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1.
J Cell Sci ; 132(1)2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30478195

RESUMO

Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF receptor 1 (FGFR1) signaling; however, they induce remarkably distinct receptor trafficking and cellular responses. The molecular basis of such a dichotomy and the role of distinct types of ligand-receptor interaction remain elusive. Number of molecules and brightness (N&B) analysis revealed that FGF2 and NCAM promote different FGFR1 assembly and dynamics at the plasma membrane. NCAM stimulation elicits long-lasting cycles of short-lived FGFR1 monomers and multimers, a behavior that might reflect a rapid FGFR1 internalization and recycling. FGF2, instead, induces stable dimerization at the dose that stimulates cell proliferation. Reducing the occupancy of FGFR1 in response to low FGF2 doses causes a switch towards cyclically exposed and unstable receptor dimers, consistently with previously reported biphasic response to FGF2 and with the divergent signaling elicited by different ligand concentrations. Similar instability was observed upon altering the endocytic pathway. Thus, FGF2 and NCAM induce differential FGFR1 clustering at the cell surface, which might account for the distinct intracellular fate of the receptor and, hence, for the different signaling cascades and cellular responses.


Assuntos
Membrana Celular/metabolismo , Proliferação de Células , Fator 2 de Crescimento de Fibroblastos/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Movimento Celular , Endocitose , Fator 2 de Crescimento de Fibroblastos/genética , Células HeLa , Humanos , Moléculas de Adesão de Célula Nervosa/genética , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
2.
Bioconjug Chem ; 26(1): 153-60, 2015 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-25494619

RESUMO

Multifunctional nanoparticles are usually produced by sequential synthesis, with long multistep protocols. Our study reports a generic modular strategy for the parallel one-step multifunctionalization of different hydrophobic nanoparticles. The method was designed and developed by taking advantage of the natural noncovalent interactions between the fatty acid binding sites of the bovine serum albumin (BSA) and the aliphatic surfactants on different inorganic nanomaterials. As a general example of the approach, three different nanoparticles-iron oxide, upconverting nanophosphors, and gold nanospheres-were nanoemulsified in water with BSA. To support specific applications, multifunctional capability was incorporated with a variety of previously modified BSA modules. These modules include different conjugated groups, such as chelating agents for (68)Ga or (89)Zr and ligand molecules for enhanced in vivo targeting. A large library of 13 multimodal contrast agents was developed with this convergent strategy. This platform allows a highly versatile and easy tailoring option for efficient incorporation of functional groups. Finally, as demonstration of this versatility, a bimodal (PET/MRI) probe including a maleimide-conjugated BSA was selectively synthesized with an RGD peptide for in vivo imaging detection of tumor angiogenesis.


Assuntos
Meios de Contraste/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Tomografia por Emissão de Pósitrons/métodos , Animais , Bovinos , Meios de Contraste/farmacocinética , Meios de Contraste/toxicidade , Ácidos Graxos/metabolismo , Fibroblastos/efeitos dos fármacos , Maleimidas/química , Camundongos , Modelos Moleculares , Conformação Molecular , Nanopartículas/toxicidade , Oligopeptídeos/química , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Distribuição Tecidual
3.
Life Sci Alliance ; 7(9)2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38886017

RESUMO

The unfolded protein response can switch from a pro-survival to a maladaptive, pro-apoptotic mode. During ER stress, IRE1α sensors dimerize, become phosphorylated, and activate XBP1 splicing, increasing folding capacity in the ER protein factory. The steps that turn on the IRE1α endonuclease activity against endogenous mRNAs during maladaptive ER stress are still unknown. Here, we show that although necessary, IRE1α dimerization is not sufficient to trigger phosphorylation. Random and/or guided collisions among IRE1α dimers are needed to elicit cross-phosphorylation and endonuclease activities. Thus, reaching a critical concentration of IRE1α dimers in the ER membrane is a key event. Formation of stable IRE1α clusters is not necessary for RNase activity. However, clustering could modulate the potency of the response, promoting interactions between dimers and decreasing the accessibility of phosphorylated IRE1α to phosphatases. The stepwise activation of IRE1α molecules and their low concentration at the steady state prevent excessive responses, unleashing full-blown IRE1 activity only upon intense stress conditions.


Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases , Proteínas Serina-Treonina Quinases , Endorribonucleases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Humanos , Estresse do Retículo Endoplasmático/fisiologia , Multimerização Proteica , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismo , Ribonucleases/metabolismo
4.
Hemasphere ; 7(8): e931, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37492437

RESUMO

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.

5.
Nat Cell Biol ; 25(1): 120-133, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36543981

RESUMO

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.


Assuntos
Cavéolas , Caveolina 1 , Cavéolas/metabolismo , Caveolina 1/metabolismo , Mecanotransdução Celular , Membrana Celular/metabolismo , Proteínas/metabolismo
6.
FASEB J ; 25(9): 2883-97, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21602447

RESUMO

We studied the molecular forms of the GPI-anchored urokinase plasminogen activator receptor (uPAR-mEGFP) in the human embryo kidney (HEK293) cell membrane and demonstrated that the binding of the amino-terminal fragment (ATF) of urokinase plasminogen activator is sufficient to induce the dimerization of the receptor. We followed the association kinetics and determined precisely the dimeric stoichiometry of uPAR-mEGFP complexes by applying number and brightness (N&B) image analysis. N&B is a novel fluctuation-based approach for measuring the molecular brightness of fluorophores in an image time sequence in live cells. Because N&B is very sensitive to long-term temporal fluctuations and photobleaching, we have introduced a filtering protocol that corrects for these important sources of error. Critical experimental parameters in N&B analysis are illustrated and analyzed by simulation studies. Control experiments are based on mEGFP-GPI, mEGFP-mEGFP-GPI, and mCherry-GPI, expressed in HEK293. This work provides a first direct demonstration of the dimerization of uPAR in live cells. We also provide the first methodological guide on N&B to discern minor changes in molecular composition such as those due to dimerization events, which are involved in fundamental cell signaling mechanisms.


Assuntos
Membrana Celular/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Algoritmos , Simulação por Computador , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Immunoblotting , Microscopia de Fluorescência/instrumentação , Multimerização Proteica , Reprodutibilidade dos Testes , Vitronectina/metabolismo
7.
Front Cell Dev Biol ; 9: 655773, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34277604

RESUMO

HS1, the hematopoietic homolog of cortactin, acts as a versatile actin-binding protein in leucocytes. After phosphorylation, it is involved in GTPase and integrin activation, and in BCR, TCR, and CXCR4 downstream signaling. In normal and leukemic B cells, HS1 is a central cytoskeletal interactor and its phosphorylation and expression are prognostic factors in chronic lymphocytic leukemia (CLL) patients. We here introduce for the first time a super-resolution imaging study based on single-cell 3D-STED microscopy optimized for revealing and comparing the nanoscale distribution of endogenous HS1 in healthy B and CLL primary cells. Our study reveals that the endogenous HS1 forms heterogeneous nanoclusters, similar to those of YFP-HS1 overexpressed in the leukemic MEC1 cell line. HS1 nanoclusters in healthy and leukemic B cells form bulky assemblies at the basal sides, suggesting the recruitment of HS1 for cell adhesion. This observation agrees with a phasor-FLIM-FRET and STED colocalization analyses of the endogenous MEC1-HS1, indicating an increased interaction with Vimentin at the cell adhesion sites. In CLL cells isolated from patients with poor prognosis, we observed a larger accumulation of HS1 at the basal region and a higher density of HS1 nanoclusters in the central regions of the cells if compared to good-prognosis CLL and healthy B cells, suggesting a different role for the protein in the cell types analyzed. Our 3D-STED approach lays the ground for revealing tiny differences of HS1 distribution, its functionally active forms, and colocalization with protein partners.

