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1.
PLoS One ; 8(3): e59578, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23555714

RESUMEN

Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their "cores". After computational reconstruction, we showed that these membranes are organized as a network of tubules which resemble the endoplasmic reticulum (ER). Our findings explain how membrane-bound proteins interact and are spatially arranged within LB "cores" and support a model for LB formation by incorporating cytoplasmic membranes of the ER, instead of the conventional view that LBs emerge from the ER leaflets. This is important to understand the functional capabilities of leukocyte LBs in health and during diverse diseases in which these organelles are functionally involved.


Asunto(s)
Tomografía con Microscopio Electrónico , Eosinófilos/citología , Imagenología Tridimensional , Lípidos , Orgánulos/ultraestructura , Adipocitos/citología , Eosinófilos/ultraestructura , Humanos , Membranas Intracelulares/diagnóstico por imagen , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Orgánulos/metabolismo , Ultrasonografía
2.
Eur J Nucl Med Mol Imaging ; 35(4): 779-89, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18057934

RESUMEN

PURPOSE: The study aims to evaluate the iodinated imidazopyridine, N',N'-diethyl-6-Chloro-(4'-[(123)I]iodophenyl)imidazo[1,2-a]pyridine-3-acetamide ([(123)I]-CLINDE) as a tracer for the study of peripheral benzodiazepine binding sites (PBBS). MATERIALS AND METHODS: In vitro studies were performed using membrane homogenates and sections from kidney, adrenals, and brain cortex of Sprague-Dawley (SD) rats and incubated with [(123)I]-CLINDE. For in vivo studies, the rats were injected with [(123)I]-CLINDE. In competition studies, PBBS-specific drugs PK11195 and Ro 5-4864 and the CBR specific drug Flumazenil were injected before the radiotracer. RESULTS: In vitro binding studies in adrenal, kidney, and cortex mitochondrial membranes indicated that [(123)I]-CLINDE binds with high affinity to PBBS, K(d) = 12.6, 0.20, and 3.84 nM, respectively. The density of binding sites was 163, 5.3, and 0.34 pmol/mg protein, respectively. In vivo biodistribution indicated high uptake in adrenals (5.4), heart (1.5), lungs (1.5), kidney (1.5) %ID/g at 6 h p.i. In the central nervous system (CNS), the olfactory bulbs displayed the highest uptake; up to six times the activity in blood. Pre-administration of unlabeled CLINDE, PK11195 and Ro 5-4864 (1 mg/kg) reduced the uptake of [(123)I]-CLINDE by 70-55% in olfactory bulbs. In the kidney and heart, a reduction of 60-80% ID/g was observed, while an increase was observed in the adrenals requiring 10 mg/kg for significant displacement. Flumazenil had no effect on uptake in peripheral organs and brain. Metabolite analysis indicated >90% of the radioactivity in the above tissues was intact [(123)I]-CLINDE. CONCLUSION: [(123)I]-CLINDE displays high and selective uptake for the PBBS and warrants further development as a probe for imaging PBBS using single photon emission computed tomography (SPECT).


Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Radioisótopos de Yodo/farmacocinética , Glándulas Suprarrenales/diagnóstico por imagen , Glándulas Suprarrenales/metabolismo , Animales , Benzodiazepinas/metabolismo , Sitios de Unión , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/metabolismo , Membranas Intracelulares/diagnóstico por imagen , Membranas Intracelulares/metabolismo , Riñón/diagnóstico por imagen , Riñón/metabolismo , Cinética , Masculino , Mitocondrias/diagnóstico por imagen , Mitocondrias/metabolismo , Cintigrafía , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Distribución Tisular
3.
Anal Chem ; 77(7): 2147-56, 2005 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-15801749

RESUMEN

In interphase eukaryotic cells, molecular transport between the cytoplasm and the nucleus is mediated by the nuclear pore complex (NPC), which perforates the double-membraned nuclear envelope (NE). Local permeability of the NE at large intact nuclei (approximately 400 microm in diameter) isolated from Xenopus laevis oocytes was studied by scanning electrochemical microscopy (SECM). Steady-state tip current versus tip-nucleus distance curves (approach curves) were measured with 10- and 2-microm-diameter Pt disk microelectrodes at the nuclei in isotonic buffer solutions containing redox-active molecules. The approach curves in the normalized form are independent of the tip diameter, indicating diffusion-limited membrane transport of the redox molecules. SECM chronoamperometry demonstrated that a decrease in the steady-state tip current at short tip-nucleus distances is due to smaller diffusion coefficients and concentrations of the redox molecules in the nucleus than those in the buffer solution. The experimental approach curves fit very well with theoretical ones for freely permeable membranes, yielding the NE permeability to the molecules that is at least 2 orders of magnitude larger than permeability of bilayer lipid membranes and cell membranes. This result indicates that passive transport of the redox molecules across the NE is facilitated by open NPC pores. The flux of the redox molecules sustainable by a single NPC channel (>9.8 x 10(6) molecules per NPC per second) and the diameter of the channel pore (>15 nm) were estimated from the SECM data by assuming the NE as an array of nanometer-sized NPC pores. The effects of the redox molecules on the nucleus and the NPC function were examined by studying signal-mediated nuclear import of rhodamine-labeled bovine serum albumin with and without nuclear localization signals by fluorescence microscopy.


