RESUMO
The role of the recruitment-derecruitment of small structures in the lung (lung units) as the lung increases and decreases in volume has been debated. The objective of this study was to develop a model to estimate the change in the number and volume of open lung units as an excised lung is inflated-deflated between minimum and maximum lung volume. The model was formulated based on the observation that the compliance of the slowly changing quasi-static pressure-volume (P-V) curve of an excised rat lung can differ from the compliance of a faster changing small sinusoidal pressure volume perturbations superimposed on the curve. In those regions of the curve where differences in compliance occur, the lung tissue properties exhibit nonlinear characteristics, which cannot be linearized using "incremental" or "small signal" analysis. The model attributes the differences between the perturbation and quasi-static compliance to an additional nonlinear compliance term that results from the sequential opening and closing of lung units. Using this approach, it was possible to calculate the normalized average volume and the normalized number of open units as the lung is slowly inflated-deflated. Results indicate that the normalized average volume and the normalized number of open units are not linearly related to normalized lung volume, and at equal lung volumes the normalized number of open units is greater and the normalized average lung unit volume is smaller during lung deflation when compared to lung inflation. In summary, a model was developed to describe the recruitment-derecruitment process in excised lungs based on the differences between small signal perturbation compliance and quasi-static compliance. Values of normalized lung unit volume and the normalized number of open lung units were shown to be nonlinear functions of both transpulmonary pressure and normalized lung volume.
Assuntos
Pulmão/anatomia & histologia , Pulmão/fisiologia , Modelos Biológicos , Animais , Tamanho do Órgão , Pressão , Ventilação Pulmonar , Ratos , Ratos Sprague-DawleyRESUMO
Our laboratory has previously demonstrated that application of an antimicrobial spray product containing titanium dioxide (TiO(2)) generates an aerosol of titanium dioxide in the breathing zone of the applicator. The present report describes the design of an automated spray system and the characterization of the aerosol delivered to a whole body inhalation chamber. This system produced stable airborne levels of TiO(2) particles with a median count size diameter of 110 nm. Rats were exposed to 314 mg/m(3) min (low dose), 826 mg/m(3) min (medium dose), and 3638 mg/m(3) min (high dose) of TiO(2) under the following conditions: 2.62 mg/m(3) for 2 h, 1.72 mg/m(3) 4 h/day for 2 days, and 3.79 mg/m(3) 4 h/day for 4 days, respectively. Pulmonary (breathing rate, specific airway resistance, inflammation, and lung damage) and cardiovascular (the responsiveness of the tail artery to constrictor or dilatory agents) endpoints were monitored 24 h post-exposure. No significant pulmonary or cardiovascular changes were noted at low and middle dose levels. However, the high dose caused significant increases in breathing rate, pulmonary inflammation, and lung cell injury. Results suggest that occasional consumer use of this antimicrobial spray product should not be a hazard. However, extended exposure of workers routinely applying this product to surfaces should be avoided. During application, care should be taken to minimize exposure by working under well ventilated conditions and by employing respiratory protection as needed. It would be prudent to avoid exposure to children or those with pre-existing respiratory disease.
Assuntos
Anti-Infecciosos/toxicidade , Artérias/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Titânio/toxicidade , Acetilcolina/farmacologia , Administração por Inalação , Aerossóis , Albuminas/metabolismo , Animais , Artérias/fisiologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , L-Lactato Desidrogenase/metabolismo , Pulmão/fisiologia , Masculino , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Tamanho da Partícula , Fenilefrina/farmacologia , Ratos , Ratos Sprague-Dawley , Testes de Função Respiratória , Cauda , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Cough is considered an early sign of many respiratory diseases. Recently, there has been increased interest in measuring, analyzing, and characterizing the acoustical properties of a cough. In most cases the main focus of those studies was to distinguish between involuntary coughs and ambient sounds over a specified time period. The objective of this study was to develop a system to measure high fidelity voluntary cough sounds to detect lung diseases. To further augment the analysis capability of the system, a non-invasive flow measurement was also incorporated into the design. One of the main design considerations was to increase the fidelity of the recorded sound characteristics by increasing the signal to noise ratio of cough sounds and to minimize acoustical reflections from the environment. To accomplish this goal, a system was designed with a mouthpiece connected to a cylindrical tube. A microphone was attached near the mouthpiece so that its diaphragm was tangent to the inner surface of the cylinder. A pneumotach at the end of the tube measured the airflow generated by the cough. The system was terminated with an exponential horn to minimize sound reflections. Custom software was developed to read, process, display, record, and analyze cough sound and airflow characteristics. The system was optimized by comparing acoustical reflections and total signal to background noise ratios across different designs. Cough measurements were also collected from volunteer subjects to assess the viability of the system. Results indicate that analysis of cough characteristics has the potential to detect lung disease.
