards higher median BTVGE-180 [32.1 (0.4-236.0) ml] compared to BTVFET [19.3 (0.7-150.2) ml; p = 0.062] with a moderate spatial overlap [median Sørensen-Dice coefficient 0.55 (0.07-0.85)]. In MRI, median VOLCE [9.7 (0.1-72.5) ml] was significantly smaller than both BTVFET and BTVGE180 (p < 0.001 each), leading to a poor spatial correlation with BTVGE-180 [0.29 (0.01-0.48)] and BTVFET [0.38 (0.01-0.68)]. CONCLUSION: PET with 18F-GE-180 and 18F-FET provides differing imaging information in HGG dependent on the IDH-mutational status, with diverging spatial overlap and vast exceedance of contrast-enhancement in MRI. Combined PET imaging might reveal new insights regarding non-invasive characterization of tumour heterogeneity and might influence patients' management.
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Carbazoles , Glioma/diagnóstico por imagen , Glioma/patología , Tomografía de Emisión de Positrones/métodos , Tirosina/análogos & derivados , Adulto , Anciano , Transporte Biológico , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Carbazoles/metabolismo , Femenino , Glioma/genética , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Proyectos Piloto , Polimorfismo Genético , Trazadores Radiactivos , Receptores de GABA/genética , Carga Tumoral , Tirosina/metabolismoRESUMEN
OBJECTIVE: The 18-kDa mitochondrial translocator protein (TSPO) was reported to be upregulated in gliomas. 18F-GE-180 is a novel 3rd generation TSPO receptor ligand with improved target-to-background contrast compared to previous tracers. In this pilot study, we compared PET imaging with 18F-GE-180 and MRI of patients with untreated and recurrent pretreated glioblastoma. METHODS: Eleven patients with histologically confirmed IDH wildtype gliomas (10 glioblastomas, 1 anaplastic astrocytoma) underwent 18F-GE-180 PET at initial diagnosis or recurrence. The PET parameters mean background uptake (SUVBG), maximal tumour-to-background ratio (TBRmax) and PET volume using different thresholds (SUVBG × 1.6, 1.8 and 2.0) were evaluated in the 60-80 min p.i. summation images. The different PET volumes were compared to the contrast-enhancing tumour volume on MRI. RESULTS: All gliomas were positive on 18F-GE-180 PET and were depicted with extraordinarily high tumour-to-background contrast (median SUVBG 0.47 (0.37-0.93), TBRmax 6.61 (3.88-9.07)). 18F-GE-180 uptake could be found even in areas without contrast enhancement on MRI, leading to significantly larger PET volumes than MRI-based volumes (median 90.5, 74.5, and 63.8 mL vs. 31.0 mL; p = 0.003, 0.004, 0.013). In percentage difference, the PET volumes were on average 179%, 135%, and 90% larger than the respective MRI volumes. The median spatial volumetric correlation (Sørensen-Dice coefficient) of PET volumes and MRI volumes prior to radiotherapy was 0.48, 0.54, and 0.58. CONCLUSION: 18F-GE-180 PET provides a remarkably high tumour-to-background contrast in untreated and pretreated glioblastoma and shows tracer uptake even beyond contrast enhancement on MRI. To what extent 18F-GE-180 uptake reflects the tumour extent of human gliomas and inflammatory cells remains to be evaluated in future prospective studies with guided stereotactic biopsies and correlation of histopathological results.
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Carbazoles , Glioblastoma/diagnóstico por imagen , Glioblastoma/metabolismo , Tomografía de Emisión de Positrones , Receptores de GABA/metabolismo , Femenino , Humanos , Ligandos , Masculino , Persona de Mediana Edad , Proyectos Piloto , RecurrenciaRESUMEN
Introduction: A promising candidate in the field of pharmacological treatment options regarding major depressive disorder (MDD) is the mitochondrial translocator protein (18 kDa) (TSPO). TSPO is crucial for neurosteroid synthesis, which is in turn important for the regulation of emotions. It has already been shown that TSPO expression in platelets of depressed patients is reduced compared to healthy subjects. Methods: We measured TSPO levels in platelets of 37 depressed patients before and after 6 weeks of pharmacological treatment to test the hypotheses that i) such treatment would increase TSPO expression and ii) that this increase would be correlated with therapeutic response. Results: Surprisingly, TSPO levels in platelets of all patients were significantly reduced after 6 weeks of treatment (p=0.044). Within the responder group, a non-significant trend towards greater TSPO level reduction could be observed. Discussion: These results challenge our hypotheses that TSPO levels might increase during antidepressant therapy along with a decrease in depressive symptoms. Thus, we assume that TSPO expression in platelets is not a suitable state marker for MDD.
