RESUMEN
Despite advances in tissue engineering and regenerative medicine, skin regeneration and cutaneous wound healing remains a significant medical challenge. A bioengineered skin that stimulates the body's natural regeneration capability is needed to address the current lack of treatment options. To this end, a biocompatible collagen wound matrix was developed using an electrochemical deposition fabrication process. The advanced collagen wound matrix has relatively high tensile strength compared to normal collagen matrix made by the heat gelation process and open porosity, and serves as an excellent platform for cellular growth and differentiation. Human adipose derived stem cells (hADSCs) were cultured on this collagen matrix and a co-culture system with primary keratinocytes and keratinocyte conditioned media was developed for differentiation of the hADSCs to keratinocyte-like cells. After fifteen days, hADSCs in co-culture began to exhibit a "cobblestone-like" morphology, indicating preliminary signs of differentiation to a keratinocyte-like cell. Based on morphological analysis at day 30, the co-culture with keratinocyte conditioned media system shows promising preliminary evidence of hADSC differentiation to a keratinocyte-like cell on an electrochemically aligned collagen wound matrix.
Asunto(s)
Tejido Adiposo/metabolismo , Diferenciación Celular , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Queratinocitos/metabolismo , Células Madre/metabolismo , Heridas y Lesiones/metabolismo , Tejido Adiposo/patología , Matriz Extracelular/patología , Femenino , Humanos , Queratinocitos/patología , Masculino , Células Madre/patología , Heridas y Lesiones/patologíaRESUMEN
Movement timing in the sub-second range engages a brain network comprising cortical and sub-cortical areas. The present study aims at investigating the functional significance of the left dorsolateral premotor cortex (dPMC) for precise movement timing as determined by sensorimotor synchronization and rhythm reproduction. To this end, 18 healthy volunteers performed an auditorily paced synchronization-continuation task with the right hand. A simple reaction time task served as control condition. Transcranial direct current stimulation (tDCS) was applied over the left dPMC in order to modulate cortical excitability either with anodal or cathodal polarity or as sham stimulation. TDCS was applied for 10 minutes, respectively on separate days. For the continuation task the analysis revealed significantly smaller inter-tap intervals (ITIs) following cathodal tDCS suggesting movement hastening as well as a trend towards larger ITIs following anodal stimulation suggesting movement slowing. No significant effect was found following sham stimulation. Neither for synchronization nor for reaction time tasks significant polarity-specific effects emerged. The data suggest the causal involvement of the dPMC in temporally precisereproduction of isochronous rhythms rather than sensorimotor synchronization. The present findings support the hypothesis that different cortical brain areas within the motor-control-network distinctively contribute to movement timing in the sub-second range.
Asunto(s)
Ondas Encefálicas , Sincronización Cortical , Corteza Motora/fisiología , Movimiento , Estimulación Transcraneal de Corriente Directa , Adulto , Femenino , Voluntarios Sanos , Humanos , Masculino , Adulto JovenRESUMEN
Studies on classification learning suggested that altered dopamine function in Parkinson's Disease (PD) specifically affects learning from feedback. In patients OFF medication, enhanced learning from negative feedback has been described. This learning bias was not seen in observational learning from feedback, indicating different neural mechanisms for this type of learning. The present study aimed to compare the acquisition of stimulus-response-outcome associations in PD patients OFF medication and healthy control subjects in active and observational learning. 16 PD patients OFF medication and 16 controls were examined with three parallel learning tasks each, two feedback-based (active and observational) and one non-feedback-based paired associates task. No acquisition deficit was seen in the patients for any of the tasks. More detailed analyses on the learning strategies did, however, reveal that the patients showed more lose-shift responses during active feedback learning than controls, and that lose-shift and win-stay responses more strongly determined performance accuracy in patients than controls. For observational feedback learning, the performance of both groups correlated similarly with the performance in non-feedback-based paired associates learning and with the accuracy of observed performance. Also, patients and controls showed comparable evidence of feedback processing in observational learning. In active feedback learning, PD patients use alternative learning strategies than healthy controls. Analyses on observational learning did not yield differences between patients and controls, adding to recent evidence of a differential role of the human striatum in active and observational learning from feedback.
