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1.
Opt Lett ; 48(5): 1088-1091, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36857220

RESUMO

Fiber optic bundles are used in narrow-diameter medical and industrial instruments for acquiring images from confined locations. Images transmitted through these bundles contain only one pixel of information per fiber core and fail to capture information from the cladding region between cores. Both factors limit the spatial resolution attainable with fiber bundles. We show here that computational imaging (CI) can be combined with spectral coding to overcome these two fundamental limitations and improve spatial resolution in fiber bundle imaging. By acquiring multiple images of a scene with a high-resolution mask pattern imposed, up to 17 pixels of information can be recovered from each fiber core. A dispersive element at the distal end of the bundle imparts a wavelength-dependent lateral shift on light from the object. This enables light that would otherwise be lost at the inter-fiber cladding to be transmitted through adjacent fiber cores. We experimentally demonstrate this approach using synthetic and real objects. Using CI with spectral coding, object features 5× smaller than individual fiber cores were resolved, whereas conventional imaging could only resolve features at least 1.5× larger than each core. In summary, CI combined with spectral coding provides an approach for overcoming the two fundamental limitations of fiber optic bundle imaging.

2.
Nat Commun ; 11(1): 5355, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097705

RESUMO

Water and lipids are key participants in many biological processes, but there are few non-invasive methods that provide quantification of these components in vivo, and none that can isolate and quantify lipids in the blood. Here we develop a new imaging modality termed shortwave infrared meso-patterned imaging (SWIR-MPI) to provide label-free, non-contact, spatial mapping of water and lipid concentrations in tissue. The method utilizes patterned hyperspectral illumination to target chromophore absorption bands in the 900-1,300 nm wavelength range. We use SWIR-MPI to monitor clinically important physiological processes including edema, inflammation, and tumor lipid heterogeneity in preclinical models. We also show that SWIR-MPI can spatially map blood-lipids in humans, representing an example of non-invasive and contact-free measurements of in vivo blood lipids. Together, these results highlight the potential of SWIR-MPI to enable new capabilities in fundamental studies and clinical monitoring of major conditions including obesity, cancer, and cardiovascular disease.


Assuntos
Raios Infravermelhos , Lipídeos/sangue , Imagem Óptica/métodos , Ondas de Rádio , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Água/análise , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Marrom/patologia , Adulto , Animais , Biomarcadores/sangue , Doenças Cardiovasculares/diagnóstico por imagem , Edema/diagnóstico por imagem , Edema/patologia , Feminino , Xenoenxertos , Humanos , Inflamação/diagnóstico por imagem , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Obesidade/diagnóstico por imagem , Imagem Óptica/instrumentação , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação
3.
Biol Sex Differ ; 11(1): 54, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32972452

