RESUMEN

We demonstrate the potential of Raman spectroscopy to detect cocaine concealed inside transparent glass bottles containing alcoholic beverages. A clear Raman signature of cocaine with good signal-to-noise was obtained from a approximately 300 g solution of adulterated cocaine (purity 75%) in a 0.7 L authentic brown bottle of rum with 1 s acquisition time. The detection limit was estimated to be of the order of 9 g of pure cocaine per 0.7 L (approximately 0.04 moles L(-1)) with 1 s acquisition time. The technique holds great promise for the fast, non-invasive, detection of concealed illicit compounds inside beverages using portable Raman instruments, thus permitting drug trafficking to be combated more effectively.

Asunto(s)

Bebidas Alcohólicas/análisis , Cocaína/análisis , Espectrometría Raman/métodos , Detección de Abuso de Sustancias/métodos , Cocaína/química , Etanol/química , Drogas Ilícitas/análisis , Drogas Ilícitas/química

RESUMEN

A simple procedure for the recovery of deep subsurface Raman spectra in stratified turbid samples by defocusing a conventional Raman instrument is presented. The method is based on effects present with spatially offset Raman spectroscopy (SORS) and, although not as efficient as the standard SORS approach, it permits a simple way of recovering subsurface Raman spectra from less challenging samples. Demonstration of the effect is performed using a standard SORS device and a commercial Raman instrument on the noninvasive measurement of paracetamol tablets held within a nontransparent white plastic bottle.

Asunto(s)

Coloides/química , Mezclas Complejas/química , Nefelometría y Turbidimetría/instrumentación , Nefelometría y Turbidimetría/métodos , Espectrometría Raman/instrumentación , Espectrometría Raman/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

We present a Raman spectroscopic method for the noninvasive detection of liquid explosives within bottles, and other packaging, of substantially higher sensitivity and wider applicability than that currently available via conventional Raman spectroscopy. The approach uses a modification of the spatially offset Raman spectroscopy (SORS) concept, which permits the interrogation of a wide range of containers, including transparent, colored, and diffusely scattering plastic and glass beverage, medicine, and cosmetic bottles, with no change in experimental geometry. The enhanced sensitivity is achieved by the technique's inherent ability to effectively suppress fluorescence and Raman contributions originating from the wall of the container. The application is demonstrated on the noninvasive detection of hydrogen peroxide solution, a critical component of a number of liquid explosives. In contrast to conventional Raman spectroscopy, the modified SORS concept enables the detection of concealed hydrogen peroxide solution in all the studied cases.

RESUMEN

A method is presented for the use of SAM layers as internal standards for calibration in surface-enhanced Raman spectroscopy. Three cyano-containing compounds were attached to gold colloids via a metal-sulfur bond and evaluated for spectral stability and normalization capacity. The results show that the analyte, rhodamine 6G, and the internal standard signal enhancement covaried, and it was possible to quantify the analyte with PLS. The fact that the enhancing substrate was chaotic assemblies with large variation in signal enhancement shows the versatility of this method.

RESUMEN

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell-type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.

Asunto(s)

Astrocitos/ultraestructura , Movimiento Celular/fisiología , Filamentos Intermedios/fisiología , Animales , Proteína Ácida Fibrilar de la Glía/genética , Ratones , Ratones Noqueados , Microscopía Electrónica , Microscopía por Video , Modelos Biológicos , Vimentina/genética

