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1.
FEBS Lett ; 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31898812

RESUMO

The Teosinte branched 1/Cycloidea/Proliferating cell factor (TCP) domain is an evolutionarily conserved DNA binding domain unique to the plant kingdom. To date, the functions of TCPs have been well studied, but the three-dimensional structure of the TCP domain is lacking. Here, we have determined the crystal structure of the TCP domain from OsPCF6. The structure reveals that the TCP domain adopts three short ß-strands followed by a helix-loop-helix structure, distinct from the canonical basic helix-loop-helix structure. This folded domain shows high structural similarity to the ribbon-helix-helix (RHH) transcriptional repressors, a family of DNA binding proteins with a conserved 3D structural motif (RHH fold), indicating that TCPs could be reclassified as RHH proteins. Our work will provide insight toward a better understanding of the mechanisms underlying TCP protein function.

2.
Neurophotonics ; 6(4): 045008, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31737743

RESUMO

ß -Amyloid ( A ß ) plaque, representing the progressive accumulation of the protein that mainly consists of A ß , is one of the prominent pathological hallmarks of Alzheimer's disease (AD). Label-free imaging of A ß plaques holds the potential to be a histological examination tool for diagnosing AD. We applied label-free multiphoton microscopy to identify extracellular A ß plaque as well as intracellular A ß accumulation for the first time from AD mouse models. We showed that a two-photon-excited fluorescence signal is a sensitive optical marker for revealing the spatial-temporal progression and the surrounding morphological changes of A ß deposition, which demonstrated that both extracellular and intracellular A ß accumulations play an important role in the progression of AD. Moreover, combined with a custom-developed image-processing program, we established a rapid method to visualize different degrees of A ß deposition by color coding. These results provide an approach for investigating pathophysiology of AD that can complement traditional biomedical procedures.

3.
Nanoscale ; 11(46): 22475-22481, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31746908

RESUMO

Silver nanowires (AgNWs), as one of the most important plasmonic waveguides, can support several different plasmonic modes. These surface plasmon polariton (SPP) modes have different electric field distributions, effective mode areas, propagation lengths and losses and thus can be used for different applications, from efficiently collecting single photons to carrying quantum entanglement. Therefore, the excitation and analysis of these different SPP modes are of pivotal importance for the development of subwavelength optical devices. In this work, we investigate different SPP modes on a suspended AgNW adhered to a fiber taper. Theoretical simulations and experimental results show that the desired SPP modes can be selectively excited by adjusting either the polarization of the excitation light or the coupling length between the fiber taper and the AgNW. Moreover, fundamental and higher-order SPP modes can be distinguished by means of a far-field method. Our results not only enable convenient and controllable excitation of the desired SPP modes but also provide unique insight into the optical properties of plasmonic waveguides.

4.
Biochim Biophys Acta Gene Regul Mech ; 1862(8): 759-770, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31269460

RESUMO

The majority of melanomas carry an oncogenic BRAF mutation (BRAFV600E), which results in constitutive kinase activity driving melanoma proliferation. While inhibitors of BRAFV600E (BRAFi) effectively lead to rapid tumor shrinkage, most patients treated with BRAFi develop acquired resistance. Identification of factors as regulators of melanoma growth and as potential sources of resistance is thus crucial for the design of improved therapies to treat advanced melanoma with more durable responses. Here, we show that KH-type splicing regulatory protein (KSRP) is critical for proliferation of melanoma cells without and with acquired resistance to vemurafenib. Silencing KSRP reduces cell proliferation and augments the growth suppressive effects of vemurafenib. We identify killin (KLLN), a p53-regulated DNA replication inhibitor, as a downstream effector of growth inhibition by KSRP silencing and demonstrate that KSRP promotes decay of KLLN mRNA through an RNA-protein interaction. Using heterologous mRNA reporters, we show that a U-rich element within the 3' untranslated region of KLLN is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of melanoma cell growth in part through controlling KLLN mRNA stability.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genética , Vemurafenib/uso terapêutico , Regiões 3' não Traduzidas , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Melanoma/genética , Camundongos , Estabilidade de RNA , RNA Mensageiro/química , Proteínas Supressoras de Tumor/química , Regulação para Cima
5.
FEBS J ; 286(21): 4294-4309, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31230405