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Antidepresivos/uso terapéutico , Trastorno Depresivo Mayor/sangre , Trastorno Depresivo Mayor/tratamiento farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Receptores de GABA/sangre , Adulto , Análisis de Varianza , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Escalas de Valoración Psiquiátrica , Estudios Retrospectivos , Factores de TiempoRESUMEN
Citrate is one of the major substrates for intracellular metabolism. The extracellular level of citrate is stable in blood but varies locally, with slightly increased levels in brain and high levels in prostate. Recent metabolomics research suggests that citrate level is a potential harbinger of different pathophysiological states; its decrease has been correlated with male infertility, brain diseases and metastatic cancer. In this review we discuss the role of citrate as an energy substrate for sperm. We also review the function of citrate released by astrocytes in the normal operation of neurons, and consequently we suggest a potential role of neuronal plasma membrane citrate transporters in mental disorders. Finally, we review recent relevant publications studying blood, urine and tissue citrate levels in cancer patients and hypothesize that extracellular citrate supports cancer cell metabolism critical for metastasis. Despite the importance of extracellular citrate in physiological and pathophysiological processes, surprisingly little is known about citrate synthesis in specialized cells, or about citrate transporters controlling citrate movement across various membranes. Determination of the molecular origin of citrate transporters in astrocytes, sperm and cancer cells could offer novel therapeutic targets and the possibility to pharmacologically regulate citrate release and uptake for preventing male infertility, treating mental diseases and targeting cancer.
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Trastorno Bipolar/metabolismo , Ácido Cítrico/metabolismo , Demencia/metabolismo , Espacio Extracelular/metabolismo , Infertilidad Masculina/metabolismo , Neoplasias de la Próstata/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Transporte Biológico , Trastorno Bipolar/patología , Trastorno Bipolar/fisiopatología , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Membrana Celular/metabolismo , Membrana Celular/patología , Demencia/patología , Demencia/fisiopatología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Infertilidad Masculina/patología , Infertilidad Masculina/fisiopatología , Masculino , Neuronas/metabolismo , Neuronas/patología , Especificidad de Órganos , Próstata/metabolismo , Próstata/patología , Próstata/fisiopatología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/fisiopatología , Espermatozoides/metabolismo , Espermatozoides/patologíaRESUMEN
Ion-induced damage and intermixing was evaluated in InGaN/GaN multi-quantum wells (MQWs) using 35 keV N(+) implantation at room temperature. In situ ion channeling measurements show that damage builds up with a similar trend for In and Ga atoms, with a high threshold for amorphization. The extended defects induced during the implantation, basal and prismatic stacking faults, are uniformly distributed across the quantum well structure. Despite the extremely high fluences used (up to 4 × 10(16) cm(-2)), the InGaN MQWs exhibit a high stability against ion beam mixing.
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INTRODUCTION: The treatment of anxiety disorders is still a challenge; novel pharmacological approaches that combine rapid anxiolytic efficacy with fewer side effects are needed. A promising target for such compounds is the mitochondrial translocator protein (18 kDa) (TSPO). TSPO plays an important role for the synthesis of neurosteroids, known to modulate GABAA receptors, thereby exerting anxiolytic effects. METHODS: We investigated the pharmacological profile of 2 well established TSPO ligands (XBD173 and etifoxine) compared to the benzodiazepine diazepam with regard to TSPO binding affinity, TSPO expression and neurosteroidogenesis. RESULTS: In BV-2 microglia and C6 glioma cells all compounds significantly enhanced TSPO protein expression. Radioligand binding assays revealed the highest binding affinity to TSPO for XBD173, followed by diazepam and etifoxine. Pregnenolone synthesis was most potently enhanced by etifoxine. DISCUSSION: Etifoxine turned out to be the most potent enhancer of neurosteroidogenesis, although its binding affinity to TSPO was lowest. These results indicate that the efficacy of TSPO ligands to stimulate neurosteroid synthesis, thereby leading to anxiolytic effects cannot be concluded from their binding affinity to TSPO.
