Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Microbiol ; 60(11): 1086-1094, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36318359

RESUMO

Fibroblast growth factor 11 (FGF11) is one of intracrine FGFs (iFGFs), which function within cells. Unlike canonical FGFs, FGF11 remains intracellularly and plays biological roles in FGF receptor (FGFR)-independent manner. Here, we established an expression system of recombinant FGF11 proteins in E. coli and investigated whether the extracellular administration of FGF11 can activate cellular signaling. Human FGF11 has two isoforms, FGF11a and FGF11b, depending on the presence of nuclear localization sequences (NLSs) in the N-terminus. Because these two isoforms are unstable, we prepared an FGF11a-Mut by substituting three cysteine residues in the NLS with serine and FGF11b-ΔC with C-terminal truncation. The introduction of mutation in the NLS improved the solubility of FGF11 prepared from E. coli. Exogenous addition of FGF11b and FGF11b-ΔC to BALB3T3 increased cell proliferation, while FGF11a-Mut exerted no effect. FGF11b-ΔC showed higher cell proliferation activity and FGFR signaling than FGF11b. The cell-proliferating activities of FGF11b and FGF11b-ΔC were blocked by an FGFR1 inhibitor or a recombinant FGFR1, confirming the FGFR1-dependent extracellular activity of FGF11b. The analysis of circular dichroism suggested that the C-terminus of FGF11 has an α-helical structure, which may affect its interaction with FGFR1. These results suggest that the N-and C-terminus of recombinant FGF11 are involved in the activation of FGFR1. The above results provide novel insights into the function and mechanism of FGF11 that may aid the development of useful ligands for FGFR regulation.


Assuntos
Escherichia coli , Fatores de Crescimento de Fibroblastos , Humanos , Escherichia coli/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Proliferação de Células , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo
2.
Mol Oncol ; 12(3): 356-372, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29316206

RESUMO

The C-type lectin-like domain of CLEC14a (CLEC14a-C-type lectin-like domain [CTLD]) is a key domain that mediates endothelial cell-cell contacts in angiogenesis. However, the role of CLEC14a-CTLD in pathological angiogenesis has not yet been clearly elucidated. In this study, through complementarity-determining region grafting, consecutive deglycosylation, and functional isolation, we generated a novel anti-angiogenic human monoclonal antibody that specifically targets CLEC14a-CTLD and that shows improved stability and homogeneity relative to the parental antibody. We found that this antibody directly inhibits CLEC14a-CTLD-mediated endothelial cell-cell contact and simultaneously downregulates expression of CLEC14a on the surface of endothelial cells. Using various in vitro and in vivo functional assays, we demonstrated that this antibody effectively suppresses vascular endothelial growth factor (VEGF)-dependent angiogenesis and tumor angiogenesis of SNU182 human hepatocellular carcinoma, CFPAC-1 human pancreatic cancer, and U87 human glioma cells. Furthermore, we also found that this antibody significantly inhibits tumor angiogenesis of HCT116 and bevacizumab-adapted HCT116 human colorectal cancer cells. These findings suggest that antibody targeting of CLEC14a-CTLD has the potential to suppress VEGF-dependent angiogenesis and tumor angiogenesis and that CLEC14a-CTLD may be a novel anti-angiogenic target for VEGF-dependent angiogenesis and tumor angiogenesis.


Assuntos
Anticorpos Monoclonais/farmacologia , Moléculas de Adesão Celular/metabolismo , Imunoglobulina G/farmacologia , Lectinas Tipo C/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Moléculas de Adesão Celular/genética , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Feminino , Células HCT116 , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoglobulina G/imunologia , Lectinas Tipo C/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/imunologia , Neovascularização Fisiológica/imunologia , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Biosens Bioelectron ; 72: 230-6, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25985198

RESUMO

We report the application of a fully automated surface-enhanced Raman scattering (SERS)-based solenoid-embedded microfluidic device to the quantitative and sensitive detection of anthrax biomarker poly-γ-D-glutamic acid (PGA) in solution. Analysis is based on the competitive reaction between PGA and PGA-conjugated gold nanoparticles with anti-PGA-immobilized magnetic beads within a microfluidic environment. Magnetic immunocomplexes are trapped by yoke-type solenoids embedded within the device, and their SERS signals were directly measured and analyzed. To improve the accuracy of measurement process, external standard values for PGA-free serum were also measured through use of a control channel. This additional measurement greatly improves the reliability of the assay by minimizing the influence of extraneous experimental variables. The limit of detection (LOD) of PGA in serum, determined by our SERS-based microfluidic sensor, is estimated to be 100 pg/mL. We believe that the defined method represents a valuable analytical tool for the detection of anthrax-related aqueous samples.