RESUMEN

Glial fibrillary acidic protein (GFAP) is the main component of intermediate filaments in astrocytes. To assess its function in astrocyte swelling, we compared astrocyte membrane properties and swelling in spinal cord slices of 8- to 10-day-old wild-type control (GFAP(+/+)) and GFAP-knockout (GFAP(-/-)) mice. Membrane currents and K(+) accumulation around astrocytes after a depolarizing pulse were studied using the whole-cell patch-clamp technique. In vivo cell swelling was studied in the cortex during spreading depression (SD) in 3 to 6-month-old animals. Swelling-induced changes of the extracellular space (ECS) diffusion parameters, i.e., volume fraction alpha and tortuosity lambda, were studied by the real-time iontophoretic tetramethylammonium (TMA(+)) method using TMA(+)-selective microelectrodes. Morphological analysis using confocal microscopy and quantification of xy intensity profiles in a confocal plane revealed a lower density of processes in GFAP(-/-) astrocytes than in GFAP(+/+) astrocytes. K(+) accumulation evoked by membrane depolarization was lower in the vicinity of GFAP(-/-) astrocytes than GFAP(+/+) astrocytes, suggesting the presence of a larger ECS around GFAP(-/-) astrocytes. Astrocyte swelling evoked by application of 50 mM K(+) or by hypotonic solution (HS) produced a larger increase in [K(+)](e) around GFAP(+/+) astrocytes than around GFAP(-/-) astrocytes. No differences in alpha and lambda in the spinal cord or cortex of GFAP(+/+) and GFAP(-/-) mice were found; however, the application of either 50 mM K(+) or HS in spinal cord, or SD in cortex, evoked a large decrease in alpha and an increase in lambda in GFAP(+/+) mice only. Slower swelling in GFAP(-/-) astrocytes indicates that GFAP and intermediate filaments play an important role in cell swelling during pathological states.

Asunto(s)

Astrocitos/metabolismo , Tamaño de la Célula/fisiología , Depresión de Propagación Cortical/fisiología , Proteína Ácida Fibrilar de la Glía/deficiencia , Presión Osmótica/efectos de los fármacos , Potasio/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Tamaño de la Célula/efectos de los fármacos , Depresión de Propagación Cortical/efectos de los fármacos , Difusión/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Colorantes Fluorescentes/farmacocinética , Proteína Ácida Fibrilar de la Glía/genética , Filamentos Intermedios/efectos de los fármacos , Filamentos Intermedios/metabolismo , Filamentos Intermedios/patología , Isoquinolinas/farmacocinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados/anatomía & histología , Ratones Noqueados/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Corteza Somatosensorial/metabolismo , Corteza Somatosensorial/fisiopatología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología

RESUMEN

Multivariate evaluation of surface-enhanced Raman spectra of doxorubicin in plasma was performed. In a principal component analysis (PCA) all spectral features were modelled into three principal components. The major variation of the data was shown to be the variation of doxorubicin Raman signal together with the doxorubicin fluorescence, whereas the variation due to plasma was of minor importance. It was also shown that the surface-enhanced Raman scattering (SERS) measurements were independent on such factors as measurement occasion and silver colloids. The presented results show that with some improvements, quantification of doxorubicin directly in plasma could be possible.

Asunto(s)

Doxorrubicina/sangre , Espectrometría Raman , Animales , Antibióticos Antineoplásicos/sangre , Antibióticos Antineoplásicos/química , Bovinos , Doxorrubicina/química , Análisis Multivariante , Plasma/química , Análisis de Componente Principal , Procesamiento de Señales Asistido por Computador

RESUMEN

Increased desmin synthesis and formation of desmin-containing intermediate filaments (IFs) is one of the hallmarks of transdifferentiation of hepatic stellate cells into myofibroblast-like cells. These desmin-enriched myofibroblast-like cells are the major sources of fibrotic extracellular matrix in chronically diseased liver. Myofibroblast-like cells are also involved in the contraction of sinusoids, which leads to increased intrahepatic pressure and portal hypertension. To address the requirements for the formation of desmin-containing IFs both in quiescent and in transdifferentiated stellate cells, we used mice deficient for glial fibrillary acidic protein (GFAP) and/or vimentin, which are additional IF proteins present in stellate cells. In this study, we show that desmin cannot form full-length bundles of IFs in the absence of both GFAP and vimentin. Quiescent and transdifferentiated GFAP(-/-)vim(-/-) stellate cells are devoid of normal bundles of IFs. Instead, they exhibit only residual IF bundles restricted to subcortical cytoplasm, although these cells contain equal desmin mRNA and protein levels as wild-type cells. The absence of vimentin alone restricts formation of desmin-containing IF bundles to the perinuclear region, while both the distal processes in quiescent stellate cells and the subcortical zone in myofibroblast-like cells remain free of desmin-containing IF bundles. The absence of GFAP alone does not interfere with the formation of desmin-containing IFs. Thus, to form normal IFs in stellate cells, desmin is required to partnerize with vimentin. In addition, these mouse models will prove to be instrumental in addressing the role of IFs in the process of stellate cell transdifferentiation.