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Diazepam/farmacología , Neurotransmisores/biosíntesis , Oxazinas/farmacología , Purinas/farmacología , Receptores de GABA/efectos de los fármacos , Receptores de GABA/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratones , Pregnenolona/metabolismo , Ensayo de Unión Radioligante , RatasRESUMEN
To improve the biocompatibility of polyurethane (PUR), we modified the surface by irradiation with different ions (Carbon; C, Oxygen; O, Nitrogen; N, or Argon; Ar) at 0.3-50 keV energy and doses of 1,00E+13 - 1,00E+15 ions/cm(2). The effects of ion implantation using different ion energies and densities were observed on adhesion, proliferation, and viability of human umbilical vein endothelial cells (HUVECs). The long-term in vitro stability of ion-implanted PUR was also investigated. Ion irradiation moderately affected the surface roughness (R(a)), but strongly enhanced the work of adhesion (W(a)). Cell adhesion was markedly improved on O-, N-, and Ar-, but not on C-implanted PUR surfaces. Medium ion energies and lower ion doses produced the best HUVEC attachment and proliferation, indicating the importance of choosing the proper range of energy applied during ion irradiation. In addition, apoptosis rates were significantly reduced when compared with unmodified PUR (uPUR). N implantation significantly protected the surface, although C implantation led to stronger surface erosions than on uPUR. In total, ion implantation on flexible PUR surfaces strongly improved the material surface characteristics and biocompatibility. Electron beam ion implantation within an appropriate energy window is thus a key to improving flexible PUR surfaces for clinical use to support endothelial cell performance. Thus, it can contribute to designing small-diameter grafts, which are in great demand, towards vascular tissue engineering applications.
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Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Implantes Experimentales , Docilidad/efectos de los fármacos , Poliuretanos/farmacología , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Recuento de Células , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Células Endoteliales/ultraestructura , Humanos , Iones , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Propiedades de Superficie/efectos de los fármacos , TermodinámicaRESUMEN
Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase C delta 1 (PLC delta 1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P(2)/IP(3) and PI(3,4,5)P(3), the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.
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Odorantes , Neuronas Receptoras Olfatorias/metabolismo , Fosfatidilinositoles/metabolismo , Transducción de Señal , Animales , Activación Enzimática , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoinositido Fosfolipasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
The aim of this study was to develop and characterize novel metal-polymer constructs to improve the biocompatibility of flexible but hydrophobic polyurethane (PUR) implants. Using a physical vapor deposition (PVD) technique, thin films (< or =100 nm) of zirconium (Zr) or titanium (Ti) were deposited on the polyurethane surface. Both coatings displayed good stability when subjected to cross-cutting test and especially Zr showed only minor and superficial cracks in the scanning electron microscopy analysis. PVD coating resulted in significantly lowered contact angles and the standard surface free energy of wetting (Delta(wet)G degrees ) turned to more favorable negative values (Ti: -40; Zr: -30; untreated PUR (uPUR): +10.1 mN/m). This may lead to the highly enhanced adhesion and proliferation properties observed with human umbilical vein endothelial cells (HUVECs). In addition, the novel coatings had no toxic effect and even drastically reduced apoptosis rates of HUVECs. Cell morphology, nitric oxide production, and mitochondrial membrane potential--both at static and flow conditions--were superior compared with uPUR, thus demonstrating intact physiological functions. Therefore, we suggest that combining PUR as a flexible material with a thin coating of Zr or Ti as the improved biocompatible surface may have advantages for use, for example, vascular graft material.
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Adhesión Celular/fisiología , Materiales Biocompatibles Revestidos/química , Células Endoteliales/fisiología , Poliuretanos/química , Titanio/química , Circonio/química , Apoptosis/fisiología , Proliferación Celular , Forma de la Célula , Células Endoteliales/citología , Humanos , Ensayo de Materiales , Resistencia al Corte , Propiedades de SuperficieRESUMEN
Recent data suggest that the 3-phosphoinositides can modulate cyclic nucleotide signaling in rat olfactory receptor neurons (ORNs). Given the ability of diverse lipids to modulate ion channels, we asked whether phosphatidylinositol 3,4,5-trisphosphate (PIP3) can regulate the olfactory cyclic nucleotide-gated (CNG) channel as a possible mechanism for this modulation. We show that applying PIP3 to the intracellular side of inside-out patches from rat ORNs inhibits activation of the olfactory CNG channel by cAMP. The effect of PIP3 is immediate and partially reversible, and reflects an increase in the EC50 of cAMP, not a reduction in the single-channel current amplitude. The effect of PIP3 is significantly stronger than that of PIP2; other phospholipids tested have no appreciable effect on channel activity. PIP3 similarly inhibits the recombinant heteromeric (A2/A4) and homomeric (A2) olfactory CNG channel expressed in HEK293 cells, suggesting that PIP3 acts directly on the channel. These findings indicate that 3-phosphoinositides can be functionally important regulators of CNG channels.