Assuntos
Antraz/diagnóstico , Bacillus anthracis/isolamento & purificação , Microfluídica/instrumentação , Ácido Poliglutâmico/análogos & derivados , Análise Espectral Raman/instrumentação , Antraz/sangue , Anticorpos Imobilizados/química , Desenho de Equipamento , Ouro/química , Humanos , Imunoensaio/economia , Imunoensaio/instrumentação , Limite de Detecção , Nanopartículas Metálicas/química , Microfluídica/economia , Ácido Poliglutâmico/análise , Ácido Poliglutâmico/sangue , Reprodutibilidade dos Testes
4.
PLoS One ; 10(4): e0120840, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25853816

RESUMO

Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.


Assuntos
Toxinas Botulínicas Tipo A/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Células da Medula Óssea/citologia , Citocinas/biossíntese , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Imunidade Inata/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Óxido Nítrico/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Transcrição Gênica/efeitos dos fármacos
5.
PLoS One ; 10(3): e0121171, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25803297

RESUMO

A number of peptide-based indicators have been identified and reported as potential apoptosis probes, offering great promise for early assessment of therapeutic efficacy in several types of cancer. Direct comparison of the newly developed probes with previously used ones would be an important step in assessing possible applications. Here, we compared the newly identified peptide-based phosphatidylserine (PS) indicator PSP1 (CLSYYPSYC) with annexin V, a common probe for molecular imaging of apoptotic cells, with respect to PS binding kinetics, apoptotic cell-targeting ability, and the efficacy of homing to apoptotic tumor cells in a mouse model after treatment with the anticancer agent camptothecin. Our results indicate that PSP1 efficiently targeted apoptotic cells and generated apoptosis/tumor-specific signals after cancer treatment in the animal model, whereas a similar dose of annexin V showed weak signals. The formation of a stable complex of PSP1 with PS might be one reason for the efficient in vivo targeting. We suggest that PSP1 has potential advantages for in vivo apoptotic cell imaging and could serve as a platform for the development of de novo peptide-based probes for apoptosis.


Assuntos
Anexina A5/química , Apoptose/fisiologia , Neoplasias Pulmonares/patologia , Imagem Molecular/métodos , Oligopeptídeos/química , Fosfatidilserinas/análise , Animais , Anexina A5/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Indicadores e Reagentes/química , Neoplasias Pulmonares/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oligopeptídeos/metabolismo , Fosfatidilserinas/metabolismo
6.
PLoS One ; 10(2): e0118310, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25705892

RESUMO

We developed and tested a multicomponent peptide-woven siRNA nanocomplex (PwSN) comprising different peptides designed for efficient cellular targeting, endosomal escape, and release of siRNA. To enhance tumor-specific cellular uptake, we connected an interleukin-4 receptor-targeting peptide (I4R) to a nine-arginine peptide (9r), yielding I4R-9r. To facilitate endosomal escape, we blended endosomolytic peptides into the I4R-9r to form a multicomponent nanocomplex. Lastly, we modified 9r peptides by varying the number and positions of positive charges to obtain efficient release of siRNA from the nanocomplex in the cytosol. Using this step-wise approach for overcoming the biological challenges of siRNA delivery, we obtained an optimized PwSN with significant biological activity in vitro and in vivo. Interestingly, surface plasmon resonance analyses and three-dimensional peptide models demonstrated that our designed peptide adopted a unique structure that was correlated with faster complex disassembly and a better gene-silencing effect. These studies further elucidate the siRNA nanocomplex delivery pathway and demonstrate the applicability of our stepwise strategy to the design of siRNA carriers capable of overcoming multiple challenges and achieving efficient delivery.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Peptídeos/química , RNA Interferente Pequeno/administração & dosagem , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Sequência de Aminoácidos , Animais , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células HT29 , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Nanopartículas/administração & dosagem , Peptídeos/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Receptores de Interleucina-4/química , Receptores de Interleucina-4/genética , Reprodutibilidade dos Testes , Ressonância de Plasmônio de Superfície , Transplante Heterólogo
7.
J Cancer Prev ; 19(3): 187-98, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25337588

RESUMO

BACKGROUND: NRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated. METHODS: Three-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity. RESULTS: Ectopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex). CONCLUSIONS: The present study shows that NRP/B acts as a transcriptional repressor by interacting with the co-repressors, HDAC1, providing new insight into the molecular mechanisms of NRP/B on tumor suppression.

