Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
1.
Nature ; 533(7601): 58-63, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-27120167

RESUMO

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.


Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Evolução Molecular Direcionada/métodos , Endotoxinas/genética , Endotoxinas/metabolismo , Variação Genética/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Resistência a Inseticidas , Mariposas/fisiologia , Controle Biológico de Vetores/métodos , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Bacteriófagos/genética , Biotecnologia , Caderinas/metabolismo , Morte Celular , Sequência Consenso , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Inseticidas/metabolismo , Dados de Sequência Molecular , Mariposas/citologia , Mutagênese/genética , Plantas Geneticamente Modificadas , Ligação Proteica/genética , Estabilidade Proteica , Seleção Genética
2.
Proc Natl Acad Sci U S A ; 111(30): 10911-6, 2014 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-25024206

RESUMO

High-throughput screening has become a mainstay of small-molecule probe and early drug discovery. The question of how to build and evolve efficient screening collections systematically for cell-based and biochemical screening is still unresolved. It is often assumed that chemical structure diversity leads to diverse biological performance of a library. Here, we confirm earlier results showing that this inference is not always valid and suggest instead using biological measurement diversity derived from multiplexed profiling in the construction of libraries with diverse assay performance patterns for cell-based screens. Rather than using results from tens or hundreds of completed assays, which is resource intensive and not easily extensible, we use high-dimensional image-based cell morphology and gene expression profiles. We piloted this approach using over 30,000 compounds. We show that small-molecule profiling can be used to select compound sets with high rates of activity and diverse biological performance.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos
3.
Bioorg Med Chem ; 20(6): 1979-89, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22230199

RESUMO

2011 marks the 10-year anniversary of milestone manuscripts describing drafts of the human genome sequence. Over the past decade, a number of new proteins have been linked to disease-many of which fall into classes that have been historically considered challenging from the perspective of drug discovery. Several of these newly associated proteins lack structural information or strong annotation with regard to function, making development of conventional in vitro functional assays difficult. A recent resurgence in the popularity of simple small molecule binding assays has led to new approaches that do not require knowledge of protein structure or function in advance. Here we briefly review selected methods for executing binding assays that have been used successfully to discover small-molecule probes or drug candidates.


Assuntos
Descoberta de Drogas , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Ligação Proteica , Proteínas/química , Ressonância de Plasmônio de Superfície/métodos
4.
Infect Immun ; 79(2): 606-16, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21078848

RESUMO

During group B streptococcal infection, the alpha C protein (ACP) on the bacterial surface binds to host cell surface heparan sulfate proteoglycans (HSPGs) and facilitates entry of bacteria into human epithelial cells. Previous studies in a Drosophila melanogaster model showed that binding of ACP to the sulfated polysaccharide chains (glycosaminoglycans) of HSPGs promotes host death and is associated with higher bacterial burdens. We hypothesized that ACP-glycosaminoglycan binding might determine infection outcome by altering host responses to infection, such as expression of antimicrobial peptides. As glycosaminoglycans/HSPGs also interact with a number of endogenous secreted signaling molecules in Drosophila, we examined the effects of host and pathogen glycosaminoglycan/HSPG-binding structures in host survival of infection and antimicrobial peptide expression. Strikingly, host survival after infection with wild-type streptococci was enhanced among flies overexpressing the endogenous glycosaminoglycan/HSPG-binding morphogen Decapentaplegic-a transforming growth factor ß-like Drosophila homolog of mammalian bone morphogenetic proteins-but not by flies overexpressing a mutant, non-glycosaminoglycan-binding Decapentaplegic, or the other endogenous glycosaminoglycan/HSPG-binding morphogens, Hedgehog and Wingless. While ACP-glycosaminoglycan binding was associated with enhanced transcription of peptidoglycan recognition proteins and antimicrobial peptides, Decapentaplegic overexpression suppressed transcription of these genes during streptococcal infection. Further, the glycosaminoglycan-binding domain of ACP competed with Decapentaplegic for binding to the soluble glycosaminoglycan heparin in an in vitro assay. These data suggest that, in addition to promoting bacterial entry into host cells, ACP competes with Decapentaplegic for binding to glycosaminoglycans/HSPGs during infection and that these bacterial and endogenous glycosaminoglycan-binding structures determine host survival and regulate antimicrobial peptide transcription.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Drosophila melanogaster/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Streptococcus/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Glicosaminoglicanos/química , Heparina/metabolismo , Interações Hospedeiro-Patógeno , Ligação Proteica , Streptococcus/genética , Fatores de Tempo
5.
Bioorg Med Chem Lett ; 21(14): 4164-9, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21696956