Asunto(s)
Electroquímica/métodos , Microscopía Electrónica de Rastreo/métodos , Membrana Nuclear/metabolismo , Oocitos/metabolismo , Transporte Activo de Núcleo Celular , Animales , Electroquímica/instrumentación , Femenino , Membranas Intracelulares/diagnóstico por imagen , Membranas Intracelulares/metabolismo , Canales Iónicos/ultraestructura , Microscopía Electrónica de Rastreo/instrumentación , Membrana Nuclear/ultraestructura , Señales de Localización Nuclear , Oocitos/citología , Oxidación-Reducción , Permeabilidad , Rodaminas/farmacocinética , Albúmina Sérica Bovina/metabolismo , Albúmina Sérica Bovina/farmacocinética , Ultrasonografía , Xenopus laevis
4.
Exp Hematol ; 30(11): 1293-301, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12423682

RESUMEN

OBJECTIVE: We evaluated the cytotoxic effect of ultrasonic irradiation at low energy on the viability of normal and leukemic cells and the potential mechanisms of action inducing this cytotoxicity. MATERIALS AND METHODS: Human leukemia cell lines (K562, HL-60, KG1a, and Nalm-6), primary leukemic cells, and normal mononuclear cells are treated by ultrasound at a frequency of 1.8 MHz during various exposure times (acoustical power of 7 mW/mL) and immediately tested for cell viability by the trypan blue exclusion assay. Apoptosis is evaluated by cell morphology, phosphatidylserine exposure, and DNA fragmentation. The mitochondrial potential, glutathione content, caspase-3 activation, PARP cleavage, and bcl-2/bax ratio are tested by flow cytometry. Cloning efficiency is evaluated by assays in methylcellulose. RESULTS: The technique we describe here, using minute amounts of energy and in the absence of any chemical synergy, specifically triggers apoptosis in leukemic cells while necrosis is significantly reduced. Ultrasonic treatment of 20 seconds' duration induces a series of successive phases showing the characteristic features of apoptosis: mitochondrial transmembrane potential disturbances, loss of phosphatidylserine asymmetry, morphological changes, and, finally, DNA fragmentation. In contrast to K562 cells, for which a significant reduction of cloning efficiency is observed, the growth of hematopoietic progenitors is totally unaffected. Ultrasound treatment is also associated with depletion of cellular glutathione content, suggesting a link with the oxidative stress. Moreover, the fact that active oxygen scavengers reduce ultrasonic-induced apoptosis suggests a sonochemical mechanism. CONCLUSION: The cell damage observed after exposure of leukemic cells to ultrasound is associated with the apoptotic process and may be a promising tool for a smooth, specific, and effective ex vivo purging of leukemic cells.


Asunto(s)
Apoptosis , Leucemia/patología , Células Madre Neoplásicas/diagnóstico por imagen , Terapia por Ultrasonido , Caspasa 3 , Caspasas/análisis , Supervivencia Celular , Fragmentación del ADN , Depuradores de Radicales Libres/farmacología , Glutatión/análisis , Células HL-60/química , Células HL-60/diagnóstico por imagen , Células HL-60/patología , Histidina/farmacología , Humanos , Radical Hidroxilo , Membranas Intracelulares/diagnóstico por imagen , Células K562/química , Células K562/diagnóstico por imagen , Células K562/patología , Manitol/farmacología , Lípidos de la Membrana/análisis , Mitocondrias/diagnóstico por imagen , Proteínas de Neoplasias/análisis , Células Madre Neoplásicas/química , Células Madre Neoplásicas/patología , Estrés Oxidativo , Fosfatidilserinas/análisis , Poli(ADP-Ribosa) Polimerasas/análisis , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Oxígeno Singlete , Ensayo de Tumor de Célula Madre , Ultrasonografía , Proteína X Asociada a bcl-2
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