8.
Proteomics ; 14(1): 93-104, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24273028

RESUMO

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/imunologia , Bacillus anthracis/enzimologia , Bacillus anthracis/imunologia , Proteínas de Bactérias/imunologia , Peroxirredoxinas/imunologia , Animais , Antraz/mortalidade , Antraz/prevenção & controle , Vacinas contra Antraz/química , Proteínas de Bactérias/química , Eletroforese em Gel Bidimensional , Feminino , Cobaias , Immunoblotting , Peroxirredoxinas/química , Proteômica , Análise de Sobrevida
9.
ACS Nano ; 7(9): 7462-71, 2013 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-23927443

RESUMO

Protein-cage nanoparticles are promising multifunctional platforms for targeted delivery of imaging and therapeutic agents owing to their biocompatibility, biodegradability, and low toxicity. The major advantage of protein-cage nanoparticles is the ability to decorate their surfaces with multiple functionalities through genetic and chemical modification to achieve desired properties for therapeutic and/or diagnostic purposes. Specific peptides identified by phage display can be genetically fused onto the surface of cage proteins to promote the association of nanoparticles with a particular cell type or tissue. Upon symmetrical assembly of the cage, peptides are clustered on the surface of the cage protein in bunches. The resulting PBNC (peptide bunches on nanocage) offers the potential of synergistically increasing the avidity of the peptide ligands, thereby enhancing their blocking ability for therapeutic purposes. Here, we demonstrated a proof-of-principle of PBNCs, fusing the interleukin-4 receptor (IL-4R)-targeting peptide, AP-1, identified previously by phage display, with ferritin-L-chain (FTL), which undergoes 24-subunit assembly to form highly stable AP-1-containing nanocage proteins (AP1-PBNCs). AP1-PBNCs bound specifically to the IL-4R-expressing cell line, A549, and their binding and internalization were specifically blocked by anti-IL-4R antibody. AP1-PBNCs exhibited dramatically enhanced binding avidity to IL-4R compared with AP-1 peptide, measured by surface plasmon resonance spectroscopy. Furthermore, treatment with AP1-PBNCs in a murine model of experimental asthma diminished airway hyper-responsiveness and eosinophilic airway inflammation along with decreased mucus hyperproduction. These findings hold great promise for the application of various PBNCs with ligand-specific peptides in therapeutics for different diseases, such as cancer.


Assuntos
Asma/tratamento farmacológico , Asma/imunologia , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Peptídeos/administração & dosagem , Peptídeos/imunologia , Receptores de Interleucina-4/imunologia , Animais , Antiasmáticos/administração & dosagem , Antiasmáticos/síntese química , Antiasmáticos/imunologia , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Nanocápsulas/ultraestrutura , Peptídeos/química , Ligação Proteica , Resultado do Tratamento
10.
Biochim Biophys Acta ; 1830(3): 2804-12, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23201204

RESUMO

BACKGROUND: The poly-gamma-D-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT. METHODS: To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K(d) values of 0.8 microM and 2.6 microM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed. RESULTS: Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen. GENERAL SIGNIFICANCE: Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.


Assuntos
Vacinas contra Antraz/administração & dosagem , Antraz/prevenção & controle , Anticorpos Antibacterianos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Antígenos de Bactérias/imunologia , Cápsulas Bacterianas/imunologia , Imunização Passiva , Ácido Poliglutâmico/análogos & derivados , Animais , Antraz/imunologia , Antraz/microbiologia , Antraz/mortalidade , Anticorpos Antibacterianos/biossíntese , Anticorpos Monoclonais/biossíntese , Bacillus anthracis/efeitos dos fármacos , Bacillus anthracis/imunologia , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/imunologia , Células Cultivadas , Feminino , Humanos , Cinética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Ácido Poliglutâmico/antagonistas & inibidores , Ácido Poliglutâmico/imunologia , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/imunologia , Esporos Bacterianos/patogenicidade , Análise de Sobrevida , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/imunologia
11.
Mol Cancer Res ; 10(8): 1010-20, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22710795