RESUMO

BACKGROUND: The incidence of acute kidney injury (AKI) during pregnancy precedes a high maternal mortality rate of 20-40%. AKI during pregnancy has multiple etiologies; however, the more common are maternal hypertensive disorders, which include preeclampsia and HELLP (hemolysis, elevated liver enzyme, low platelet) syndrome. Therefore, we sought to assess the impact of AKI on blood pressure, kidney injury, and anti-angiogenic factors during pregnancies with and without HELLP syndrome. METHODS: On gestational day (GD) 12, mini-osmotic pumps were inserted into a subset of normal pregnant (NP) rats infusing 4.7 µg/kg soluble fms-like tyrosine kinase-1 (sFlt-1) and 7 µg/kg soluble endoglin (sEng) to induce HELLP syndrome. On GD18, the renal pedicles were occluded for 45 min to induce AKI via bilateral ischemia reperfusion in a subset of NP (n = 18) or HELLP (n = 20) rats. Control NP (n = 20) and HELLP (n = 20) rats underwent a SHAM surgery on GD18. Plasma, urine, and maternal organs were saved for further analysis. Renal injury was assessed via renal histopathology, glomerular filtration rate (GFR), T cell infiltration, and assessment of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Data was measured via two-way analysis of variance with Tukey's test for post hoc analysis. RESULTS: Blood pressures were increased in HELLP+AKI rats (p = 0.0001); both NP+AKI and HELLP+AKI rats had increased lactate dehydrogenase (p < 0.0001) and aspartate aminotransferase levels (p < 0.0001), and decreased platelet levels (p < 0.001) vs. NP rats. HELLP+AKI (p = 0.002) and HELLP rats (p = 0.0002) had evidence of renal fibrosis vs. NP rats. GFR was decreased in HELLP+AKI (p = 0.01) rats vs. NP rats. Urinary KIM-1 was increased in NP+AKI rats vs. NP (p = 0.003) and HELLP rats (p = 0.01). HELLP+AKI rats had increased urinary KIM-1 vs. NP (p = 0.0008) and HELLP rats (p = 0.004) and increased NGAL vs. HELLP rats (p = 0.002). HELLP+AKI rats had increased sFlt-1 (p = 0.009) vs. NP rats. NP+AKI (p = 0.02) and HELLP+AKI (p = 0.007) rats had increased sEng vs. NP rats. CD3+CD4+ T cells were significantly increased in HELLP+AKI rats vs. NP (p = 0.0002) and NP+AKI (p = 0.05) rats. T regulatory cells were significantly decreased in HELLP+AKI (p = 0.03) and NP+AKI (p = 0.02) rats vs. NP rats; there were no changes between groups in T helper 17 cells (p = 0.34). CONCLUSION: The findings in this study suggest that AKI during pregnancy contributes to increased blood pressure and biochemical markers for HELLP syndrome, creates an anti-angiogenic imbalance, and exacerbates kidney injury as shown on histopathology, GFR, and kidney injury markers.


Assuntos
Endoglina/metabolismo , Síndrome HELLP , Nefropatias/etiologia , Rim/citologia , Linfócitos T Reguladores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/sangue , Biomarcadores/urina , Peso ao Nascer , Pressão Sanguínea , Endoglina/genética , Feminino , Nefropatias/patologia , Gravidez , Ratos , Ratos Sprague-Dawley , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética
4.
Am J Physiol Regul Integr Comp Physiol ; 319(2): R195-R202, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32640833

RESUMO

Neutralization of FasL is linked to suppression of hypertension, placental inflammation, and endothelin system activation in an animal model of hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. During HELLP syndrome the placenta has been reported to serve as the primary source of Fas ligand (FasL), which has an impact on inflammation and hypertension during pregnancy and is dysregulated in women with severe preeclampsia and HELLP syndrome. We hypothesize that neutralization of FasL during pregnancy in an animal model of HELLP syndrome decreases inflammation and placental apoptosis, improves endothelial damage, and improves hypertension. On gestational day (GD) 12, rats were chronically infused with placental antiangiogenic factors sFlt-1 and sEng to induce HELLP syndrome. To neutralize FasL, MFL4 or FasL antibody was infused into a subset of HELLP or normal pregnant rats on GD13. IgG infusion into another group of NP and HELLP rats on GD13 was used as a control for FasL antibody, and all rats were euthanized on GD19 after blood pressure measurement. Plasma and placentas were collected to assess inflammation, apoptosis, and the degree of placental debris activation of endothelial cells. Administration of MFL4 to HELLP rats significantly decreased blood pressure compared with untreated HELLP rats and HELLP rats infused with IgG and improved the biochemistry of HELLP syndrome. Both circulating and placental FasL were significantly attenuated in response to MFL4 infusion, as were levels of placental and circulating TNFα when compared with untreated HELLP rats and HELLP rats infused with IgG. Endothelial cells exposed to placental debris and media from HP + MFL4 rats secreted significantly less endothelin-1 compared with stimulated endothelial cells from HELLP placentas. Neutralization of FasL is associated with decreased MAP and improvement in placental inflammation and endothelial damage in an animal model of HELLP syndrome.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Endotelina-1/sangue , Proteína Ligante Fas/imunologia , Síndrome HELLP/tratamento farmacológico , Placenta/fisiopatologia , Animais , Modelos Animais de Doenças , Proteína Ligante Fas/sangue , Feminino , Síndrome HELLP/sangue , Síndrome HELLP/imunologia , Síndrome HELLP/fisiopatologia , Imunoglobulina G , Placenta/imunologia , Gravidez , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangue , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangue
5.
J Biomed Opt ; 24(12): 1-11, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31884745