8.
Cells ; 9(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32028690

RESUMO

Membrane-bound proteases play a key role in biology by degrading matrix proteins or shedding adhesion receptors. MT1-MMP metalloproteinase is critical during cancer invasion, angiogenesis, and development. MT1-MMP activity is strictly regulated by internalization, recycling, autoprocessing but also through its incorporation into tetraspanin-enriched microdomains (TEMs), into invadopodia, or by its secretion on extracellular vesicles (EVs). We identified a juxtamembrane positively charged cluster responsible for the interaction of MT1-MMP with ERM (ezrin/radixin/moesin) cytoskeletal connectors in breast carcinoma cells. Linkage to ERMs regulates MT1-MMP subcellular distribution and internalization, but not its incorporation into extracellular vesicles. MT1-MMP association to ERMs and insertion into TEMs are independent phenomena, so that mutation of the ERM-binding motif in the cytoplasmic region of MT1-MMP does not preclude its association with the tetraspanin CD151, but impairs the accumulation and coalescence of CD151/MT1-MMP complexes at actin-rich structures. Conversely, gene deletion of CD151 does not impact on MT1-MMP colocalization with ERM molecules. At the plasma membrane MT1-MMP autoprocessing is severely dependent on ERM association and seems to be the dominant regulator of the enzyme collagenolytic activity. This newly characterized MT1-MMP/ERM association can thus be of relevance for tumor cell invasion.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Células MCF-7 , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/genética , Microdomínios da Membrana/metabolismo , Mutação/genética , Ligação Proteica , Domínios Proteicos , Frações Subcelulares/metabolismo , Tetraspanina 24/metabolismo
9.
J Vis Exp ; (153)2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31762461

RESUMO

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis. N&B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.


Assuntos
Microscopia de Fluorescência/métodos , Receptores de Superfície Celular/fisiologia , Difusão , Corantes Fluorescentes , Células HeLa , Humanos , Fótons , Espectrometria de Fluorescência/métodos
10.
Biophys J ; 94(2): L14-6, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17981902

RESUMO

Changing the data representation from the classical time delay histogram to the phasor representation provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we can easily recognize the presence of different molecular species in a pixel or the occurrence of fluorescence resonance energy transfer. The analysis of the fluorescence lifetime imaging microscopy (FLIM) data in the phasor space is done observing clustering of pixels values in specific regions of the phasor plot rather than by fitting the fluorescence decay using exponentials. The analysis is instantaneous since is not based on calculations or nonlinear fitting. The phasor approach has the potential to simplify the way data are analyzed in FLIM, paving the way for the analysis of large data sets and, in general, making the FLIM technique accessible to the nonexpert in spectroscopy and data analysis.


Assuntos
Microscopia de Fluorescência/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Fibroblastos/citologia , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Paxilina/metabolismo , Transfecção
11.
Chem Biol ; 14(4): 431-41, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17462578

RESUMO

High-mobility group box 1 protein (HMGB1) is a nuclear component, but extracellularly it serves as a signaling molecule involved in acute and chronic inflammation, for example in sepsis and arthritis. The identification of HMGB1 inhibitors is therefore of significant experimental and clinical interest. We show that glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, inhibits HMGB1 chemoattractant and mitogenic activities, and has a weak inhibitory effect on its intranuclear DNA-binding function. NMR and fluorescence studies indicate that glycyrrhizin binds directly to HMGB1 (K(d) approximately 150 microM), interacting with two shallow concave surfaces formed by the two arms of both HMG boxes. Our results explain in part the anti-inflammatory properties of glycyrrhizin, and might direct the design of new derivatives with improved HMGB1-binding properties.


Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Citocinas/antagonistas & inibidores , Ácido Glicirrízico/metabolismo , Proteína HMGB1/antagonistas & inibidores , Proteína HMGB1/metabolismo , Células 3T3 , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Sítios de Ligação , Citocinas/metabolismo , DNA/metabolismo , Fluorescência , Ácido Glicirrízico/química , Ácido Glicirrízico/farmacologia , Proteína HMGB1/química , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Mitógenos/antagonistas & inibidores , Mitógenos/farmacologia , Modelos Moleculares , Ligação Proteica
12.
J Biomed Opt ; 13(3): 031215, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18601539

RESUMO

The oligomerization of glycosylphosphatidylinositol-anchored proteins is thought to regulate their association with membrane microdomains, subcellular sorting, and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in human embryo kidnay 293 (HEK293) cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, the diffusion coefficient decreased in monomer-enriched fractions only for the active receptor, suggesting that uPAR monomers might be preferentially engaged in multiprotein transmembrane signaling complexes. Our approach helps in limiting the alteration of the data due to out-of-focus effects and in minimizing the overestimation of the molecular brightness. In addition to a careful design of the cellular model, it gives reliable estimates of diffusion coefficients and oligomerization of GPI-anchored proteins, in steady-state conditions, at low expression levels, and in live, unperturbed cells.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Glicosilfosfatidilinositóis/metabolismo , Rim/metabolismo , Fotometria/métodos , Receptores de Superfície Celular/metabolismo , Algoritmos , Linhagem Celular , Humanos , Cinética , Proteínas de Membrana/metabolismo , Fótons , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Espectrometria de Fluorescência
13.
PLoS One ; 12(5): e0177596, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28542327