Asunto(s)

Desmina/fisiología , Filamentos Intermedios/fisiología , Hígado/fisiología , Vimentina/fisiología , Animales , Western Blotting , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/deficiencia , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Vimentina/deficiencia , Vimentina/metabolismo

RESUMEN

The importance of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP) and vimentin for astrocyte function was studied by investigating astrocytes prepared from GFAP-/- and/or vimentin-/- mice. The rate of glucose uptake through facilitative hexose transporters was not affected by depletion of GFAP or vimentin. Similarly, the absence of these IF proteins did not affect ascorbate uptake, under control or cyclic AMP-stimulated conditions, or ascorbate efflux through volume-sensitive organic anion channels. However, compared with wild-type astrocytes, glutamine concentrations were increased up to 200% in GFAP-/- astrocytes and up to 150% in GFAP+/- astrocytes and this increase was not dependent on the presence of vimentin. GFAP-/- astrocytes in culture still contain IFs (made of vimentin and nestin), whereas GFAP-/- vim-/- cultured astrocytes lack IFs. Thus, glutamine levels appear to correlate inversely with GFAP, rather than depend on the presence of IFs per se. Furthermore, the effect of GFAP is dose-dependent since the glutamine concentration in GFAP+/- astrocytes falls between those in wild-type and GFAP-/- astrocytes.

Asunto(s)

Ácido Ascórbico/metabolismo , Astrocitos/metabolismo , Proteína Ácida Fibrilar de la Glía/deficiencia , Glucosa/metabolismo , Glutamina/metabolismo , Vimentina/deficiencia , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Transporte Biológico , Bucladesina/farmacología , Células Cultivadas , AMP Cíclico/farmacología , Proteína Ácida Fibrilar de la Glía/fisiología , Canales Iónicos/fisiología , Ratones , Ratones Endogámicos C57BL , Vimentina/fisiología

RESUMEN

Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.

Asunto(s)

Astrocitos/química , Proteínas de Filamentos Intermediarios/análisis , Proteínas del Tejido Nervioso , Animales , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/fisiología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Nestina , Fosforilación , Vimentina/análisis , Vimentina/inmunología

RESUMEN

In response to injury of the central nervous system, astrocytes become reactive and express high levels of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP), vimentin, and nestin. We have shown that astrocytes in mice deficient for both GFAP and vimentin (GFAP-/-vim-/-) cannot form IFs even when nestin is expressed and are thus devoid of IFs in their reactive state. Here, we have studied the reaction to injury in the central nervous system in GFAP-/-, vimentin-/-, or GFAP-/-vim-/- mice. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP-/- or vimentin-/- mice, but was impaired in GFAP-/-vim-/- mice that developed less dense scars frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured central nervous system and that some degree of functional overlap exists between these IF proteins.

Asunto(s)

Astrocitos/fisiología , Lesiones Encefálicas/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Proteínas del Tejido Nervioso , Traumatismos de la Médula Espinal/metabolismo , Vimentina/genética , Animales , Astrocitos/química , Astrocitos/ultraestructura , Lesiones Encefálicas/genética , División Celular/fisiología , Cicatriz/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Epéndimo/citología , Epéndimo/metabolismo , Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Nestina , Traumatismos de la Médula Espinal/genética , Vasodilatación/fisiología , Vimentina/metabolismo , Heridas Punzantes/genética , Heridas Punzantes/metabolismo

RESUMEN

Astrocytes maintain their volume in response to changes in osmotic pressure in their environment by an afflux/influx of ions and organic osmoequivalents. The initial swelling of an astrocyte transferred to a hypoosmotic medium is thus reversed within minutes. The mechanisms which trigger this process as well as the sensors for cell volume are largely unknown, however, the cytoskeleton appears to be involved. We have addressed the role of one component of the cytoskeleton, the intermediate filaments, in the maintenance of astrocytic cell volume. Astrocytes from wild type mice were compared with cells from mice deficient for either glial fibrillary acidic protein (GFAP-/-) or vimentin (vimentin-/-) and with astrocytes from mice deficient for both proteins (GFAP-/-vim-/-). Whereas GFAP-/- and vimentin-/- cultured or reactive astrocytes retain intermediate filaments, the GFAP-/-vim-/- astrocytes are completely devoid of these structures. The rate of efflux of the preloaded osmoequivalent 3H-taurine from primary and passaged cultures of astrocytes was monitored. A reduction of NaCl (25 mM) in the perfusion medium led to a 400-900% increase of 3H-taurine afflux in astrocytes from wild type mice. The stimulated efflux was not significantly affected in astrocytes from GFAP-/- or vimentin-/- mice. However, the efflux from astrocytes from GFAP-/-vim-/- mice was 25-46% lower than the wild type levels. The results strengthen the role of the cytoskeleton in astrocyte volume regulation and suggest an involvement of intermediate filaments in the process.