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AMP Cíclico/metabolismo , Canales Iónicos/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Línea Celular , Expresión Génica , Humanos , Canales Iónicos/genética , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
In olfactory receptor neurons (ORNs), ligand-odorant receptor interactions cause G protein-mediated activation of adenylate cyclase and a subsequent increase in concentration of the intracellular messenger cAMP. Odorant-evoked elevation in cAMP is thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca2+ influx, leading to membrane depolarization and the generation of action potentials. Our data show that in freshly dissociated rat ORNs, odorant-induced elevation in cAMP also activates cAMP-dependent protein kinase (PKA), which is then able to phosphorylate various protein targets in the olfactory signal transduction pathway, specifically voltage-gated sodium and calcium channels. The presence of PKI (PKA inhibitor peptide) blocked the modulatory action of cAMP on voltage-gated ion channels. By modulating the input/output properties of the sensory neurons, this mechanism could take part in the complex adaptation process in odorant perception. In addition, we found modulation of voltage-gated sodium and calcium channel currents by 5-hydroxytryptamine and the dopamine D1 receptor agonist SKF 38393. These findings suggest that in situ ORNs might also be a target for efferent modulation.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Canales Iónicos/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/metabolismo , Transducción de Señal/fisiología , Olfato/fisiología , Animales , Bario/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Colforsina/farmacología , Inhibidores Enzimáticos/farmacología , Canales Iónicos/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neurotransmisores/metabolismo , Neurotransmisores/farmacología , Odorantes , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/efectos de los fármacos , Técnicas de Placa-Clamp , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/metabolismo , Receptores Odorantes/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Olfato/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismoRESUMEN
Odorant receptors (ORs) constitute the molecular basis for the detection of volatile odorous molecules and the perception of smell. Our understanding of chemical senses has been greatly expanded by the discovery of the OR gene families in vertebrates and in the nematode Caenorhabditis elegans. Recently, candidate Drosophila OR genes have been identified. The putative ORs do not possess any primary sequence identity with known vertebrate or C. elegans receptors, but belong to the family of G protein-coupled receptors according to their predicted seven transmembrane topology. To prove olfactory function of these proteins, we expressed a member of the putative Drosophila OR gene family, Or43a, in Xenopus laevis oocytes. Using two-electrode voltage-clamp recording we identified four odors (cyclohexanone, cyclohexanol, benzaldehyde, and benzyl alcohol) that activated the receptor at low micromolar concentration and structurally related substances that did not. This report shows the function and specificity of a member of the recently identified family of Drosophila ORs expressed in a heterologous system.
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Drosophila/metabolismo , Receptores Odorantes/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Femenino , Potenciales de la Membrana , Técnicas de Placa-Clamp , Receptores Odorantes/efectos de los fármacos , Receptores Odorantes/genética , Transducción de Señal , Xenopus laevisRESUMEN
We have previously shown that bacterially expressed p53 protein or p53 protein isolated from cis-diamminedichloroplatinum II (cisplatin)-damaged cells is capable of binding to double-stranded platinated DNA molecules lacking any p53 DNA binding sites. Here we report using various p53 mutants that two separate domains of p53 protein affect p53 binding to platinated DNA. Mutations within the central core of p53, the domain responsible for sequence-specific DNA binding activity, completely eliminated p53 binding to platinated DNA. Based on competition experiments p53 preferred binding to sequence-specific DNA molecules over platinated DNA molecules. However, p53 binding to platinated DNA molecules was significantly stronger than p53 interactions with DNA molecules lacking damage and a p53 consensus site. Finally, an antibody specific to the C-terminal domain of p53 (pAb421) which activates sequence-specific DNA binding activity inhibited p53 binding to platinated DNA. Taken together, these results suggest that in addition to binding to p53 DNA binding sites, p53 also interacts with cisplatin-damaged DNA molecules.