RESUMO

The VirB/D type IV secretion system (T4SS) plays an essential role in materials transport between host cells and pathogenic Helicobacter pylori and is considered the major pathogenic mediator of H. pylori-associated gastric disease. VirB8, an inner membrane protein that interacts with many other proteins, is a crucial component for secretory function. Here, we present a crystal structure of the periplasmic domain of CagV, the VirB8 counterpart in the H. pylori Cag-T4SS. The structure reveals a fold similar to that of other VirB8 members except for the absence of the α5 helix, a discontinuous ß1 strand, a larger angle between the α2 and α3 helices, a more hydrophobic surface groove, but exhibits a different dimer interface. Whether the dimerization occurs in solution was proved by mutagenesis, size-exclusion chromatography and cross-linking assays. Unlike the classical dimerization mode, the interface of the CagV dimer is principally formed by several hydrogen bonds, which indicates instability of dimerization. The structure here demonstrates the difference in dimerization among VirB8 homologues and indicates the considerable compositional and functional diversity of them in T4SS. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6IQT.

6.
FEBS J ; 286(14): 2809-2821, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30974511

RESUMO

The phosphate starvation response 1 (PHR1) protein has a central role in mediating the response to phosphate starvation in plants. PHR1 is composed of a number of domains including a MYB domain involved with DNA binding and a coiled-coil domain proposed to be involved with dimer formation. PHR1 binds to the promoter of phosphate starvation-induced genes to control the levels of phosphate required for nutrition. Previous studies have shown that both the MYB domain and the coiled-coil domain of PHR1 are required for binding the target DNA. Here, we describe the crystal structure of the PHR1 MYB domain and two structures of its complex with the PHR1-binding DNA sequence (P1BS). Structural and isothermal titration calorimetry has been carried out showing that the MYB domain of PHR1 alone is sufficient for target DNA recognition and binding. Two copies of the PHR1 MYB domain bind to the same major groove of the P1BS DNA with few direct interactions between the individual MYB domains. In addition, the PHR1 MYB-P1BS DNA complex structures reveal amino acid residues involved in DNA recognition and binding. Mutagenesis of these residues results in lost or impaired ability of PHR1 MYB to bind to its target DNA. The results presented reveal the structural basis for DNA recognition by the PHR1 MYB domain and demonstrate that two PHR1 MYB domains attach to their P1BS DNA targeting sequence. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6J4K (PHR1 MYB), 6J4R (PHR1 MYB-R-P1BS), 6J5B (MYB-CC-R2-P1BS).

7.
Talanta ; 199: 336-346, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952268

RESUMO

Highly stable and multifunctional fluorescent quantum dots are particularly attractive in practical applications. Here, a new kind of ultra-small-sized silicon quantum dot-gadolinium (SiQD-Gd) was successfully fabricated by a newly-designed facile hydrothermal growth and chelating method. The obtained SiQD-Gd exhibited outstanding water dispersibility, stability and good fluorescent property with the quantum yield of 11.6%. SiQD-Gd displayed a low cytotoxicity in normal cell lines (HELF, HEK293F) and tumor cell lines (H1299, A549). Meanwhile, SiQD-Gd showed excellent magnetic resonance response with r1 relaxation rate of 10.5 mmol L-1·s-1 and r2 relaxation rate of 47.5 mmol L-1·s-1, which are 2.5 and 7.4 times enhanced comparing to that of the commercial MR agent Magnevist. In vivo studies showed significant contrast enhancement effect of its T1- and T2-weighted MR imaging. In addition, in vivo fluorescent imaging for mice and zebrafish indicated its potential applications in fluorescent tracking. Thus, the excellent multimodal imaging capacity and biocompatibility of SiQD-Gd make it a potential imaging agent for clinic applications.