Assuntos
Auscultação/instrumentação , Tosse/diagnóstico , Tosse/fisiopatologia , Diagnóstico por Computador/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Espectrografia do Som/instrumentação , Adulto , Idoso , Desenho de Equipamento , Análise de Falha de Equipamento , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A major goal of this study was to characterize the cervicocollic reflexes (CCRs) in awake squirrel monkeys and compare it to observations in cat. This was carried out by stabilizing the head in space while rotating the lower body. The magnitude and phase of the torque produced between the head and the restraint system was used as an indicator of the CCR. Many properties of the squirrel monkey's CCR were found to be similar to those of the cat. The torque decreased as a function of frequency and amplitude. In addition, the static level of torque increased with head eccentricity. One difference was that the torque was 90x smaller in squirrel monkeys. Biomechanical differences, such as differences in head inertia, could account for these differences. The second goal was to determine if the CCR was sensitive to increases in the head's inertia. To test this, we increased the head's inertia by a factor of 36 and allowed the reflexes to adapt by rotating the whole body while the head was free to move. The CCR was rapidly assessed by periodically stabilizing the head in space during whole-body rotations. The magnitude of the torque increased by nearly 60%, suggesting that the CCR may adapt when changes in the head's inertia are imposed. Changes in the torque were also consistent with changes in head-movement kinematics during whole-body rotation. This suggests that the collic reflexes may dynamically adapt to maintain the performance and kinematics of reflexive head movement.
Assuntos
Adaptação Fisiológica/fisiologia , Movimentos da Cabeça/fisiologia , Cabeça/fisiologia , Músculos do Pescoço/fisiologia , Reflexo de Estiramento/fisiologia , Estimulação Acústica , Animais , Comportamento Animal , Eletromiografia , Análise de Fourier , Macaca mulatta , Psicofísica , Rotação , TorqueRESUMO
The head's inertia produces forces on the neck when the body moves. One collective function of the vestibulocollic and cervicocollic reflexes (VCR and CCR) is thought to be to stabilize the head with respect to the trunk during whole body movements. Little is known as to whether their head-movement kinematics produced by squirrel monkeys during whole body rotations are similar to those of cats and humans. Prior experiments with cats and human subjects have shown that yaw head-movement kinematics are unaffected by changes in the head's inertia when the whole body is rotated. These observations have led to the hypothesis that the combined actions of the VCR and CCR accommodate for changes in the head's inertia. To test this hypothesis in squirrel monkeys, it was imperative to first characterize the behavior of head movements produced during whole body rotation and then investigate their sensitivity to changes in the head's inertia. Our behavioral studies show that squirrel monkeys produce only small head movements with respect to the trunk during whole body rotations over a wide range of stimulus frequencies and velocities (0.5-4.0 Hz; 0-100 degrees /s). Similar head movements were produced when only small additional changes in the head's inertia occurred. Electromyographic recordings from the splenius muscle revealed that an active process was utilized such that increases in muscle activation occurred when the inertia of the head was increased. These results are consistent with prior cat and human studies, suggesting that squirrel monkeys have a similar horizontal VCR and CCR.