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Cisplatino/toxicidad , Daño del ADN , ADN/efectos de los fármacos , ADN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , ADN/genética , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Genes p53 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Células Tumorales CultivadasRESUMEN
Desensitization of ligand-gated ion channels shapes synaptic responses and provides critical neuroprotection at central synapses, yet the molecular mechanisms underlying the desensitization process are poorly understood. Using the whole-cell voltage-clamp technique, we investigated desensitization kinetics of recombinant human and guinea pig alpha-homomeric 5-hydroxytryptamine type 3 (5-HT(3A)) receptors heterologously expressed in human embryonic kidney 293 cells. Human 5-HT(3A) receptors desensitize 3.5 times faster than does the homologous receptor from guinea pigs. By constructing various chimeras and through site-directed mutagenesis, we have identified a single serine in the M1 region of the human 5-HT(3A) receptor sequence (S248) that, when substituted with threonine found in the equivalent guinea pig sequence (T254), conferred guinea pig-like kinetics on the time course of desensitization of the human receptor. Correspondingly, the reverse mutation (guinea pig T254S) resulted in a fast, human-like time constant of desensitization. Thus, the primary structure of the M1 region is an important determinant of desensitization kinetics of recombinant 5-HT(3A) receptors.
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Riñón/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Cobayas , Humanos , Riñón/citología , Cinética , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/fisiología , Receptores de Serotonina 5-HT3 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serotonina/metabolismo , Serotonina/farmacología , Relación Estructura-ActividadRESUMEN
1. The non-genomic effects of tetrahydrodeoxycorticosterone (THDOC; 5-alpha-pregnane-3-alpha, 21-diol-20-one) were studied in cultured hypothalamic neurons of the rat. 2. The effects of THDOC (10 nM - 1 microM) on responses to different concentrations of exogenously applied GABA and on spontaneous inhibitory postsynaptic currents (IPSCs) were measured with whole-cell voltage clamp recordings. 3. Application of GABA induced inward currents with dose-dependently increasing amplitudes (up to 3.9 nA at a holding potential of -20 mV). High doses of THDOC (100 nM-1 microM) induced small inward currents on its own (14+/-3 and 24+/-3 pA, respectively). 4. Simultaneous application of 10 microM GABA with 100 nM or 1 microM THDOC increased current amplitudes by 125 and 128%, respectively. At 10 nM THDOC exerted no consistent effects on GABA currents. 5. Responses to 1 microM of GABA were modulated in a bidirectional manner by different doses of THDOC: 10 nM THDOC reduced the amplitude of GABA responses to 80% (P=0.018, n=15), whereas 100 nM and 1 microM THDOC enhanced the GABA response to 115 and 180% (P=0.0007, n = 15), respectively. 6. The time constant of decay of spontaneous inhibitory postsynaptic currents (IPSCs) was reversibly increased from 91+/-10 to 314+/-34 ms (n=3) by the application of THDOC (1 microM). The amplitudes of the IPSCs were not affected by THDOC. 7. These data indicate that THDOC modulates GABA responses of hypothalamic neurons in a bidirectional manner, resulting in a complex tuning of neuronal excitability in the hypothalamus.
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Canales de Cloruro/efectos de los fármacos , Desoxicorticosterona/análogos & derivados , Hipotálamo/efectos de los fármacos , Sinapsis/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacología , Animales , Células Cultivadas , Desoxicorticosterona/farmacología , Hipotálamo/fisiología , Ratas , Sinapsis/fisiologíaRESUMEN
Here, we provide the first evidence for functional expression of a human olfactory receptor protein (OR17-40) and show that recombinant olfactory receptors can be functionally expressed in heterologous systems. A mixture of 100 different odorants (Henkel 100) elicited a transient increase in intracellular [Ca(2+)] in human embryonic kidney 293 (HEK293) cells stably or transiently transfected with the plasmid pOR17-40. By subdividing the odorant mixture into progressively smaller groups, we identified a single component that represented the only effective substance: helional. Only the structurally closely related molecule heliotroplyacetone also activated the receptor. Other compounds, including piperonal, safrole, and vanillin, were completely ineffective. Mock-transfected cells and cells transfected with other receptors showed no change in intracellular [Ca(2+)] in response to odor stimulation. We were also able to functionally express OR17-40 in Xenopus laevis oocytes. Coexpression of a "reporter" channel allowed measurement of the response of oocytes injected with the cRNA of the human receptor to the odor mixture Henkel 100. The effective substances were the same (helional, heliotropylacetone) as those identified by functionally expressing the receptor in HEK293 cells and were active at the same, lower micromolar concentration. These findings open the possibility of now characterizing the sensitivity and specificity of many, if not all, of the hundreds of different human olfactory receptors.