Asunto(s)
Encéfalo/fisiopatología , Retroalimentación Formativa , Aprendizaje/fisiología , Enfermedad de Parkinson/fisiopatología , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
For music and language processing, memory for relative pitches is highly important. Functional imaging studies have shown activation of a complex neural system for pitch memory. One region that has been shown to be causally involved in the process for nonmusicians is the supramarginal gyrus (SMG). The present study aims at replicating this finding and at further examining the role of the SMG for pitch memory in musicians. Nonmusicians and musicians received cathodal transcranial direct current stimulation (tDCS) over the left SMG, right SMG, or sham stimulation, while completing a pitch recognition, pitch recall, and visual memory task. Cathodal tDCS over the left SMG led to a significant decrease in performance on both pitch memory tasks in nonmusicians. In musicians, cathodal stimulation over the left SMG had no effect, but stimulation over the right SMG impaired performance on the recognition task only. Furthermore, the results show a more pronounced deterioration effect for longer pitch sequences indicating that the SMG is involved in maintaining higher memory load. No stimulation effect was found in both groups on the visual control task. These findings provide evidence for a causal distinction of the left and right SMG function in musicians and nonmusicians.
Asunto(s)
Memoria/fisiología , Música , Lóbulo Parietal/fisiología , Percepción de la Altura Tonal/fisiología , Estimulación Transcraneal de Corriente Directa , Estimulación Acústica , Adolescente , Adulto , Análisis de Varianza , Femenino , Lateralidad Funcional , Humanos , Imagen por Resonancia Magnética , Masculino , Estimulación Luminosa , Competencia Profesional , Tiempo de Reacción/fisiología , Adulto JovenRESUMEN
Motor learning results from practice but also between practice sessions. After skill acquisition early consolidation results in less interference with other motor tasks and even improved performance of the newly learned skill. A specific significance of the primary motor cortex (M1) for early consolidation has been suggested. Since synchronized oscillatory activity is assumed to facilitate neuronal plasticity, we here investigate alterations of motor-cortical oscillations by means of event-related desynchronization (ERD) at alpha (8-12 Hz) and beta (13-30 Hz) frequencies in healthy humans. Neuromagnetic activity was recorded using a 306-channel whole-head magnetoencephalography (MEG) system. ERD was investigated in 15 subjects during training on a serial reaction time task and 10 min after initial training. The data were compared with performance during a randomly varying sequence serving as control condition. The data reveal a stepwise decline of alpha-band ERD associated with faster reaction times replicating previous findings. The amount of beta-band suppression was significantly correlated with reduction of reaction times. While changes of alpha power have been related to lower cognitive control after initial skill acquisition, the present data suggest that the amount of beta suppression represents a neurophysiological marker of early cortical reorganization associated with motor learning.
Asunto(s)
Potenciales Evocados Motores/fisiología , Aprendizaje/fisiología , Actividad Motora/fisiología , Corteza Motora/fisiología , Neuronas/fisiología , Adulto , Femenino , Humanos , Magnetoencefalografía , Masculino , Movimiento/fisiología , Tiempo de Reacción/fisiologíaRESUMEN
OBJECTIVES: Severe hepatic encephalopathy gives rise to asterixis, a striking motor symptom also called flapping tremor, which is characterized by a sudden ceasing of muscle tone in all muscles of a limb. In this study, we aimed at scrutinizing the cortical activation associated with asterixis and unraveling the underlying pathophysiological mechanisms. MATERIAL AND METHODS: We recorded simultaneously neural activity with magnetoencephalography (MEG) and muscle activity with surface EMG in nine patients with manifest hepatic encephalopathy showing asterixis. Asterixis events were detected semiautomatically and served as triggers for averaging MEG signals. Evoked responses averaged time-locked to asterixis events were subjected to equivalent current dipole (ECD) modeling. Additionally, we localized the strongest cortico-muscular coherence in the frequency of the co-occurring tremulousness. RESULTS: Evoked fields averaged time-locked to asterixis events were best explained by a single dipolar source in the contralateral primary motor cortex (M1, Talairach coordinates of mean localization: -40, -20, and 64; Brodmann area 4). This dipole showed a twofold field reversal, that is biphasic wave, with frontal dipole orientation at 49 ms before flap onset and 99 ms after flap onset. Conversely, two maxima with occipital dipole orientation were observed 2 ms and 160 ms after flap onset. Cortico-muscular coherence for the tremulousness was likewise localized in the contralateral M1 confirming earlier findings in the present patient cohort. CONCLUSIONS: Our results reveal an involvement of M1 in the generation of asterixis. As also tremulousness, also called mini-asterixis, was shown to originate in M1, asterixis and mini-asterixis may share common pathophysiological mechanisms.