RESUMO

It is known that VEGF receptors (VEGFR) and integrins interact with each other to regulate angiogenesis. We reported previously that the fasciclin 1 (FAS1) domain-containing protein, TGFBIp/ßig-h3 (TGF-ß-induced protein) is an angiogenesis regulator that inhibits both endothelial cell migration and growth via αvß3 integrin. In an attempt to target the interaction between VEGFR-2 and αvß3 integrin, we determined whether the FAS1 domain region of TGFBIp/ßig-h3 (FAS1 domain protein) can block the interaction between the two receptors, leading to the suppression of angiogenesis. In this study, we showed that FAS1 domain protein inhibits VEGF165-induced endothelial cell proliferation and migration via αvß3 integrin, resulting in the inhibition of VEGF165-induced angiogenesis. We also defined a molecular mechanism by which FAS1 domain protein blocks the association between αvß3 integrin and VEGFR-2, showing that it binds to αvß3 integrin but not to VEGFR-2. Blocking the association of these major angiogenic receptors with FAS1 domain protein inhibits signaling pathways downstream of VEGFR-2. Collectively, our results indicate that FAS1 domain protein, in addition to its inhibitory effect on αvß3 integrin-mediated angiogenesis, also inhibits VEGF165-induced angiogenesis. Thus, FAS1 domain protein can be further developed into a potent anticancer drug that targets two principal angiogenic pathways.


Assuntos
Proteínas da Matriz Extracelular , Integrina alfaVbeta3/metabolismo , Neovascularização Patológica , Fator de Crescimento Transformador beta , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Movimento Celular , Proliferação de Células , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina alfaVbeta3/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor fas/química , Receptor fas/metabolismo
12.
J Biol Chem ; 285(12): 9202-10, 2010 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-20032458

RESUMO

Binding of the 4-1BB ligand (4-1BBL) to its receptor, 4-1BB, provides the T lymphocyte with co-stimulatory signals for survival, proliferation, and differentiation. Importantly, the 4-1BB-4-1BBL pathway is a well known target for anti-cancer immunotherapy. Here we present the 2.3-A crystal structure of the extracellular domain of human 4-1BBL. The ectodomain forms a homotrimer with an extended, three-bladed propeller structure that differs from trimers formed by other members of the tumor necrosis factor (TNF) superfamily. Based on the 4-1BBL structure, we modeled its complex with 4-1BB, which was consistent with images obtained by electron microscopy, and verified the binding site by site-directed mutagenesis. This structural information will facilitate the development of immunotherapeutics targeting 4-1BB.


Assuntos
Ligante 4-1BB/química , Ligante 4-1BB/fisiologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/fisiologia , Ligante 4-1BB/genética , Sítios de Ligação , Proliferação de Células , Clonagem Molecular , Cristalografia por Raios X/métodos , Dimerização , Citometria de Fluxo , Humanos , Microscopia Eletrônica/métodos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/química
13.
Acta Otolaryngol ; 127(7): 687-92, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17573563

RESUMO

CONCLUSION: Diverse expression of the different subtypes of aquaporins in different parts of the Eustachian tube and middle ear suggests region-specific functions of the aquaporins in the normal physiology of the tubotympanum and also suggests that they may play roles in the pathophysiology of otitis media. OBJECTIVES: The epithelial cells of the middle ear and Eustachian tube must maintain adequate water balance for normal function of the mucociliary system. Since aquaporins (AQPs) are known to play critical roles in water homeostasis, we investigated their expression in the tubotympanum of the rat. METHODS: The expression of AQP subtypes 1, 2, 4, 5, and 7 were examined in the rat Eustachian tube and middle ear using RT-PCR, Western blotting, and immunohistochemistry. RESULTS: Transcripts for AQP 1, 4, and 5 were detected in the Eustachian tube and middle ear. Expression of these molecules at the protein level was confirmed by Western blot analysis. Immunohistochemical analysis demonstrated that AQP 4 was localized to the basolateral membranes of ciliated epithelial cells while AQP 5 was localized to the apical surface of serous gland cells, but not goblet cells, in the rat Eustachian tube. AQP 1 was found to be expressed by the subepithelial fibroblasts.