RESUMO

We demonstrate the possibility of measuring FRET efficiency with a low-cost frequency-domain fluorescence lifetime imaging microscope (FD-FLIM). The system utilizes single-frequency-modulated excitation, which enables the use of cost-effective laser sources and electronics, simplification of data acquisition and analysis, and a dual-channel detection capability. Following calibration with coumarin 6, we measured the apparent donor lifetime in mTFP1-mVenus FRET standards expressed in living cells. We evaluated the system's sensitivity by differentiating the short and long lifetimes of mTFP1 corresponding to the known standards' high and low FRET efficiency, respectively. Furthermore, we show that the lifetime of the vinculin tension sensor, VinTS, at focal adhesions (2.30 ± 0.16 ns) is significantly (p < 10 - 6) longer than the lifetime of the unloaded TSMod probe (2.02 ± 0.16 ns). The pixel dwell time was 6.8 µs for samples expressing the FRET standards, with signal typically an order of magnitude higher than VinTS. The apparent FRET efficiency (EFRETapp) of the standards, calculated from the measured apparent lifetime, was linearly related to their known FRET efficiency by a factor of 0.92 to 0.99 (R2 = 0.98). This relationship serves as a calibration curve to convert apparent FRET to true FRET and circumvent the need to measure multiexponential lifetime decays. This approach yielded a FRET efficiency of 18% to 19.5%, for VinTS, in agreement with published values. Taken together, our results demonstrate a cost-effective, fast, and sensitive FD-FLIM approach with the potential to facilitate applications of FLIM in mechanobiology and FRET-based biosensing.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/química , Animais , Linhagem Celular , Desenho de Equipamento , Transferência Ressonante de Energia de Fluorescência/instrumentação , Adesões Focais/fisiologia , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência/instrumentação , Processamento de Sinais Assistido por Computador , Vinculina/química
6.
Opt Lett ; 44(16): 3968-3971, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31415524

RESUMO

This Letter presents a framework for computational imaging (CI) in fiber-bundle-based endoscopy systems. Multiple observations are acquired of objects spatially modulated with different random binary masks. Sparse-recovery algorithms then reconstruct images with more resolved pixels than individual fibers in the bundle. Object details lying within the diameter of single fibers are resolved, allowing images with 41,663 resolvable points to be generated through a bundle with 2,420 fibers. Computational fiber bundle imaging of micro- and macro-scale objects is demonstrated using fluorescent standards and biological tissues, including in vivo imaging of a human fingertip. In each case, CI recovers details that conventional endoscopy does not provide.

7.
Skelet Muscle ; 8(1): 34, 2018 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-30368252

RESUMO

BACKGROUND: Myostatin antagonists are being developed as therapies for Duchenne muscular dystrophy due to their strong hypertrophic effects on skeletal muscle. Engineered follistatin has the potential to combine the hypertrophy of myostatin antagonism with the anti-inflammatory and anti-fibrotic effects of activin A antagonism. METHODS: Engineered follistatin was administered to C57BL/6 mice for 4 weeks, and muscle mass and myofiber size was measured. In the mdx model, engineered follistatin was dosed for 12 weeks in two studies comparing to an Fc fusion of the activin IIB receptor or an anti-myostatin antibody. Functional measurements of grip strength and tetanic force were combined with tissue analysis for markers of necrosis, inflammation, and fibrosis to evaluate improvement in dystrophic pathology. RESULTS: In wild-type and mdx mice, dose-dependent increases in muscle mass and quadriceps myofiber size were observed for engineered follistatin. In mdx, increases in grip strength and tetanic force were combined with improvements in muscle markers for necrosis, inflammation, and fibrosis. Improvements in dystrophic pathology were greater for engineered follistatin than the anti-myostatin antibody. CONCLUSIONS: Engineered follistatin generated hypertrophy and anti-fibrotic effects in the mdx model.