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Corteza Cerebral/fisiopatología , Discinesias/etiología , Discinesias/fisiopatología , Encefalopatía Hepática/complicaciones , Encefalopatía Hepática/fisiopatología , Anciano , Electromiografía , Femenino , Humanos , Magnetoencefalografía , Masculino , Persona de Mediana Edad , Músculo Esquelético/fisiopatologíaRESUMEN
Synchronized oscillatory activity at alpha (8-12 Hz) and beta (13-30 Hz) frequencies plays a key role in motor control. Nevertheless, its exact functional significance has yet to be solved. Transcranial alternating current stimulation (tACS) allows the frequency-specific modulation of ongoing oscillatory activity. The goal of the present study was to investigate the effect of 10 and 20 Hz tACS over left primary motor cortex (M1) on motor functions and cortical excitability in healthy subjects. To this end, tACS was applied for 10 min. Sham stimulation served as control condition. Movement speed and accuracy of the right hand were assessed in 15 right-handed subjects before and after (0, 30 and 60 min) tACS of M1. Cortical silent period (CSP) and motor evoked potentials (MEPs) were determined as measures of M1 excitability. While 10 Hz tACS particularly increased movement variability, especially in tasks requiring internal pacing, 20 Hz tACS resulted in movement slowing. Behavioural effects occurred in distinct time windows. While 10 Hz effects developed over 30 min after stimulation, 20 Hz tACS effects were found immediately after stimulation. Following 10 Hz tACS these effects were significantly correlated with CSP duration, indicating interference with inhibitory pathways. The present findings suggest differential effects of stimulation frequency on motor behaviour and M1 excitability.
Asunto(s)
Estimulación Eléctrica/métodos , Potenciales Evocados Motores/fisiología , Corteza Motora/fisiología , Movimiento/fisiología , Adulto , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Estimulación Magnética TranscranealRESUMEN
Pathophysiological changes in basal ganglia-thalamo-cortical circuits are well established in idiopathic Parkinson's disease (PD). However, it remains open whether such alterations already occur at early stages representing a characteristic neurophysiological marker of PD. Therefore, the present study aims at elucidating changes of synchronised oscillatory activity in early PD patients. In this study, we performed whole-head magnetoencephalography (MEG) in a resting condition and during steady state contraction of the more severely affected forearm in 10 drugnaive, de novo patients, in 10 early-stage patients with chronic medication and in 10 age-matched control subjects. While cortico-muscular coherence (CMC) did not differ between groups, patients showed increased sensori-motor cortical power at beta frequency (1330 Hz) during rest as well as during isometric contraction compared to controls. In healthy control subjects the power of the contralateral hemisphere was significantly suppressed during isometric contraction. By contrast, both hemispheres were activated equally strongly in de novo patients. In medicated patients, the pattern was found to be reversed. Contralateral beta power was significantly correlated with motor impairment during isometric contraction but not during rest. The present results suggest that the reduced ability of the primary motor cortex to disengage from increased beta band oscillations during the execution of movements is an early marker of PD.
Asunto(s)
Corteza Motora/fisiopatología , Enfermedad de Parkinson/fisiopatología , Adulto , Anciano , Electromiografía , Femenino , Antebrazo , Humanos , Contracción Isométrica , Magnetoencefalografía , Masculino , Persona de Mediana Edad , Músculo Esquelético/fisiopatologíaRESUMEN
AIM: Manifest hepatic encephalopathy (HE) goes along with motor symptoms such as ataxia, mini-asterixis, and asterixis. The relevance of motor impairments in cirrhotics without and with minimal HE (mHE) is still a matter of debate. PATIENTS AND METHODS: We tested three different groups of patients with liver cirrhosis: no signs of HE (HE 0), mHE, and manifest HE grade 1 according to the West Haven criteria (HE 1). All patients (n = 24) and 11 healthy control subjects were neuropsychometrically tested including critical flicker frequency (CFF), a reliable measure for HE. Motor abilities were assessed using Fahn Tremor Scale and International Ataxia Rating Scale. Fastest alternating index finger movements were analyzed for frequency and amplitude. RESULTS: Statistical analyses showed an effect of HE grade on tremor and ataxia (P < 0.01). Additionally, both ratings yielded strong negative correlation with CFF (P < 0.01, R = -0.5). Analysis of finger movements revealed an effect of HE grade on movement frequency (P < 0.03). Moreover, decreasing movement frequency and increasing movement amplitude parallel decreasing CFF (P < 0.01, R = 0.6). CONCLUSION: Our results indicate that ataxia, tremor, and slowing of finger movements are early markers for cerebral dysfunction in HE patients even prior to neuropsychometric alterations becoming detectable.