Assuntos
Movimentos da Cabeça/fisiologia , Cabeça/fisiologia , Animais , Fenômenos Biomecânicos , Vértebras Cervicais/fisiologia , Interpretação Estatística de Dados , Eletromiografia , Gravitação , Vias Neurais/citologia , Vias Neurais/fisiologia , Dinâmica não Linear , Reflexo Vestíbulo-Ocular/fisiologia , Rotação , Saimiri , Terminologia como AssuntoRESUMO
Evidence suggests that pregnant animals are more sensitive than nonpregnant animals to the systemic administration of endotoxin. Studies were undertaken to assess whether an enhanced sensitivity of the pulmonary system to aerosolized endotoxin might exist during pregnancy. Pregnant Sprague-Dawley female rats (17 d of gestation) or age-matched virgin female rats were exposed to air or endotoxin (lipopolysaccharide) by inhalation for 3 h. At 18 h following exposure to endotoxin, lactate dehydrogenase activity levels in bronchoalveolar lavage (BAL) fluid samples from pregnant rats were 1.5-fold greater than those from endotoxin-exposed virgin rats. BAL polymorphonuclear leukocyte (PMN) numbers were also approximately twofold greater in pregnant rats than in virgins following the inhalation of endotoxin. The increases in BAL PMNs in pregnant rats following endotoxin exposure were observed just following exposure to endotoxin as well as at 18 h following exposure. These results indicate that an increased pulmonary inflammatory response to inhaled endotoxin occurs during pregnancy in rats. Additional findings suggest that these pregnancy-linked pulmonary responses to endotoxin cannot be explained by the following potential mechanisms: changes in the inhaled dose of endotoxin, or alterations in the responsiveness of alveolar macrophages to endotoxin. To our knowledge this is the first study that has evaluated pulmonary responses to inhaled endotoxin during pregnancy. Our finding that pregnancy is associated with an increased lung inflammatory response to aerosolized endotoxin raises the possibility that there may be a generalized enhancement of pulmonary responses to inhaled toxic agents during pregnancy.
Assuntos
Endotoxinas/toxicidade , Inflamação , Exposição por Inalação , Pneumopatias/etiologia , Aerossóis , Animais , Endotoxinas/administração & dosagem , Feminino , Gravidez , Complicações na Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
As the result of a high prevalence of fixed airways obstruction in workers at a microwave popcorn manufacturing plant, we examined the hypothesis that vapors of butter flavoring used in the manufacture of microwave popcorn and other foods can produce airway injury in rats. Rats were exposed to vapors liberated from heated butter flavoring. Rats were exposed for 6 h by inhalation and were necropsied 1 day after exposure. The exposure was found by GC-MS analysis to be a complex mixture of various organic gases with the major peaks consisting of diacetyl (2,3-butanedione), acetic acid, acetoin (3-hydroxy-2-butanone), butyric acid, acetoin dimers, 2-nonanone, and delta-alkyl lactones. Diacetyl was used as a marker of exposure concentration. In the lung, butter flavoring vapors containing 285-371 ppm diacetyl caused multifocal, necrotizing bronchitis, which was most consistently present in the mainstem bronchus. Alveoli were unaffected. Butter flavoring vapors containing 203-371 ppm diacetyl caused necrosuppurative rhinitis, which affected all four levels of the nose. Within the posterior two nasal levels (T3 and T4), necrosis and inflammation was principally localized to the nasopharyngeal duct. Control rats were unaffected. Therefore, concentrations of butter flavoring vapors that can occur during the manufacture of foods are associated with epithelial injury in the nasal passages and pulmonary airways of rats.
Assuntos
Brônquios/patologia , Diacetil/toxicidade , Aromatizantes/toxicidade , Mucosa Nasal/patologia , Animais , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Histocitoquímica , Exposição por Inalação , Masculino , Microscopia Eletrônica , Líquido da Lavagem Nasal/citologia , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/metabolismo , Necrose , Ratos , Ratos Sprague-Dawley , Organismos Livres de Patógenos EspecíficosRESUMO
The risk of lung injury from ozone exposure has been well documented. It is also known that various factors may significantly influence the susceptibility of animals to the toxic effects of ozone. In the present study, we investigated the possibility that hyperthyroidism might be associated with increases in ozone-induced pulmonary toxicity. To create a hyperthyroid condition, mature male Sprague--Dawley rats were given injections of thyroxine (dose range: 0.1 to 1 mg/kg body wt daily for 7 days). Control rats received vehicle injections. The animals were then exposed to air or ozone (dose range: 0.5 to 3 ppm for 3 h). At 18 h postexposure, bronchoalveolar lavage fluid and cells were harvested. In hyperthyroid animals, ozone exposure was associated with three- to sixfold increases in bronchoalveolar lavage fluid lactate dehydrogenase activities and albumin levels as well as the number of polymorphonuclear leukocytes harvested by bronchoalveolar lavage above levels observed in ozone-exposed control rats. Additional results from the present study suggest that these thyroid hormone-linked effects cannot be fully explained by differences in whole-body metabolic rate or changes in the inhaled dose of ozone. These findings indicate that the risk of ozone-induced lung toxicity is substantially increased in a hyperthyroid state and suggest that the susceptibility of the lung to damage from ozone exposure may be significantly influenced by individual thyroid hormone status.