Assuntos
Aquaporinas/metabolismo , Orelha Média/metabolismo , Tuba Auditiva/metabolismo , Mucosa/metabolismo , Animais , Membrana Basal/metabolismo , Western Blotting , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Imuno-Histoquímica , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
J Mol Med (Berl) ; 84(9): 726-36, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16924475

RESUMO

It is perhaps rare to encounter among the various immunologically competent receptor-ligand pairs that a single cell surface determinant unleashes both a hidden suppressive function and costimulation. 4-1BB, an activation-induced tumor necrosis factor receptor family member chiefly viewed as a powerful T-cell costimulatory molecule, is one such example. Accumulated evidence in recent years uncovered an unknown facet of in vivo 4-1BB signaling (i.e., "active suppression"). Although in vitro signaling via 4-1BB is shown to support both CD4(+) and CD8(+) T-cell responses, the same induces a predominant CD8(+) T-cell response suppressing CD4(+) T-cell function when applied in vivo. How, when, and why such dual immunoregulatory effect of anti-4-1BB monoclonal antibody (MAB) comes into play is currently the focus of intense research. Existing data, although not complete, uncover several important aspects of in vivo 4-1BB signaling in the amelioration or exacerbation of various immune disorders. Despite minor disagreements, a majority agree that upregulation of interferon (IFN)-gamma is critical to anti-4-1BB MAB therapy in addition to immune modulators such as interleukin 2, transforming growth factor beta, and indolamine 2,3-dioxygenase(5), all of which contribute greatly to the success of anti-4-1BB MAB-based immunotherapy. Anti-4-1BB MAB-mediated expansion of novel CD11c(+)CD8(+) T cells is additional weaponry that appears critical for its in vivo suppressive function. These CD11c(+)CD8(+) T cells express high levels of IFN-gamma, become effective killers, and mediate selective suppression of CD4(+) T cells. In this review, we discuss the dual nature (costimulatory and suppressive) of 4-1BB-mediated immune regulation, its current status, future direction, and its impact on the immune system, with special reference to its immunotherapy.


Assuntos
Ligante 4-1BB/imunologia , Transdução de Sinais/imunologia , Ligante 4-1BB/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Humanos , Transporte Proteico
15.
Acta Otolaryngol ; 126(6): 564-9, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16720438

RESUMO

CONCLUSION: Spiral ligament fibrocytes (SLFs) may be involved in the innate immune response of the inner ear by producing chemoattractants for recruiting inflammatory cells such as neutrophils and monocytes. OBJECTIVE: The purpose of this study was to investigate the cellular responses of SLFs when challenged by inflammatory stimuli such as components of otitis media pathogens or proinflammatory cytokines. MATERIALS AND METHODS: To detect released inflammatory cytokines and chemokines, cells were treated for 48 h with whole lysates of nontypable Haemophilus influenzae (NTHi), Streptococcus pneumoniae, or with interleukin 1 alpha (IL-1alpha). The culture medium was then collected and applied to protein arrays. To compare mRNA levels of chemokines, total RNA was extracted after 3 h of treatment with the above agents, and quantitative real-time PCR was performed. RESULTS: Protein array analysis showed that in response to NTHi or S. pneumoniae, rat SLFs released monocyte chemotactic protein 1, macrophage inflammatory protein 3 alpha, TNF-alpha, and cytokine-induced neutrophil chemoattractant 2 and 3. Treatment with IL-1alpha, on the other hand, resulted in release of MCP-1 but not the other molecules. Tissue inhibitor of metalloproteinase 1 and vascular endothelial growth factor were released regardless of the inflammatory stimulus used.


Assuntos
Quimiocinas/metabolismo , Infecções por Haemophilus/imunologia , Haemophilus influenzae/imunologia , Imunidade Inata/imunologia , Mediadores da Inflamação/metabolismo , Órgão Espiral/imunologia , Otite Média/imunologia , Infecções Pneumocócicas/imunologia , Gânglio Espiral da Cóclea/imunologia , Lâmina Espiral/imunologia , Animais , Linhagem Celular Transformada , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocinas/genética , Fibroblastos/imunologia , Expressão Gênica/fisiologia , Humanos , Interleucina-1/fisiologia , Monócitos/imunologia , Infiltração de Neutrófilos/imunologia , Reação em Cadeia da Polimerase , Análise Serial de Proteínas , RNA Mensageiro/genética , Ratos
16.
BMC Infect Dis ; 6: 12, 2006 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-16433908

RESUMO

BACKGROUND: We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi) and that interleukin 1 alpha (IL-1 alpha) up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization) in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. METHODS: The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM). Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. RESULTS: Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. CONCLUSION: We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway.