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Receptores Odorantes/fisiología , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Línea Celular , Membrana Celular/fisiología , ADN Complementario , Femenino , Humanos , Riñón , Potenciales de la Membrana/efectos de los fármacos , Modelos Neurológicos , Datos de Secuencia Molecular , Odorantes , Oocitos/fisiología , Estructura Secundaria de Proteína , ARN Complementario/metabolismo , Receptores Odorantes/química , Receptores Odorantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Transfección , Xenopus laevisRESUMEN
BACKGROUND: This study evaluates the effects of individual high-density exposure (2-3 weeks, all day) plus response prevention therapy on 85 unselected inpatients suffering from obsessive-compulsive disorder (OCD). METHODS: Twenty-eight therapist practitioners performed treatment. RESULTS: At 1-year follow-up, self-ratings indicated that patients on average felt much improved. Impairment by both obsessions and compulsions was significantly reduced (p < 0.001 in each case), and OCD symptoms as assessed by two OCD questionnaires had also decreased significantly, as had depressiveness and overall symptomatology (all p < 0.001). Defining improvements as >30% successes, as done in other OCD treatment outcome studies, success rates were 68 and 69%, respectively, for the two OCD questionnaire scores, 75% for self-rated impairment by obsessions and 84.5% for compulsions, 85.6% for overall psychopathology (General Symptom Index), and 75% for depressiveness (Beck Depression Inventory). Effect sizes were >1 SD for all of the measures included. CONCLUSIONS: We conclude that long-term effects for high-density treatment of unselected OCD patients bring about as good results as treatments with exposure and response prevention performed with selected patient samples in a research context.
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Trastorno Obsesivo Compulsivo/terapia , Psicoterapia/métodos , Adulto , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Trastorno Obsesivo Compulsivo/psicología , Escalas de Valoración PsiquiátricaRESUMEN
The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed and characterized the first human olfactory receptor (OR 17-40). Application of a mixture of hundred different odorants elicited a transient increase in intracellular calcium at HEK 293-cells which were transfected with a plasmid containing the receptor encoding DNA and a membrane import sequence. By subdividing the odorant mixture in smaller groups we could identify a single component which represented the only effective substance: helional. Testing some structurally closely related molecules we found only one other compound which also could activate the receptor: heliotropyl acetone. All other compounds tested were completely ineffective. These findings represent the beginning of molecular understanding of odorant recognition in humans.
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Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Benzaldehídos/química , Benzaldehídos/metabolismo , Benzaldehídos/farmacología , Benzodioxoles , Calcio/metabolismo , Línea Celular , Clonación Molecular , Estrenos/farmacología , Humanos , Datos de Secuencia Molecular , Odorantes , Inhibidores de Fosfodiesterasa/farmacología , Pirrolidinonas/farmacología , Receptores Odorantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Safrol/química , Safrol/metabolismo , Safrol/farmacología , Transducción de Señal , Relación Estructura-Actividad , TransfecciónRESUMEN
Nuclear gene-induced variegation mutants provide a powerful system to dissect interactions between the genetic systems of the nucleus-cytoplasm, the chloroplast, and the mitochondrion. The immutans (im) variegation mutation of Arabidopsis is nuclear and recessive and results in the production of green- and white-sectored leaves. The green sectors contain cells with normal chloroplasts, whereas the white sectors are heteroplastidic and contain cells with abnormal, pigment-deficient plastids as well as some normal chloroplasts. White sector formation can be promoted by enhanced light intensities, but sectoring becomes irreversible early in leaf development. The white sectors accumulate the carotenoid precursor phytoene. We have positionally cloned IM and found that the gene encodes a 40.5-kD protein with sequence motifs characteristic of alternative oxidase, a mitochondrial protein that functions as a terminal oxidase in the respiratory chains of all plants. However, phylogenetic analyses revealed that the IM protein is only distantly related to these other alternative oxidases, suggesting that IM is a novel member of this protein class. We sequenced three alleles of im, and all are predicted to be null. Our data suggest a model of variegation in which the IM protein functions early in chloroplast biogenesis as a component of a redox chain responsible for phytoene desaturation but that a redundant electron transfer function is capable of compensating for IM activity in some plastids and cells.