Assuntos
Hipertireoidismo/complicações , Pneumopatias/induzido quimicamente , Ozônio/toxicidade , Albuminas/análise , Animais , Metabolismo Basal , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Edema/induzido quimicamente , Hipertireoidismo/induzido quimicamente , L-Lactato Desidrogenase/análise , Contagem de Leucócitos , Masculino , Neutrófilos , Ozônio/administração & dosagem , Ratos , Ratos Sprague-Dawley , Respiração/efeitos dos fármacos , Tiroxina/administração & dosagem , Tiroxina/sangue , Volume de Ventilação PulmonarRESUMO
We present in vivo fluorescent, near-infrared (NIR), reflectance images of indocyanine green (ICG) and carotene-conjugated 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide (HPPH-car) to discriminate spontaneous canine adenocarcinoma from normal mammary tissue. Following intravenous administration of 1.0 mg kg-1 ICG or 0.3 mg kg-1 HPPH-car into the canine, a 25 mW, 778 nm or 70 mW, 660 nm laser diode beam, expanded by a diverging lens to approximately 4 cm in diameter, illuminated the surface of the mammary tissue. Successfully propagating to the tissue surface, ICG or HPPH-car fluorescence generated from within the tissue was collected by an image-intensified, charge-coupled device camera fitted with an 830 or 710 nm bandpass interference filter. Upon collecting time-dependent fluorescence images at the tissue surface overlying both normal and diseased tissue volumes, and fitting these images to a pharmacokinetic model describing the uptake (wash-in) and release (wash-out) of fluorescent dye, the pharmacokinetics of fluorescent dye was spatially determined. Mapping the fluorescence intensity owing to ICG indicates that the dye acts as a blood pool or blood persistent agent, for the model parameters show no difference in the ICG uptake rates between normal and diseased tissue regions. The wash-out of ICG was delayed for up to 72 h after intravenous injection in tissue volumes associated with disease, because ICG fluorescence was still detected in the diseased tissue 72 h after injection. In contrast, HPPH-car pharmacokinetics illustrated active uptake into diseased tissues, perhaps owing to the overexpression of LDL receptors associated with the malignant cells. HPPH-car fluorescence was not discernable after 24 h. This work illustrates the ability to monitor the pharmacokinetic delivery of NIR fluorescent dyes within tissue volumes as great as 0.5-1 cm from the tissue surface in order to differentiate normal from diseased tissue volumes on the basis of parameters obtained from the pharmacokinetic models.
Assuntos
Clorofila/análogos & derivados , Verde de Indocianina/farmacocinética , Neoplasias Mamárias Animais/diagnóstico , Fármacos Fotossensibilizantes/farmacocinética , Adenocarcinoma/diagnóstico , Animais , Carotenoides/farmacocinética , Clorofila/farmacocinética , Doenças do Cão/diagnóstico , Cães , Feminino , Espectrometria de Fluorescência , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
GAP1(IP4BP) is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)]. We have studied Ins(1,3,4,5)P(4) binding to GAP1(IP4BP), and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P(4) binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P(4) binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P(4) has shown that the binding site is located in a partially buried pocket between the beta 1/beta 2- and beta 3/beta 4-loops. Many of the residues involved in the binding are conserved in GAP1(IP4BP). Therefore we generated a model of the PH domain of GAP1(IP4BP) in complex with Ins(1,3,4,5)P(4) based on the Btk-Ins(1,3,4,5)P(4) complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P(4), indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.
Assuntos
Proteínas Sanguíneas/química , Fosfatos de Inositol/química , Fosfoproteínas/química , Receptores Citoplasmáticos e Nucleares/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Lisina/química , Metionina/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Triptofano/químicaRESUMO
The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).