Assuntos
Ativinas/antagonistas & inibidores , Folistatina/uso terapêutico , Distrofias Musculares/tratamento farmacológico , Miostatina/antagonistas & inibidores , Animais , Folistatina/administração & dosagem , Força da Mão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Contração Muscular , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico
8.
Opt Express ; 24(6): 6145-55, 2016 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-27136808

RESUMO

This paper investigates a highly parallel extension of the single-pixel camera based on a focal plane array. It discusses the practical challenges that arise when implementing such an architecture and demonstrates that system-specific optical effects must be measured and integrated within the system model for accurate image reconstruction. Three different projection lenses were used to evaluate the ability of the system to accommodate varying degrees of optical imperfection. Reconstruction of binary and grayscale objects using system-specific models and Nesterov's proximal gradient method produced images with higher spatial resolution and lower reconstruction error than using either bicubic interpolation or a theoretical system model that assumes ideal optical behavior. The high-quality images produced using relatively few observations suggest that higher throughput imaging may be achieved with such architectures than with conventional single-pixel cameras. The optical design considerations and quantitative performance metrics proposed here may lead to improved image reconstruction for similar highly parallel systems.

9.
Physiol Meas ; 28(6): 611-23, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17664616

RESUMO

Failure of cardiac antiarrhythmic ablation to block action potential conduction produces poor outcomes which lead to repeat procedures. To overcome this, an intraoperative index of the quality of an ablation lesion is needed. We hypothesized that a rise in the translesion stimulus-excitation delay (TED) can indicate a continuous, transmural, linear lesion, and that the TED is related to the path length in the viable tissue around the lesion. Rabbit hearts were isolated, perfused with a warm physiological solution and stained with transmembrane potential-sensitive fluorescent dye. Radiofrequency (RF) ablation was performed on ventricular epicardium with a vacuum-assisted coagulation device to produce either a complete or incomplete lesion. Complete lesions were both transmural and continuous. Incomplete lesions were noncontinuous or nontransmural. The TED was determined with bipolar stimulation at one side of the lesion and either a bipolar electrogram at the other side or optical mapping on both sides. Hearts were then stained with tetrazolium chloride and examined histologically to estimate minimum path lengths of viable tissue from the stimulation site to the recording site. Complete lesions increased the TED by factors of 2.6-3.1 (p < 0.05), whereas incomplete lesions did not significantly increase the TED. Larger minimum path lengths were found for cases that had an increased TED. The TED was quantitatively predictable based on a conduction velocity of 0.38-0.49 m s(-1), which is typical of rabbit hearts. The TED significantly increases when a linear lesion is complete, suggesting that an intraoperative measurement of the TED may help to improve ablation lesions and outcomes. Predictability of the TED based on the viable tissue path suggests that quantitative TEDs for clinical lesions may be anticipated provided that the conduction velocity is considered.


Assuntos
Ablação por Cateter , Técnicas Eletrofisiológicas Cardíacas/métodos , Coração/fisiopatologia , Potenciais de Ação , Animais , Eletrocardiografia , Eletrodos , Coelhos
10.
Ann Biomed Eng ; 33(12): 1802-7, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16389528

RESUMO

Cardiac action potentials have been measured with single-photon excitation (SPE) of transmembrane voltage-sensitive fluorescent dye. Two-photon excitation (TPE) may have advantages for localization and depth of the tissue region from which the action potential is measured. However measurements of action potentials with SPE have not been demonstrated. We sought to develop a method for TPE of di-4-ANEPPS and test whether the method yields voltage-dependent fluorescence in cardiac tissue. We modified our SPE and ratio-metric fluorescence recording system to use a femtosecond pulsed near-infrared laser. Modifications were made to enhance fluorescence collection efficiency and to block infrared laser light from entering the fluorescence collection system. Fluorescence was collected simultaneously in green (510-570 nm) and red (590-700 nm) wavelength bands. Action potentials were observed in the ratio of the green signal to the red signal, but were not observed above the noise level in either of the individual signals. Incorporation of a common-mode noise subtraction method revealed action potentials in green and red signals. We also found that the di-4-ANEPPS fluorescence emission spectrum for TPE at 930 nm was similar to the emission spectrum for SPE at 488 nm. The multiphoton method may be beneficial for highly localized cardiac optical measurements.