Assuntos
Corantes Fluorescentes/química , Gadolínio/química , Imagem Multimodal , Pontos Quânticos/química , Silício/química , Animais , Linhagem Celular , Humanos , Camundongos , Peixe-Zebra
8.
ACS Appl Mater Interfaces ; 10(32): 26964-26971, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30035532

RESUMO

Herein, new near-infrared (NIR) luminescent ruthenium complexes were prepared for detecting Cu2+ ions. Then, ruthenium complex hybrid nanomaterials (Ru-LPMSNs) were fabricated successfully by imbedding the ruthenium complex into mesoporous silica nanoparticles. Benefiting from the novel large-pore mesoporous structure and good adsorbility of LPMSNs, Ru-LPMSN hybrid materials showed a significantly enhanced fluorescence intensity and stability. NIR fluorescence of Ru-LPMSNs was rapidly quenched by Cu2+ ions. Ru-LPMSNs also showed high Cu2+ ion selectivity and sensitivity as a sensor. The detection limit of Cu2+ ions was 10 nM with a wide linear relationship between the fluorescence intensity of Ru-LPMSNs and the concentration of Cu2+ ions. The mechanism of fluorescence quenching might be that the combination of the ruthenium complex and Cu2+ ions constrained the photoinduced electron-transfer process. Furthermore, Ru-LPMSNs dramatically increased the fluorescence signals in cells and achieved Cu2+-ion detection. Ru-LPMSNs had different tissue affinities and could monitor distribution of exogenous Cu2+ ions in vivo. Moreover, Ru-LPMSNs realized direct and rapid detection of Cu2+-ion content in serum. These results indicated the potential applications of the prepared nanomaterials as Cu2+ detection agents.


Assuntos
Rutênio/química , Cátions Bivalentes , Cobre , Limite de Detecção , Nanopartículas , Dióxido de Silício
9.
Int J Nanomedicine ; 13: 2973-2987, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29861631

RESUMO

Vaccination is one of the most effective approaches in the prevention and control of disease worldwide. Oral vaccination could have wide applications if effective protection cannot be achieved through traditional (eg, parenteral) routes of vaccination. However, oral administration is hampered by the difficulties in transferring vaccines in vivo. This has led to the development of materials such as carriers with potential adjuvant effects. Considering the requirements for selecting adjuvants for oral vaccines as well as the advantages of nanoparticle/microparticle materials as immune effectors and antigen conveyors, synthetic materials could improve the efficiency of oral vaccination. In this review, nanoparticles and microparticles with adjuvant characteristics are described with regard to their potential importance for oral immunization, and some promising and successful modification strategies are summarized.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Nanopartículas/administração & dosagem , Vacinas/administração & dosagem , Administração Oral , Animais , Humanos , Imunização , Nanopartículas/química , Vacinação , Vacinas/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia
10.
Sci Rep ; 8(1): 509, 2018 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-29323236

RESUMO

The large-conductance calcium-activated K+ (BK) channel contains two intracellular tandem Ca2+-sensing RCK domains (RCK1 and RCK2), which tetramerize into a Ca2+ gating ring that regulates channel opening by conformational expansion in response to Ca2+ binding. Interestingly, the gating ring's intersubunit assembly interface harbors the RCK2 Ca2+-binding site, known as the Ca2+ bowl. The gating ring's assembly interface is made in part by intersubunit coordination of a Ca2+ ion between the Ca2+ bowl and an RCK1 Asn residue, N449, and by apparent intersubunit electrostatic interactions between E955 in RCK2 and R786 and R790 in the RCK2 of the adjacent subunit. To understand the role of the intersubunit assembly interface in Ca2+ gating, we performed mutational analyses of these putative interacting residues in human BK channels. We found that N449, despite its role in Ca2+ coordination, does not set the channel's Ca2+ sensitivity, whereas E955 is a determinant of Ca2+ sensitivity, likely through intersubunit electrostatic interactions. Our findings provide evidence that the intersubunit assembly interface contains molecular determinants of Ca2+-sensitivity in BK channels.


Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Sítios de Ligação , Cloreto de Cálcio/farmacologia , Células HEK293 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Potenciais da Membrana/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Domínios Proteicos , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Eletricidade Estática
11.
Biosci Rep ; 37(3)2017 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-28442601

RESUMO

A novel alkylsulfatase from bacterium Pseudomonas sp. S9 (SdsAP) was identified as a thermostable alkylsulfatases (type III), which could hydrolyze the primary alkyl sulfate such as sodium dodecyl sulfate (SDS). Thus, it has a potential application of SDS biodegradation. The crystal structure of SdsAP has been solved to a resolution of 1.76 Å and reveals that SdsAP contains the characteristic metallo-ß-lactamase-like fold domain, dimerization domain, and C-terminal sterol carrier protein type 2 (SCP-2)-like fold domain. Kinetic characterization of SdsAP to SDS by isothermal titration calorimetry (ITC) and enzymatic activity assays of constructed mutants demonstrate that Y246 and G263 are important residues for its preference for the hydrolysis of 'primary alkyl' chains, confirming that SdsAP is a primary alkylsulfatase.


Assuntos
Proteínas de Bactérias/química , Pseudomonas/metabolismo , Sulfatases/química , Hidrólise , Cinética , Dodecilsulfato de Sódio/química , Especificidade por Substrato , Sulfatos/química , Tensoativos/química , beta-Lactamases/química
12.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 3): 167-173, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28291753

RESUMO

Helicobacter pylori, a Gram-negative bacterial pathogen prevalent in the human population, is the causative agent of severe gastric diseases. An H. pylori type IV secretion (T4S) system encoded by the cytotoxin-associated gene pathogenicity island (cagPAI) is responsible for communication with host cells. As a component of the cagPAI T4S system core complex, CagX plays an important role in virulence-protein translocation into the host cells. In this work, the crystal structure of the C-terminal domain of CagX (CagXct), which is a homologue of the VirB9 protein from the VirB/D4 T4S system, is presented. CagXct is only the second three-dimensional structure to be elucidated of a VirB9-like protein. Another homologue, TraO, which is encoded on the Escherichia coli conjugative plasmid pKM101, shares only 19% sequence identity with CagXct; however, there is a remarkable similarity in tertiary structure between these two ß-sandwich protein domains. Most of the residues that are conserved between CagXct and TraO are located within the protein core and appear to be responsible for the preservation of this domain fold. The studies presented here will contribute to our understanding of different bacterial T4S systems.


Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Helicobacter pylori/química , Sistemas de Secreção Tipo IV/química , Fatores de Virulência/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Ilhas Genômicas , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Modelos Moleculares , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
13.
ACS Appl Mater Interfaces ; 8(51): 34933-34950, 2016 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-27935687

RESUMO

Oral vaccines have become a recent focus because of their potential significance in disease prevention and therapy. In the development of oral vaccine-based therapeutics, synthetic materials with tailorable structures and versatile functions can act as antigen conveyers with adjuvant effects, reduce the time cost for vaccine optimization, and provide high security and enhanced immunity. This review presents an overview of the current status of tailoring synthetic adjuvants for oral vaccination, modification strategies for producing effectors with specific structures and functions, enhancement of immune-associated efficiencies, including the barrier-crossing capability to protect antigens in the gastrointestinal tract, coordination of the antigens penetrating mucosa and cell barriers, targeting of concentrated antigens to immune-associated cells, and direct stimulation of immune cells. Finally, we focus on prospective synthetic adjuvants that facilitate the use of oral vaccines via two approaches, namely, in vivo antigen expression and cancer immunotherapy.