Assuntos
Membrana Celular/metabolismo , Fosfatos de Inositol/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células COS , Núcleo Celular/metabolismo , Células HeLa , Humanos , Lipossomos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/metabolismo , Sacarose , TransfecçãoRESUMO
Ozone (O(3)) is toxic to respiratory epithelium and causes airway inflammation and hyperreactivity. To evaluate the role of the epithelium in the development of hyperreactivity, we examined in guinea pigs the effects of inhaled O(3) (3 ppm for 1 h; 0-24 h after exposure) on 1) reactivity to inhaled methacholine (MCh), 2) reactivity of the isolated, perfused trachea (IPT) to MCh, 3) epithelium-derived relaxing factor (EpDRF)-mediated relaxations of IPT induced by mucosal hyperosmolar solutions, 4) neurogenic contraction and relaxation responses, 5) transepithelial potential difference, and 6) microscopic analysis of nitrotyrosine immunofluorescence, substance P fiber density, and tracheal morphology. At 0 h, O(3) caused hyperreactivity to inhaled MCh and mucosally but not serosally applied MCh in IPT (only in the presence of the epithelium) and a decrease in transepithelial potential difference. Inhibition of EpDRF-induced relaxation responses occurred at 2 h. All of these changes returned to control by 12 to 18 h. O(3) had no effect on neurogenic responses. Nitrotyrosine immunofluorescence appeared in the trachea at 0 h in detached epithelial cell ghosts and in intrapulmonary airways by 6 h. Substance P fiber density was elevated in smooth muscle at 0 and 18 h but not in epithelium or lamina propria of intrapulmonary and extrapulmonary bronchi. Loss of cilia and mucosubstances in the mucosa occurred at 0 h; the epithelium became markedly attenuated over 12 to 24 h. A reversible increase in epithelial permeability and a decrease in EpDRF production may contribute to O(3)-induced hyperreactivity to MCh.
Assuntos
Óxido Nítrico/biossíntese , Ozônio/toxicidade , Traqueia/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Epitélio/fisiologia , Cobaias , Técnicas In Vitro , Masculino , Cloreto de Metacolina/farmacologia , Perfusão , Traqueia/patologia , Traqueia/fisiologiaRESUMO
We present near-infrared frequency-domain photon migration imaging for the lifetime sensitive detection and localization of exogenous fluorescent contrast agents within tissue-simulating phantoms and actual tissues. We employ intensity-modulated excitation light that is expanded and delivered to the surface of a tissue or tissue-simulating phantom. The intensity-modulated fluorescence generated from within the volume propagates to the surface and is collected using a gain-modulated image-intensified charge-coupled device camera. From the spatial values of modulation amplitude and phase of the detected fluorescent light, micromolar volumes of diethylthiatricarbocyanine iodide (tau = 1.17 ns) and indocyanine green (ICG) (tau = 0.58 ns) embedded 1.0 cm deep in a tissue phantom are localized and discriminated on the basis of their lifetime differences. To demonstrate the utility of frequency-domain fluorescent measurements for imaging disease, we image the fluorescence emitted from the surface of in vivo and ex vivo canine mammary gland tissues containing lesions with preferential uptake of ICG. Pathology confirms the ability to detect spontaneous mammary tumors and regional lymph nodes amidst normal mammary tissue and fat as deep as 1.5 cm from the tissue surface.
Assuntos
Doenças do Cão/diagnóstico por imagem , Neoplasias Mamárias Animais/diagnóstico por imagem , Animais , Meios de Contraste , Doenças do Cão/patologia , Cães , Corantes Fluorescentes , Metástase Linfática/diagnóstico por imagem , Neoplasias Mamárias Animais/patologia , Modelos Anatômicos , RadiografiaRESUMO
GAP1(IP4BP) and GAP1(m) belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP4 receptors. Here we show they are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1(IP4BP) is primarily localised to the plasma membrane, whereas GAP1(m) appears localised to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation did not indicate a specific co-localisation between membrane-bound GAP1(m) and several Ca2+ store markers, consistent with the lack of co-localisation between GAP1(m) and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1(m) does not reside at a site where it could regulate the ability of InsP4 to release intracellular Ca2+. As GAP1(m) is primarily localised to the cytosol of unstimulated cells it may be spatially regulated in order to interact with Ras at the plasma membrane.
Assuntos
Proteínas de Transporte/metabolismo , Fosfatos de Inositol/metabolismo , Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Ativadoras de ras GTPase , Animais , Células COS , ATPases Transportadoras de Cálcio/biossíntese , Proteínas de Transporte/biossíntese , Técnica Indireta de Fluorescência para Anticorpo , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Leucemia Basofílica Aguda , Especificidade de Órgãos , Biossíntese de Proteínas , Ratos , Frações Subcelulares/metabolismo , Células Tumorais CultivadasRESUMO
A method is presented to determine fluorescence decay lifetimes within tissuelike scattering media. Fluorescence lifetimes are determined for micromolar concentrations of the dyes 3,3'-Diethylthiatricarbocyanine Iodide and Indocyanine Green by frequency-domain investigations of light propagating in turbid media. Dual-wavelength photon-migration measurements that use intensity-modulated sources at excitation and emission wavelengths of the fluorophores provide optical parameters of the media as well as fluorescence properties of the dyes. The deduction of fluorescence lifetimes requires no calibration with reference fluorophores, and the results are shown to be independent of dye concentration.