RESUMO

Yb and Er codoped NaT(XO4)2 (T = Y, La, Gd, Lu and X = Mo, W) disordered oxides show a green (Er3+ related) up-conversion (UC) efficiency comparable to that of Yb:Er:ß-NaYF4 compound and unless 3 times larger UC ratiometric thermal sensitivity. The similar UC efficiency of Yb:Er doped NaT(XO4)2 and ß-NaYF4 compounds allowed testing equal subcutaneous depths of ex-vivo chicken tissue in both cases. This extraordinary behavior for NaT(XO4)2 oxides with large cutoff phonon energy (hω≈ 920 cm-1) is ascribed to 4F9/2 electron population recycling to higher energy 4G11/2 level by a phonon assisted transition. Crystalline nanoparticles of Yb:Er:NaLu(MoO4)2 have been synthesized by sol-gel with sizes most commonly in the 50-80 nm range, showing a relatively small reduction of the UC efficiency with regards to bulk materials. Fluorescence lifetime and multiphoton imaging microscopies show that these nanoparticles can be efficiently distributed to all body organs of a perfused mouse.


Assuntos
Érbio/química , Fluorescência , Nanopartículas/química , Imagem Óptica , Óxidos/química , Temperatura , Itérbio/química , Animais , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Camundongos , Óxidos/metabolismo , Tamanho da Partícula , Perfusão
14.
Biomaterials ; 76: 157-72, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26524536

RESUMO

The generation of functional, vascularized tissues is a key challenge for both tissue engineering applications and the development of advanced in vitro models analyzing interactions among circulating cells, endothelium and organ-specific microenvironments. Since vascularization is a complex process guided by multiple synergic factors, it is critical to analyze the specific role that different experimental parameters play in the generation of physiological tissues. Our goals were to design a novel meso-scale model bridging the gap between microfluidic and macro-scale studies, and high-throughput screen the effects of multiple variables on the vascularization of bone-mimicking tissues. We investigated the influence of endothelial cell (EC) density (3-5 Mcells/ml), cell ratio among ECs, mesenchymal stem cells (MSCs) and osteo-differentiated MSCs (1:1:0, 10:1:0, 10:1:1), culture medium (endothelial, endothelial + angiopoietin-1, 1:1 endothelial/osteo), hydrogel type (100%fibrin, 60%fibrin+40%collagen), tissue geometry (2 × 2 × 2, 2 × 2 × 5 mm(3)). We optimized the geometry and oxygen gradient inside hydrogels through computational simulations and we analyzed microvascular network features including total network length/area and vascular branch number/length. Particularly, we employed the "Design of Experiment" statistical approach to identify key differences among experimental conditions. We combined the generation of 3D functional tissue units with the fine control over the local microenvironment (e.g. oxygen gradients), and developed an effective strategy to enable the high-throughput screening of multiple experimental parameters. Our approach allowed to identify synergic correlations among critical parameters driving microvascular network development within a bone-mimicking environment and could be translated to any vascularized tissue.


Assuntos
Osso e Ossos/irrigação sanguínea , Simulação por Computador , Engenharia Tecidual , Técnicas de Cocultura , Matriz Extracelular , Humanos , Técnicas In Vitro
15.
Mol Cancer Ther ; 2(1): 29-40, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12533670