Asunto(s)

Astrocitos/citología , Astrocitos/fisiología , Proteína Ácida Fibrilar de la Glía/deficiencia , Taurina/metabolismo , Vimentina/deficiencia , Animales , Células Cultivadas , Cruzamientos Genéticos , Proteína Ácida Fibrilar de la Glía/genética , Homeostasis/fisiología , Filamentos Intermedios/fisiología , Líquido Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Mutantes , Concentración Osmolar , Taurina/análisis , Factores de Tiempo , Tritio , Vimentina/genética

RESUMEN

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein predominantly expressed in cells of astroglial origin. To allow for the study of the biological functions of GFAP we have previously generated GFAP-negative mice by gene targeting [Pekny et al. (1995) EMBO J. 14, 1590-1598]. Astrocytes in culture, similar to reactive astrocytes in vivo, express three intermediate filament proteins: GFAP, vimentin, and nestin. Using primary astrocyte-enriched cultures from GFAP-negative mice, we now report on the effect of GFAP absence on (i) the synthesis of other intermediate filament proteins in astrocytes, (ii) intermediate filament formation, (iii) astrocyte process formation (stellation) in response to neurons in mixed cerebellar astrocyte/neuron cultures, and (iv) saturation cell density in vitro. GFAP-/- astrocytes were found to produce both nestin and vimentin. At the ultrastructural level, the amount of intermediate filaments as revealed by transmission electron microscopy was reduced in GFAP-/- astrocytes compared to that in GFAP+/+ astrocytes. GFAP-/- astrocytes retained the ability to form processes in response to neurons in mixed astrocyte/neuron cultures from the cerebellum. GFAP-/- astrocyte-enriched primary cultures exhibited an increased final cell saturation density. The latter leads us to speculate that the loss of GFAP expression observed focally in a proportion of human malignant gliomas may reflect tumor progression toward a more rapidly growing and malignant phenotype.

Asunto(s)

Astrocitos/citología , Proteína Ácida Fibrilar de la Glía/fisiología , Filamentos Intermedios/ultraestructura , Proteínas del Tejido Nervioso , Neuronas/citología , Animales , Astrocitos/ultraestructura , Neoplasias Encefálicas/patología , Recuento de Células , Células Cultivadas , Cerebelo/citología , Técnicas de Cocultivo , Inhibición de Contacto , Genotipo , Proteína Ácida Fibrilar de la Glía/deficiencia , Proteína Ácida Fibrilar de la Glía/genética , Glioma/patología , Humanos , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Nestina , Vimentina/metabolismo

RESUMEN

Cell culture models have been extensively used for studies of blood-brain barrier (BBB) function. However, most in vitro models fail to reproduce the peculiar physiological and morphological properties of in situ brain microvascular endothelial cells. A recently developed, tridimensional and dynamic model of the BBB has permitted studies of glial-endothelial interactions in hollow fibers exposed to intraluminal flow. We have taken advantage of this technique and have investigated the ability of glial fibrillary acidic protein (GFAP)-deficient (GFAP-/-) astrocytes to induce BBB properties in aortic endothelial cells (BAEC) cultured in vitro. BAEC exposed to flow were seeded intraluminally in hollow fibers and co-cultured with extraluminally seeded mouse astrocytes. Under these conditions, astrocytes have been shown to induce blood-brain barrier properties in non-brain endothelial cells. We followed induction of a BBB phenotype by measuring the transendothelial resistance, as well as endothelial permeability to potassium, theophylline, 8-sulphophenyl-theophylline (8-SPT), sucrose, and Evans blue. Wild-type mouse astrocytes induced BBB properties in aortic endothelial cells following 3-4 weeks of co-culturing. Thus, these endothelial cells restricted passage of K+ ions into the extracapillary space and selectively excluded hydrophilic molecules, such as 8-SPT and 14C-sucrose. GFAP-/- astrocytes failed to induce a significant restriction to the passage of potassium and hydrophilic drugs (sucrose, 8-SPT), failed to induce transendothelial resistance values comparable to control co-cultures, but were capable of inducing exclusion of Evans blue by endothelial cells. These results suggest that GFAP (and intermediate filaments) may play a role in the induction of BBB properties in non-BBB endothelial cells.