RESUMO
Previously we have purified and cloned a high affinity isomerically specific inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4)-binding protein which, because it is clearly a member of the GAP1 family of Ras GTPase-activating proteins (GAP), we have termed GAP1(IP4BP). Here we show that expressed full-length GAP1(IP4BP) binds Ins(1,3,4, 5)P4 with an affinity and specificity similar to that of the originally purified protein, a binding activity which is dependent on a functional PH/Btk domain. Furthermore, we highlight a fundamental distinction between GAP1(IP4BP) and its homologue GAP1(m), namely that both proteins function as Ras GAPs but only GAP1(IP4BP) displays Rap GAP activity.
Assuntos
Fosfatos de Inositol/metabolismo , Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Ativadoras de ras GTPase , Sequência de Bases , Sítios de Ligação , Primers do DNA , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas/química , Proteínas/genética , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Frequency-domain measurements of photon migration are coupled with a model of fluorescence generation and propagation in order to develop a method for reconstructing maps of fluorescent properties within interior tissue volumes from exterior measurements at the air-tissue interface. Simulation results confirm the feasibility of optical imaging through the use of exogenously administered contrast agents on the basis of fluorophore decay kinetics and yield. Experimental measurements using single-pixel and multipixel devices illustrate that the contrast owing to exogenous fluorescence exceeds that owing to absorption or scattering caused by endogenous chromophores or tissue structure and owing to absorption caused by exogenous contrast agents.
Assuntos
Diagnóstico por Imagem , Fótons , Espectrometria de Fluorescência , HumanosRESUMO
The ability to map interior optical properties of a highly scattering medium from exterior measurements of light propagation is afforded by optical tomography. In this communication, we describe the problem of optical tomography, the techniques of photon migration measurements necessary to accomplish it, and the development of multipixel measurements for rapid collection of optical signals. These multipixel measurements are shown to provide detection of contrast owing to the optical properties of absorption and fluorescence associated with dye-laden heterogeneities embedded in a tissue-like scattering medium. From these rapid measurements, successful reconstruction of an interior optical property map may now be possible with clinically realistic data acquisition times. Applications for the technology arise for biomedical optical imaging for the in vivo detection of disease and the diagnosis of tissue (bio-) chemistry.
Assuntos
Fótons , Tomografia/métodos , Absorção , Fluorescência , Matemática , Óptica e FotônicaRESUMO
The ability to optically image or detect diseased tissue volumes located deep within tissues depends upon the degree of contrast provided by differences in local optical properties. In this report, we show that the exogenous contrast offered by fluorescent compounds is superior to that provided by nonfluorescing, light-absorbing compounds when time-dependent measurements are employed. In addition, we show that the induced contrast is not only moderated by the preferential uptake of fluorescent agents into diseased tissue volumes of interest but also by the fluorescent optical properties and the fluorescence dynamics in the specific tissue volume. Using tissue phantom studies, we demonstrated experimentally that near-infrared-absorbing and fluorescent dyes such as indocyanine green can provide detection of diseased tissue volumes from fluorescence measurements made at the periphery of tissue when there is perfect, 100-fold and 10-fold partitioning in diseased tissues over that in surrounding normal tissues. Experimental results of common laser dyes show the contrast is also mediated by the quantum yield and lifetime parameters that may be dependent upon the local tissue environment.
Assuntos
Imagens de Fantasmas , Espectrometria de Fluorescência/métodos , Espectrofotometria/métodos , Diagnóstico , Corantes Fluorescentes , Verde de Indocianina , Lasers , Espectrometria de Fluorescência/instrumentação , Espectrofotometria/instrumentaçãoRESUMO
We present a straightforward procedure for frequency domain modeling of reradiation in a highly scattering medium with an arbitrary, finite three-dimensional geometry. We use a finite difference numerical solver to determine the fluence distribution at the excitation wavelength, which is then coupled to the emission wavelength with an array of equivalent reradiating sources. We then calculate the fluence distribution at the emission wavelength with a second, independent numerical simulation with new optical parameters appropriate to the emission wavelength, using the distributed reradiating sources as the excitation. We compare three-dimensional simulations of a fluorophore distributed in a scattering medium with experimental data. We also compare simulations of the Raman reradiation of small diamonds in a scattering medium with experiment.