RESUMO

Soluble copolymers of camptothecin (CPT), based on poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA), were obtained by conjugation through the degradable spacers -Gly-Phe-Leu-Gly- or -Gly-6-aminohexanoyl-Gly-. We investigated to what extent passive accumulation and retention of hydroxypropyl methacrylamide copolymer of CPT (pHPMA-CPT) in tumors and modulation of the drug release influence efficacy. Release of CPT in vivo was detected by time-resolved phase-shift fluorescence imaging on tumor specimens, based on the evidence that free and bound drug had different fluorescence lifetimes in solution. HT-29 murine specimens, obtained at several times after treatment with (3)H-labeled free CPT, pHPMA-Gly-Phe-Leu-Gly-CPT, or pHPMA-Gly-6-aminohexanoyl-Gly-CPT, were either imaged for time-resolved phase-shift fluorescence or subjected to autoradiography. Phase shifts of CPT conjugates were equal or longer than those of free CPT, indicating the presence of both free and polymer-bound drug in the tumor, in agreement with autoradiograms. pHPMA-Gly-Phe-Leu-Gly-CPT underwent relevant intratumor hydrolysis during the first 24 h, whereas the hydrolysis of pHPMA-Gly-6-aminohexanoyl-Gly-CPT was slow. The latter showed antitumor activity at doses from 10 to 22.5 mg/kg/day against s.c. HT-29, A2780, M14, and A549 s.c. xenografts. Moreover, inhibition of tumor growth lasted for up to 73-88 days, and cures were observed on mice with orthotopic implanted HT-29; pHPMA-Gly-Phe-Leu-Gly-CPT was 2-fold more potent than pHPMA-Gly-6-aminohexanoyl-Gly-CPT but less tolerated. Our data suggest that the efficacy of pHPMA-CPT copolymers is related to their intratumor accumulation, and in vivo properties of releasing CPT by esterolytic and proteolytic degradation.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Ésteres , Metacrilatos , Acrilamidas , Animais , Antineoplásicos Fitogênicos/farmacocinética , Camptotecina/farmacocinética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/tratamento farmacológico , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Distribuição Tecidual , Transplante Heterólogo
16.
Biophys Chem ; 110(3): 281-95, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15228964

RESUMO

Copolymers of camptothecin (CPT) and [N-(2-hydroxypropyl) methacrylamide] (HPMA) are novel anticancer drugs that show improved pharmacological profile in animal models as compared to the free drug CPT. We investigate here the aggregation properties of a HPMA-glycyl-6-aminohexanoyl-glycyl-CPT copolymer ( approximately 20,000 Da). The molecular size of HPMA-copolymer CPT is followed over 5 orders of magnitudes of concentration in isotonic buffer by measuring either the time resolved fluorescence anisotropy (FA) of CPT or the autocorrelation function of the light scattered by the copolymer. A detailed analysis of these data suggests the presence of elongated structures with axial ratio approximately 3 in the range 0.1-0.5 microg/ml and aggregates with association number higher than 2 in more concentrated solutions (up to 10 mg/ml). The binding affinity of HPMA-copolymer CPT for serum albumin is inversely dependent on the degree of aggregation of the copolymer. We also show that the copolymer concentration in plasma from mice treated with an active, non-toxic, dose of HPMA-copolymer CPT, decreases from 3 to 0.01 mg/ml in 72 h. In the same range of concentrations in vitro, we do not detect hydrophobic aggregates of polymers with high (>3) association number. Our study indicates that the circulating HPMA-copolymer CPT in mice should not undergo extensive aggregation and should interact with serum albumin more weakly than free CPT.


Assuntos
Camptotecina/química , Metacrilatos/química , Polímeros/química , Anisotropia , Técnicas de Diluição do Indicador , Soluções Isotônicas/química , Estrutura Molecular , Albumina Sérica/farmacologia , Espectrometria de Fluorescência , Fatores de Tempo
17.
Microsc Res Tech ; 76(11): 1135-46, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23934660

RESUMO

Number of molecules and Brightness (N&B) has been proposed for measuring the molecular brightness and number of fluorophores in time-sequence of images, in live cells. If the fluorescently tagged-proteins are mobile in the illumination volume, the stoichiometry of their oligomers can be derived from the increase of the brightness of the fluorescent dyes due to clustering. We examine aspects concerning extra-fluctuation effects induced by cell shifts and photobleaching, which yield large overestimates of the clusters size and sub-unit counts. We develop an offline corrective approach consisting in frame re-alignment and boxcar filtering for recovering precision of the analysis. Using simulations we derive general criteria for approaching this analysis, and assess the application limits of the corrective procedure. We tested the approach in extreme experimental conditions (few pixels, large extra-variance perturbations), in which we analyzed the minimal increases of brightness as that expected between a monomeric and dimeric GPI-mEGFP constructs. We show how most of the perturbing effects can be abolished, and obtain the correct the brightness of GPI-mEGFP monomers and dimers.