Asunto(s)
Discinesias/diagnóstico , Discinesias/etiología , Encefalopatía Hepática/complicaciones , Cirrosis Hepática/complicaciones , Anciano , Alcoholismo/complicaciones , Ataxia/diagnóstico , Ataxia/etiología , Femenino , Dedos/fisiología , Lateralidad Funcional , Humanos , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Temblor/diagnóstico , Temblor/etiologíaRESUMEN
The neural mechanisms subserving uni- and bimanual control of movements are not well understood. Nevertheless, recent studies indicate a functional role of oscillatory activity in movement control and point towards a hemispheric asymmetry in motor control. This study specifically addresses the issues of (i) task-relatedness, (ii) hemispheric symmetry, and (iii) frequency specificity of the measures power, cerebro-muscular coherence, and cerebro-cerebral coherence in bilateral primary motor cortex and supplementary motor area (SMA). We have studied 10 right-handed subjects with simultaneous recordings of magnetoencephalography and surface electromyography during different unimanual and bimanual tasks. Using the analysis technique Dynamic Imaging of Coherent Sources (DICS), left and right primary motor cortex and SMA were functionally localized. Power, cerebro-musclar coherence, and cerebro-cerebral coherence between these areas were computed for four frequency bands in each condition and subjected to ANOVA. Results show a task-specific modulation of power and coherence, and further indicate a hemispheric asymmetry in the control of unimanual and bimanual movements. In addition, different frequency bands showed different task-dependent variations. The gamma band (26-40 Hz) showed strongest modulation for cerebro-muscular coherence and was strongest for the isometric contraction conditions. In contrast, the beta band (13-24 Hz) showed the strongest variations between static and dynamic conditions, and seems to play a particular role in movement control. In summary, our results indicate a differential functional role of oscillatory activity and coupling in the motor system.
Asunto(s)
Lateralidad Funcional/fisiología , Corteza Motora/fisiología , Movimiento/fisiología , Adulto , Ritmo beta , Electromiografía , Femenino , Humanos , Interpretación de Imagen Asistida por Computador , Contracción Isométrica/fisiología , Magnetoencefalografía , MasculinoRESUMEN
In this study cerebral reorganization was investigated in thalidomide-damaged subjects who use their feet to compensate for their malformed upper extremities. Tactile localization across toes was combined with fMRI to study use-dependent plasticity of the human somatosensory cortex. The manner of compensatory foot use was assessed by a questionnaire. In the behavioural experiment toes were stimulated with above threshold monofilaments and subjects had to report which toe was stimulated. When feet were employed for all everyday actions subjects made significantly fewer errors in the localization task. In subjects who use their feet only for specific actions such as grasping objects there were as many localization errors as in the control group of thalidomide-affected subjects with normal extremities. However, the patterns of mislocalizations were different with less errors occurring for the toe of the dominant foot involved in these actions. Functional MRI showed stronger haemodynamic responses to electrical stimulation of the toes in subjects using their feet for everyday actions as compared to controls. Our data show that long-term use of the feet for fine sensorimotor skills leads to better performance in tactile localization and changes in cerebral SI representation supporting the notion of use-dependent plasticity in the somatosensory cortex.