Assuntos
Vacinação , Adjuvantes Imunológicos , Administração Oral , Antígenos , Estudos Prospectivos , Vacinas
14.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 8): 586-90, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27487921

RESUMO

White spot syndrome virus (WSSV) is a major shrimp pathogen known to infect penaeid shrimp and other crustaceans. VP24 is one of the major envelope proteins of WSSV. In order to facilitate purification, crystallization and structure determination, the predicted N-terminal transmembrane region of approximately 26 amino acids was truncated from VP24 and several mutants were prepared to increase the proportion of selenomethionine (SeMet) residues for subsequent structural determination using the SAD method. Truncated VP24, its mutants and the corresponding SeMet-labelled proteins were purified, and the native and SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of VP24 were obtained using a reservoir consisting of 0.1 M Tris-HCl pH 8.5, 2.75 M ammonium acetate with a drop volume ratio of two parts protein solution to one part reservoir solution. Notably, ATP was added as a critical additive to the drop with a final concentration of 10 mM. Crystals of SeMet-labelled VP24 mutant diffracted to 3.0 Šresolution and those of the native diffracted to 2.4 Šresolution; the crystals belonged to space group I213, with unit-cell parameters a = b = c = 140 Å.


Assuntos
Plasmídeos/química , Proteínas do Envelope Viral/genética , Vírus da Síndrome da Mancha Branca 1/química , Sequência de Aminoácidos , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Mutação , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Selenometionina/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírus da Síndrome da Mancha Branca 1/metabolismo
15.
Sci Rep ; 6: 32309, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27572278

RESUMO

White spot syndrome virus (WSSV) is one of the major and most serious pathogen in the shrimp industry. As one of the most abundant envelope protein, VP24 acts as a core protein interacting with other structure proteins and plays an important role in virus assembly and infection. Here, we have presented the crystal structure of VP24 from WSSV. In the structure, VP24 consists of a nine-stranded ß-barrel fold with mostly antiparallel ß-strands, and the loops extending out the ß-barrel at both N-terminus and C-terminus, which is distinct to those of the other two major envelope proteins VP28 and VP26. Structural comparison of VP24 with VP26 and VP28 reveals opposite electrostatic surface potential properties of them. These structural differences could provide insight into their differential functional mechanisms and roles for virus assembly and infection. Moreover, the structure reveals a trimeric assembly, suggesting a likely natural conformation of VP24 in viral envelope. Therefore, in addition to confirming the evolutionary relationship among the three abundant envelope proteins of WSSV, our structural studies also facilitate a better understanding of the molecular mechanism underlying special roles of VP24 in WSSV assembly and infection.


Assuntos
Mutação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vírus da Síndrome da Mancha Branca 1/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cristalografia por Raios X , Modelos Moleculares , Penaeidae/virologia , Conformação Proteica , Multimerização Proteica , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/metabolismo , Vírus da Síndrome da Mancha Branca 1/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia
16.
Plant Cell ; 28(5): 1025-34, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27081181

RESUMO

GRAS proteins belong to a plant-specific protein family with many members and play essential roles in plant growth and development, functioning primarily in transcriptional regulation. Proteins in the family are minimally defined as containing the conserved GRAS domain. Here, we determined the structure of the GRAS domain of Os-SCL7 from rice (Oryza sativa) to 1.82 Å. The structure includes cap and core subdomains and elucidates the features of the conserved GRAS LRI, VHIID, LRII, PFYRE, and SAW motifs. The structure is a dimer, with a clear groove to accommodate double-stranded DNA. Docking a DNA segment into the groove to generate an Os-SCL7/DNA complex provides insight into the DNA binding mechanism of GRAS proteins. Furthermore, the in vitro DNA binding property of Os-SCL7 and model-defined recognition residues are assessed by electrophoretic mobility shift analysis and mutagenesis assays. These studies reveal the structure and preliminary DNA interaction mechanisms of GRAS proteins and open the door to in-depth investigation and understanding of the individual pathways in which they play important roles.


Assuntos
Cristalografia por Raios X/métodos , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , DNA de Plantas/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/genética , Proteínas de Plantas/genética , Estrutura Terciária de Proteína
17.
Biomaterials ; 77: 307-19, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26624805