Assuntos
Citosol/química , Corantes Fluorescentes/análise , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Linhagem Celular , Fluorescência , Humanos
18.
J Cell Biol ; 183(3): 527-42, 2008 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-18955551

RESUMO

VCAM-1 and ICAM-1, receptors for leukocyte integrins, are recruited to cell-cell contact sites on the apical membrane of activated endothelial cells. In this study, we show that this recruitment is independent of ligand engagement, actin cytoskeleton anchorage, and heterodimer formation. Instead, VCAM-1 and ICAM-1 are recruited by inclusion within specialized preformed tetraspanin-enriched microdomains, which act as endothelial adhesive platforms (EAPs). Using advanced analytical fluorescence techniques, we have characterized the diffusion properties at the single-molecule level, nanoscale organization, and specific intradomain molecular interactions of EAPs in living primary endothelial cells. This study provides compelling evidence for the existence of EAPs as physical entities at the plasma membrane, distinct from lipid rafts. Scanning electron microscopy of immunogold-labeled samples treated with a specific tetraspanin-blocking peptide identify nanoclustering of VCAM-1 and ICAM-1 within EAPs as a novel mechanism for supramolecular organization that regulates the leukocyte integrin-binding capacity of both endothelial receptors during extravasation.


Assuntos
Adesão Celular/fisiologia , Endotélio Vascular/fisiologia , Leucócitos/fisiologia , Proteínas de Membrana/fisiologia , Antígenos CD/fisiologia , Adesão Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Endotélio Vascular/ultraestrutura , Humanos , Integrinas/genética , Integrinas/fisiologia , Molécula 1 de Adesão Intercelular/fisiologia , Leucócitos/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/fisiologia , Veias Umbilicais/ultraestrutura , Molécula 1 de Adesão de Célula Vascular/fisiologia
19.
J Cell Biol ; 179(5): 1067-82, 2007 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-18056417

RESUMO

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer-oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein-uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA-plasminogen activator inhibitor type 1-mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents.


Assuntos
Membrana Celular/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Receptores de Superfície Celular/metabolismo , Linhagem Celular , Difusão , Dimerização , Endocitose , Matriz Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Biológicos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ligação Proteica , Transporte Proteico , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Soro , Vitronectina/metabolismo
20.
Biophys J ; 82(5): 2652-64, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11964252

RESUMO

Fibroblast growth factor-2 (basic FGF), a potent inducer of angiogenesis, and the naphthalene sulfonic distamycin A derivative, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate) (PNU145156E), which exhibits in vivo antiangiogenic activity, form a tight reversible (1:1) complex. PNU145156E binds to the heparin and the selenate-binding sites on bFGF. The cis bFGF-heparin (2:1) complex, essential for the activation of the angiogenic process, is thus prevented. The nature of the forces involved in bFGF:PNU145156E complex, using the wild-type and the K128Q, K138Q, K134Q, and K128Q-K138Q point mutated bFGFs was sought. Based on thermodynamic analysis of the complexation constants, protein temperature stability profiles by ultraviolet absorption, circular dichroism measurements, fluorescence Förster energy-transfer, and anisotropy studies, in harmony with the published x-ray crystallographic structure, the following molecular interactions are proposed: reduced coulombic interactions, hence loosening of the complex by the removal of charged polar groups from the bFGF-heparin binding cleft resulted in decreased binding constants and in a change in the binding mode from polar to nonpolar. Concomitantly, upon mutation, the protein was rendered more compact, less flexible, and less aqueously exposed compared with the wild type. These were further pronounced with the double mutant: weaker dominantly nonpolar protein-drug interactions were accompanied by conspicuous folding. With heparin, however, wild-type bFGF forms a tighter complex with a more compact structure.


Assuntos
Inibidores da Angiogênese/farmacocinética , Distamicinas/farmacocinética , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Dobramento de Proteína , Substituição de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Distamicinas/química , Distamicinas/farmacologia , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neovascularização Fisiológica/efeitos dos fármacos , Conformação Proteica , Desnaturação Proteica , Termodinâmica , Triptofano , Tirosina
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