Assuntos
Potenciais de Ação/fisiologia , Diagnóstico por Imagem , Fluorescência , Corantes Fluorescentes/farmacologia , Sistema de Condução Cardíaco/fisiologia , Compostos de Piridínio/farmacologia , Animais , Diagnóstico por Imagem/instrumentação , Diagnóstico por Imagem/métodos , Técnicas de Cultura de Órgãos , Coelhos
11.
Ann Biomed Eng ; 32(9): 1202-10, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15493508

RESUMO

Major effects of stimulation on cardiac transmembrane potentials (Vm) are thought to occur under the electrode, however these have not been optically mapped due to blockage of light by electrodes. Here we optically mapped under translucent indium tin oxide (ITO) electrodes in hearts stained with transmembrane voltage sensitive fluorescent dye, di-4-ANEPPS excited at 488 nm. Emissions in wavelength bands 510-570 nm and >590 nm were similarly affected by changes in ITO transmittance due to electrochemical effects of current at the electrode interface. Dual-wavelength ratiometric mapping with the two emission bands revealed Vm under the electrode during plateau-phase stimulation (220 mA). Changes in Vm were heterogeneous under the electrode, and were anisotropic with larger values along the fiber axis. These results explain early excitation sites for sufficiently strong diastolic stimulation, and agree with theoretical predictions based on summation of anisotropic effects of point stimulation and a linear 3-d cardiac bidomain computer model. The bidomain model and experiments disagree under the edge of the electrode, where modeled Vm is much larger. Thus, changes in Vm under an electrode are anisotropic with greater Vm in the direction parallel to fibers. Nonlinear effects of stimulation in hearts may limit changes in Vm under the electrode edge.


Assuntos
Mapeamento Potencial de Superfície Corporal/métodos , Cardioversão Elétrica/métodos , Estimulação Elétrica/métodos , Eletrodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Modelos Cardiovasculares , Animais , Mapeamento Potencial de Superfície Corporal/instrumentação , Simulação por Computador , Cardioversão Elétrica/instrumentação , Estimulação Elétrica/instrumentação , Sistema de Condução Cardíaco/fisiopatologia , Técnicas In Vitro , Potenciais da Membrana/fisiologia , Compostos de Piridínio , Coelhos
12.
J Biol Chem ; 279(22): 23327-34, 2004 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-15039442

RESUMO

The adhesion of platelets to the subendothelium of blood vessels at sites of vascular injury under high shear conditions is mediated by a direct interaction between the platelet receptor glycoprotein Ibalpha (GpIbalpha) and the A1 domain of the von Willebrand factor (VWF). Here we report the 2.6-A crystal structure of a complex comprised of the extracellular domain of GpIbalpha and the wild-type A1 domain of VWF. A direct comparison of this structure to a GpIbalpha-A1 complex containing "gain-of-function" mutations, A1-R543Q and GpIbalpha-M239V, reveals specific structural differences between these complexes at sites near the two GpIbalpha-A1 binding interfaces. At the smaller interface, differences in interaction show that the alpha1-beta2 loop of A1 serves as a conformational switch, alternating between an open alpha1-beta2 isomer that allows faster dissociation of GpIbalpha-A1, as observed in the wild-type complex, and an extended isomer that favors tight association as seen in the complex containing A1 with a type 2B von Willebrand Disease (VWD) mutation associated with spontaneous binding to GpIbalpha. At the larger interface, differences in interaction associated with the GpIbalpha-M239V platelet-type VWD mutation are minor and localized but feature discrete gamma-turn conformers at the loop end of the beta-hairpin structure. The beta-hairpin, stabilized by a strong classic gamma-turn as seen in the mutant complex, relates to the increased affinity of A1 binding, and the beta-hairpin with a weak inverse gamma-turn observed in the wild-type complex corresponds to the lower affinity state of GpIbalpha. These findings provide important details that add to our understanding of how both type 2B and platelet-type VWD mutations affect GpIbalpha-A1 binding affinity.


Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas/química , Fator de von Willebrand/química , Sítios de Ligação/genética , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Doenças de von Willebrand/genética , Fator de von Willebrand/genética
13.
Conf Proc IEEE Eng Med Biol Soc ; 2004: 2102-4, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-17272137

RESUMO

The aim of this study is to develop a system for the first simultaneous measurements using 31P NMR and optical transmembrane potential-sensitive fluorescence. 31P NMR is used to evaluate 5 metabolic markers (pH, sugar phosphates, phosphocreatine, phospholipid intermediates and ATP). Action potential duration is measured with a transmembrane potential-sensitive fluorescent dye, di-4ANEPPS. The system is intended to correlate the action potentials with the metabolic markers and their changes during the time course of cardiac ischemia. The requirements of this system include fabrication of a NMR probe large enough for rabbit heart experiments, incorporation of light guides for excitation of dye and collection of fluorescence in hearts located within a NMR magnet and with minimal disturbance of the magnetic field. The quality factor (Q) of the probe's coil was measured. Control NMR spectra were then acquired with phosphorus test solution. Further spectra were obtained after addition of the optical elements. Results show the ability to use our new probe to acquire a spectrum in the presence of the optical elements within the magnet, suggesting the possibility that 31P NMR spectroscopy and optical transmembrane potential measurements can be performed simultaneously in hearts.

14.
Science ; 301(5630): 222-6, 2003 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-12855811

RESUMO

Direct interaction between platelet receptor glycoprotein Ibalpha (GpIbalpha) and thrombin is required for platelet aggregation and activation at sites of vascular injury. Abnormal GpIbalpha-thrombin binding is associated with many pathological conditions,including occlusive arterial thrombosis and bleeding disorders. The crystal structure of the GpIbalpha-thrombin complex at 2.6 angstrom resolution reveals simultaneous interactions of GpIbalpha with exosite I of one thrombin molecule,and with exosite II of a second thrombin molecule. In the crystal lattice,the periodic arrangement of GpIbalpha-thrombin complexes mirrors a scaffold that could serve as a driving force for tight platelet adhesion. The details of these interactions reconcile GpIbalpha-thrombin binding modes that are presently controversial,highlighting two distinct interfaces that are potential targets for development of novel antithrombotic drugs.


Assuntos
Agregação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombina/química , Trombina/metabolismo , Sítios de Ligação , Plaquetas/química , Plaquetas/fisiologia , Cristalização , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Adesividade Plaquetária , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
15.
J Biol Chem ; 277(10): 8611-7, 2002 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-11602609

RESUMO

Early endosome antigen 1 (EEA1) is a 170-kDa polypeptide required for endosome fusion in mammalian cells. The COOH terminus of EEA1 contains a FYVE domain that interacts specifically with phosphatidylinositol 3-phosphate (PtdIns-3-P) and a Rab5 GTPase binding region adjacent to the FYVE domain. The dual interaction of EEA1 with both PtdIns-3-P and Rab5 has been hypothesized to provide the specificity required to target EEA1 to early endosomes. To test this hypothesis, we generated truncated (amino acids 1277--1411) and full-length EEA1 constructs containing point mutations in the COOH terminus that impair Rab5 but not PtdIns-3-P binding. These constructs localized to endosomes in intact cells as efficiently as their wild-type counterparts. Furthermore, overexpression of the truncated constructs, both wild-type and mutated, impaired the function of endogenous EEA1 resulting in the accumulation of small, untethered endosomes. These results suggest that association with Rab5 is not necessary for the initial binding and tethering functions of EEA1. A role for Rab5 binding was revealed, however, upon comparison of endosomes in cells expressing full-length wild-type or mutated EEA1. The mutant full-length EEA1 caused the accumulation of endosome clusters and suppressed the enlargement of endosomes caused by a persistently active form of Rab5 (Rab5Q79L). In contrast, expression of wild-type EEA1 with Rab5Q79L enhanced this enlargement. Thus, endosome tethering depends on the interaction of EEA1 with PtdIns-3-P, and its interaction with Rab5 appears to regulate subsequent fusion.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/fisiologia , Proteínas rab5 de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Aminoácidos/química , Animais , Células COS , Citosol/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Fusão de Membrana , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Transfecção , Transferrina/farmacocinética , Proteínas de Transporte Vesicular
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