Asunto(s)
Adaptación Fisiológica/fisiología , Pie/inervación , Plasticidad Neuronal/fisiología , Corteza Somatosensorial/crecimiento & desarrollo , Tacto/fisiología , Deformidades Congénitas de las Extremidades Superiores/fisiopatología , Actividades Cotidianas , Adulto , Femenino , Pie/crecimiento & desarrollo , Pie/fisiología , Lateralidad Funcional/fisiología , Fuerza de la Mano/fisiología , Humanos , Imagen por Resonancia Magnética , Masculino , Destreza Motora/fisiología , Movimiento/fisiología , Aptitud Física/fisiología , Estimulación Física , Umbral Sensorial/fisiología , Corteza Somatosensorial/anatomía & histología , Corteza Somatosensorial/fisiología , Encuestas y Cuestionarios , Talidomida/efectos adversos , Deformidades Congénitas de las Extremidades Superiores/inducido químicamente , Deformidades Congénitas de las Extremidades Superiores/patologíaRESUMEN
Proximal spinal muscular atrophy (SMA) is a common motor neuron disorder caused by mutation of the telomeric survival of motor neuron gene SMN1. The centromeric survival of motor neuron SMN2 gene is retained in all SMA patients but does not produce sufficient SMN protein to prevent the development of clinical symptoms. The SMN1 and SMN2 genes differ functionally by a single nucleotide change. This change affects the efficiency with which exon 7 is incorporated into the mRNA transcript. Thus, SMN2 produces less full-length mRNA and protein than SMN1. We have screened a library of compounds in order to identify ones that can alter the splicing pattern of the SMN2 gene. Here, we report that the compound aclarubicin increases the retention of exon 7 into the SMN2 transcript. We show that aclarubicin effectively induces incorporation of exon 7 into SMN2 transcripts from the endogenous gene in type I SMA fibroblasts as well as into transcripts from a SMN2 minigene in the motor neuron cell line NSC34. In type I fibroblasts, treatment resulted in an increase in SMN protein and gems to normal levels. Our results suggest that alteration of splicing pattern represents a new approach to modification of gene expression in disease treatment and demonstrate the feasibility of high throughput screens to detect compounds that affect the splicing pattern of a gene.
Asunto(s)
Aclarubicina/farmacología , Proteínas del Tejido Nervioso/fisiología , Atrofias Musculares Espinales de la Infancia/tratamiento farmacológico , Empalme Alternativo/efectos de los fármacos , Animales , Western Blotting , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Exones , Estudios de Factibilidad , Fibroblastos , Humanos , Inmunohistoquímica , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas del Complejo SMN , Atrofias Musculares Espinales de la Infancia/genética , Proteína 1 para la Supervivencia de la Neurona Motora , Proteína 2 para la Supervivencia de la Neurona Motora , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
BACKGROUND: We describe a rapid flow cytometric assay that correlates cell cycle with apoptotic cell death in a cell line expressing a tandem green fluorescent protein (GFP). METHODS: A Jurkat cell line was transfected with a gene construct coding for constitutive expression of a tandem GFP molecule carrying a consensus cleavage site (DEVD) for group II caspases (C-2-Y). Cells were treated with CD95 antibody (Ab), then incubated with annexin V-phycoerythrin (PE), propidium iodide (PI), and Hoechst 33342. RESULTS: After CD95 treatment, the C-2-Y cell line had twice the number of nonapoptotic cells compared with both control cell lines. This proportion of viable, nonapoptotic cells after treatment was unaffected by the level of GFP (DEVD) expression in the cells, as confirmed by sorted populations. The early apoptotic cells in the C-2-Y cell line had an increased G0-G1 phase population compared with the control cell lines. CONCLUSIONS: Apoptosis is delayed in the C-2-Y cell line and the early apoptotic cells have a higher G0-G1 cell cycle frequency. The artificial substrate competes with the natural substrate(s), thereby slowing the apoptotic process. The expression level of DEVD-GFP does not alter the delayed induction of apoptosis. Caspase activation occurs prior to phosphatidylserine translocation.