Asunto(s)

Astrocitos/fisiología , Barrera Hematoencefálica/fisiología , Endotelio Vascular/fisiología , Proteína Ácida Fibrilar de la Glía/deficiencia , Animales , Aorta Torácica/citología , Aorta Torácica/fisiología , Bovinos , Permeabilidad de la Membrana Celular/fisiología , Conductividad Eléctrica , Endotelio Vascular/citología , Azul de Evans , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Potasio/metabolismo

RESUMEN

In the prion diseases, extensive reactive gliosis is often found to be out of proportion to the degree of apparent neuronal damage. To evaluate the role of astrocytic gliosis in experimental scrapie of the mouse, we inoculated mice deficient in apolipoprotein E (apoE) or the glial fibrillary acidic protein (GFAP) with mouse prions. The expression of both apoE and GFAP in astrocytes increases as part of the reactive gliosis that accompanies scrapie. Null mice deficient in either apoE or GFAP inoculated with prions exhibited incubation times indistinguishable from untargeted control mice. The level of PrPSc and its regional deposition in the brains of ill mice deficient in either protein were also similar to control mice. Our findings demonstrate that neither apoE nor GFAP participates in the pathogenesis of the disease or in the production of PrPSc.

Asunto(s)

Apolipoproteínas E/análisis , Proteína Ácida Fibrilar de la Glía/análisis , Scrapie/metabolismo , Animales , Ratones

RESUMEN

Glial fibrillary acidic protein (GFAP) is the main component of the intermediate filaments in cells of astroglial lineage, including astrocytes in the CNS, nonmyelin forming Schwann cells and enteric glia. To address the function of GFAP in vivo, we have disrupted the GFAP gene in mice via targeted mutation in embryonic stem cells. Mice lacking GFAP developed normally, reached adulthood and reproduced. We did not find any abnormalities in the histological architecture of the CNS, in their behavior, motility, memory, blood-brain barrier function, myenteric plexi histology or intestinal peristaltic movement. Comparisons between GFAP and S-100 immunohistochemical staining patterns in the hippocampus of wild-type and mutant mice suggested a normal abundance of astrocytes in GFAP-negative mice, however, in contrast to wild-types, GFAP-negative astrocytes of the hippocampus and in the white matter of the spinal cord were completely lacking intermediate filaments. This shows that the loss of GFAP intermediate filaments is not compensated for by the up-regulation of other intermediate filament proteins, such as vimentin. The GFAP-negative mice displayed post-traumatic reactive gliosis, which suggests that GFAP up-regulation, a hallmark of reactive gliosis, is not an obligatory requirement for this process.

Asunto(s)

Astrocitos/fisiología , Proteína Ácida Fibrilar de la Glía/deficiencia , Filamentos Intermedios/fisiología , Ratones Mutantes , Tejido Nervioso/fisiología , Animales , Secuencia de Bases , Barrera Hematoencefálica/fisiología , Encéfalo/ultraestructura , Química Encefálica , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Gliosis , Hipocampo/química , Hipocampo/ultraestructura , Histocitoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes/embriología , Ratones Mutantes/crecimiento & desarrollo , Ratones Mutantes/psicología , Datos de Secuencia Molecular , Plexo Mientérico/química , Plexo Mientérico/ultraestructura , Tejido Nervioso/anatomía & histología , Tejido Nervioso/química , Desempeño Psicomotor , ARN Mensajero/análisis , Proteínas S100/aislamiento & purificación , Médula Espinal/química , Médula Espinal/ultraestructura

Asunto(s)

Antibacterianos/uso terapéutico , Utilización de Medicamentos , Registros de Hospitales , Humanos