RESUMO

Conventional oral vaccines with simple architecture face barriers with regard to stimulating effective immunity. Here we describe oral vaccines with an intelligent phase-transitional shielding layer, poly[(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid)]-poly(D,L-lactide-co-glycolide) (PMMMA-PLGA), which can protect antigens in the gastro-intestinal tract and achieve targeted vaccination in the large intestine. With the surface immunogenic protein (SIP) from group B Streptococcus (GBS) entrapped as the antigen, oral administration with PMMMA-PLGA (PTRBL)/Trx-SIP nanoparticles stimulated robust immunity in tilapia, an animal with a relatively simple immune system. The vaccine succeeded in protecting against Streptococcus agalactiae, a pathogen of worldwide importance that threatens human health and is transmitted in water with infected fish. After oral vaccination with PTRBL/Trx-SIP, tilapia produced enhanced levels of SIP specific antibodies and displayed durability of immune protection. 100% of the vaccinated tilapia were protected from GBS infection, whereas the control groups without vaccines or vaccinated with Trx-SIP only exhibited respective infection rates of 100% or >60% within the initial 5 months after primary vaccination. Experiments in vivo demonstrated that the recombinant antigen Trx-SIP labeled with FITC was localized in colon, spleen and kidney, which are critical sites for mounting an immune response. Our results revealed that, rather than the size of the nanoparticles, it is more likely that the negative charge repulsion produced by ionization of the carboxyl groups in PMMMA shielded the nanoparticles from uptake by small intestinal epithelial cells. This system resolves challenges arising from gastrointestinal damage to antigens, and more importantly, offers a new approach applicable for oral vaccination.


Assuntos
Antígenos/administração & dosagem , Preparações de Ação Retardada/química , Sistemas de Liberação de Medicamentos/métodos , Vacinas/administração & dosagem , Administração Oral , Animais , Antígenos/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Linhagem Celular , Reservatórios de Doenças , Fibroblastos , Doenças dos Peixes/prevenção & controle , Radicais Livres , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Absorção Intestinal , Tamanho da Partícula , Peritonite/prevenção & controle , Peritonite/veterinária , Poliésteres , Polimerização , Ácidos Polimetacrílicos , Eletricidade Estática , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/transmissão , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae , Tilápia/imunologia , Tilápia/microbiologia , Tripsina/farmacologia , Vacinas/química , Vacinas/imunologia
18.
Monoclon Antib Immunodiagn Immunother ; 34(4): 246-50, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26301927

RESUMO

Human DAB2 interaction protein (DAB2IP) is a member of Ras-GTPase activating protein family and functions as a tumor suppressor, implying it could serve as a prognostic biomarker in cancers. Here we generated a mouse monoclonal antibody, 2A4, directed against human DAB2IP. This antibody was identified as IgG1 and specifically recognizes DAB2IP in both its native and denatured forms. It will serve as a useful and versatile tool for further mechanistic study and development of the potential prognostic significance of DAB2IP.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas Ativadoras de ras GTPase/imunologia , Animais , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoglobulina G/imunologia , Camundongos Endogâmicos BALB C , Prognóstico
19.
Int J Nanomedicine ; 10: 2871-84, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25926732

RESUMO

Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression.


Assuntos
Vetores Genéticos/genética , Inositol/química , Transfecção/métodos , Transgenes/genética , Glicerol , Células HEK293 , Humanos , Polietilenoimina/química , Polímeros
20.
PLoS One ; 9(8): e104609, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25101777

RESUMO

Helicobacter pylori is a well-known pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Recently, there has been more considerable interest in strain-specific genes located in plasticity regions with great genetic variability. However, little is known about many of these genes. Studies suggested that certain genes in this region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. JHP933, a conserved putative protein of unknown function, is encoded by the gene in plasticity region of H. pylori strain J99. Here we have determined the structure of JHP933. Our work demonstrates that JHP933 is a nucleotidyltransferase superfamily protein with a characteristic αßαßαßα topology. A superposition demonstrates overall structural homology of the JHP933 N-terminal fragment with lincosamide antibiotic adenylyltransferase LinA and identifies a possible substrate-binding cleft of JHP933. Furthermore, through structural comparison with LinA and LinB, we pinpoint conservative active site residues which may contribute to divalent ion coordination and substrate binding.


Assuntos
Proteínas de Bactérias/química , Helicobacter pylori/enzimologia , Nucleotidiltransferases/química , Cristalografia por Raios X , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína
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