Asunto(s)
Apoptosis/genética , Caspasas/metabolismo , Fase G1/fisiología , Fase de Descanso del Ciclo Celular/fisiología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales de Origen Murino , Apoptosis/efectos de los fármacos , Citometría de Flujo/métodos , Fase G1/efectos de los fármacos , Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Células Jurkat , Proteínas Luminiscentes/metabolismo , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Estadística como Asunto , Especificidad por Sustrato/fisiología , Secuencias Repetidas en Tándem/fisiología , TransfecciónRESUMEN
The cellular chaperone, HSP90, is identified here as an essential factor for the activity of NS2/3 protease of hepatitis C virus. The cleavage activity of NS2/3 protease synthesized in reticulocyte lysate is ATP-dependent, as evidenced by ATP depletion experiments and inhibition with nonhydrolyzable ATP analogs. Geldanamycin and radicicol, ATP-competitive inhibitors of the chaperone HSP90, also inhibit the cleavage of in vitro-synthesized NS2/3. Furthermore, these HSP90 inhibitors prevent NS2/3 cleavage when the protease is expressed in mammalian cells. The physical association of NS2/3 with HSP90 is demonstrated by immunoprecipitation. Thus, by way of a chaperone/folding activity, an HSP90-containing complex is required for maturation of the polyprotein that encodes the enzymes essential for hepatitis C virus replication.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Hepacivirus/enzimología , Procesamiento Proteico-Postraduccional , Adenosina Trifosfato/metabolismo , Benzoquinonas , Cisteína Endopeptidasas/genética , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Células Jurkat , Lactamas Macrocíclicas , Lactonas/farmacología , Macrólidos , Quinonas/metabolismo , Quinonas/farmacologíaRESUMEN
Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner. A key determinant of the intracellular concentration of a given protein is its stability or "half-life." We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells. The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (UbG76V) that cannot be cleaved by ubiquitin hydrolases. We show that increasing the number of UbG76V moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins. Cells expressing a destabilized beta-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Ubiquitinas/química , Ubiquitinas/metabolismo , Biopolímeros/genética , Biopolímeros/metabolismo , Estabilidad de Enzimas , Genes Reporteros , Semivida , Humanos , Células Jurkat , Plásmidos/genética , Poliubiquitina , Complejo de la Endopetidasa Proteasomal , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Ubiquitinas/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green fluorescent protein (GFP) stably expressed within cells can serve as a genetically encoded sensor of protease activity. Using this technology, we have developed a stable cellular system for the screening of agents that modulate activation of the caspase cascade. This assay technology allows for the real-time monitoring of apoptosis in situ, using conventional fluorescent plate reader detection. By applying this assay system to an actual compound screen, small-molecule inducers of cell apoptosis were reliably identified. Follow-up pharmacology confirmed that the rank-order potency of primary hits using the intracellular GFP assay corresponded to that found using a conventional, cell lysis-based assay method.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Proteínas Luminiscentes/metabolismo , Secuencia de Aminoácidos , Anticuerpos/inmunología , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Técnicas Biosensibles , Caspasa 3 , Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Transferencia de Energía , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Fluorescencia , Genes Reporteros/genética , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Células Jurkat , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes de Fusión , Reproducibilidad de los Resultados , Transfección , Receptor fas/inmunologíaRESUMEN
The development of object permanence was investigated in black-billed magpies (Pica pica), a food-storing passerine bird. The authors tested the hypothesis that food-storing development should be correlated with object-permanence development and that specific stages of object permanence should be achieved before magpies become independent. As predicted, Piagetian Stages 4 and 5 were reached before independence was achieved, and the ability to represent a fully hidden object (Piagetian Stage 4) emerged by the age when magpies begin to retrieve food. Contrary to psittacine birds and humans, but as in dogs and cats, no "A-not-B error" occurred. Although magpies also mastered 5 of 6 invisible displacement tasks, evidence of Piagetian Stage 6 competence was ambiguous.
Asunto(s)
Conducta Animal , Aves , Cognición , Alimentos , Memoria , AnimalesRESUMEN
Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for Gi protein-coupled receptors. Two Gi-GPCRs, mu-opioid receptor (mu-OPR) and 5-hydroxytryptamine receptor la (5HT1aR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric Gq/i5 protein (which re-directs a negative Gi-type signal to a positive Gq-type response), (2) a given Gi-GPCR, and (3) a beta-lactamase (beta1a) reporter gene responsive to Gi-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Nucleares , Receptores de Superficie Celular/metabolismo , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Colorantes Fluorescentes , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Genes Reporteros , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Ligandos , Factores de Transcripción NFATC , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Transfección , beta-Lactamasas/genéticaRESUMEN
The use of green fluorescent protein (GFP) is a powerful technology that has recently enabled investigators to study dynamic molecular events within living cells. One method for detecting molecular interactions involves fluorescence resonance energy transfer (FRET) between two GFPs or between GFP and a second fluorophore. This review summarizes the use of GFP for FRET and illustrates the theme with specific examples on how GFP has been employed as an intracellular molecular sensor.
Asunto(s)
Proteínas Luminiscentes , Espectrometría de Fluorescencia/métodos , Endopeptidasas/metabolismo , Proteínas Fluorescentes Verdes , Especificidad por SustratoRESUMEN
Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.