Assuntos
Orelha Média/citologia , Células Epiteliais/metabolismo , Haemophilus influenzae/fisiologia , Interleucina-1/metabolismo , Regulação para Cima , beta-Defensinas/metabolismo , Animais , Linhagem Celular , Haemophilus influenzae/classificação , Humanos , Interleucina-1/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , beta-Defensinas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
J Cell Sci ; 118(Pt 23): 5537-48, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16306221

RESUMO

The neuronal nuclear matrix protein, NRP/B, contains a BTB domain and kelch repeats and is expressed in primary neurons but not in primary glial cells. To examine the function of NRP/B in neurons, we analyzed the structure/function of the NRP/B-BTB domain and its role in neurite outgrowth. Based on three-dimensional modeling of NRP/B, we generated an NRP/B-BTB mutant containing three mutations in the conserved amino acids D47A, H60A and R61D that was termed BTB mutant A. BTB mutant A significantly reduced the dimerization of NRP/B compared to wild-type NRP/B. The NRP/B-BTB domain was required for nuclear localization and mediated the association of NRP/B with p110RB through the TR subdomain within the B pocket of p110RB. Overexpression of wild-type NRP/B and NRP/B-BTB domain significantly induced neurite outgrowth in PC12 cells and enhanced the G0-G1 cell population by approximately 23% compared to the control cells, whereas NRP/B-BTB mutant A reduced neurite outgrowth by 70-80%, and inhibited NRP/B-p110RB association. Single cell microinjection of NRP/B-specific antibodies also blocked the neurite outgrowth of PC12 cells upon NGF stimulation. Interference of NRP/B expression by small interfering RNA (NRP/B-siRNA) inhibited neurite outgrowth and suppressed the NGF-induced outgrowth of neurites in PC12 cells. Additionally, p110RB phosphorylation at serine residue 795 was significantly reduced in PC12 cells treated with NRP/B siRNA compared to those treated with control GFP-siRNA, indicating that p110RB is a downstream target of NRP/B. Thus, the BTB domain of NRP/B regulates neurite outgrowth through its interaction with the TR subdomain within the B pocket of p110RB, and the conserved amino acids D47A, H60A and R61D within this domain of NRP/B are crucial residues for neurite extension in neuronal cells. These findings support a role for the BTB-domain of NRP/B as an important regulator of neuronal differentiation.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas dos Microfilamentos/genética , Neuritos/fisiologia , Neuropeptídeos/genética , Proteínas Nucleares/genética , Animais , Anticorpos/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Regulação da Expressão Gênica , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/farmacologia , Mutação , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura , Neurônios/citologia , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/farmacologia , Células PC12 , Fosforilação , Conformação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/farmacologia , Ratos
18.
Int J Oncol ; 24(4): 847-51, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15010821

RESUMO

To define the molecular changes associated with ovarian cancer, DNA microarray analysis has been adapted to detect differentially expressed genes in human normal ovary tissue, borderline, and invasive epithelial ovarian tumors. The differential expression of genes in the tumor tissues and normal tissues was confirmed by Northern and/or semi-quantitative RT-PCR analysis. Analysis of the differential gene-expression profiles of the normal and neoplastic ovary allowed us to detect previously unidentified genes in ovarian tissues. We observed up-regulation of the following genes in ovarian cancer: catechol-O-methyltransferase (COMT), the autocrine motility factor neuroleukin (NLK), the transcription regulator high mobility group I proteins (HMGI), the tyrosine kinase receptor ErbB-3, S100-alpha protein and Acyl-CoA-binding protein (ACBP). The transcription factor, chicken ovalbumin up-stream promoter transcription factor II (COUP-TFII), was the only gene down-regulated in ovarian cancer. Comparable gene-expression profiles were previously reported in breast cancer, suggesting that similar molecular events also exist in ovarian cancer. Our microarray analysis showed that most differentially expressed genes in ovarian cancer are linked to glucose/insulin metabolism, providing a possible molecular link between the glucose/insulin signaling pathway and the neoplasms of ovarian cancer.


Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Feminino , Glucose/metabolismo , Humanos , Insulina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...