RESUMO

Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can serve as probes to study the relevant biological roles of HDACs, novel scaffolds are necessary to develop more efficient, selective drug candidates. Screening libraries of molecules may yield structurally diverse probes that bind these enzymes and modulate their functions in cells. Here we report a small molecule with a novel hydroxy-pyrimidine scaffold that inhibits multiple HDAC enzymes and modulates acetylation levels in cells. Analogs were synthesized in an effort to evaluate structure-activity relationships.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Pirimidinas/química , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Pirimidinas/síntese química , Pirimidinas/farmacologia , Relação Estrutura-Atividade
6.
Clin Cancer Res ; 26(8): 1953-1964, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31964784

RESUMO

PURPOSE: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. EXPERIMENTAL DESIGN: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-A2/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Interleucina-2/imunologia , Neoplasias/terapia , Proteínas E7 de Papillomavirus/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Voluntários Saudáveis , Humanos , Leucócitos Mononucleares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Neoplasias/virologia
7.
Anal Biochem ; 385(1): 57-64, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18992215

RESUMO

Liquid chromatography-mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A.


Assuntos
Condroitina ABC Liase/metabolismo , Condroitina Liases/metabolismo , Arthrobacter/enzimologia , Bacteroides/enzimologia , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Ativação Enzimática , Espectrometria de Massas , Proteus vulgaris/enzimologia , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Viscosidade
8.
Biomacromolecules ; 10(3): 589-95, 2009 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-19226107

RESUMO

Metal nanoparticles have been studied for their anticoagulant and anti-inflammatory efficacy in various models. Specifically, gold and silver nanoparticles exhibit properties that make these ideal candidates for biological applications. The typical synthesis of gold and silver nanoparticles incorporates contaminants that could pose further problems. Here we demonstrate a clean method of synthesizing gold and silver nanoparticles that exhibit biological functions. These nanoparticles were prepared by reducing AuCl(4) and AgNO(3) using heparin and hyaluronan as both reducing and stabilizing agents. The particles show stability under physiological conditions and narrow size distributions for heparin particles and wider distribution for hyaluronan particles. Studies show that the heparin nanoparticles exhibit anticoagulant properties. Additionally, either gold- or silver-heparin nanoparticles exhibit local anti-inflammatory properties without any significant effect on systemic hemostasis upon administration in carrageenan-induced paw edema models. In conclusion, gold and silver nanoparticles complexed with heparin demonstrated effective anticoagulant and anti-inflammatory efficacy, having potential in various local applications.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Anticoagulantes/farmacologia , Edema/tratamento farmacológico , Glicosaminoglicanos/química , Nanopartículas Metálicas/química , Agregação Plaquetária/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Anticoagulantes/síntese química , Anticoagulantes/química , Carragenina , Modelos Animais de Doenças , Edema/induzido quimicamente , Ouro/química , Ouro/farmacologia , Heparina/química , Humanos , Ácido Hialurônico/química , Masculino , Tamanho da Partícula , Ratos , Prata/química , Prata/farmacologia , Propriedades de Superfície
9.
Nanotechnology ; 20(45): 455104, 2009 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-19822927

RESUMO

Silver and gold nanoparticles display unique physical and biological properties that have been extensively studied for biological and medical applications. Typically, gold and silver nanoparticles are prepared by chemical reductants that utilize excess toxic reactants, which need to be removed for biological purposes. We utilized a clean method involving a single synthetic step to prepare metal nanoparticles for evaluating potential effects on angiogenesis modulation. These nanoparticles were prepared by reducing silver nitrate and gold chloride with diaminopyridinyl (DAP)-derivatized heparin (HP) polysaccharides. Both gold and silver nanoparticles reduced with DAPHP exhibited effective inhibition of basic fibroblast growth factor (FGF-2)-induced angiogenesis, with an enhanced anti-angiogenesis efficacy with the conjugation to DAPHP (P<0.01) as compared to glucose conjugation. These results suggest that DAPHP-reduced silver nanoparticles and gold nanoparticles have potential in pathological angiogenesis accelerated disorders such as cancer and inflammatory diseases.


Assuntos
Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Ouro/química , Heparina/farmacologia , Nanopartículas Metálicas , Neovascularização Fisiológica/efeitos dos fármacos , Prata/química , Animais , Embrião de Galinha , Fator 2 de Crescimento de Fibroblastos/farmacologia , Compostos de Ouro/química , Heparina/química , Masculino , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Nitrato de Prata/química
10.
European J Org Chem ; 2009(16)2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24223493

RESUMO

α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazole derivatives of sialic acid using the copper-catalyzed azide-alkyne Huisgen cycloaddition ("click chemistry"). Our approach is to generate non-natural N-glycosides of sialic acid that are resistant to neuraminidase catalyzed hydrolysis as opposed to the natural O-glycosides. These N-glycosides would act as neuraminidase inhibitors to prevent the release of new virions. As a preliminary study, a small library of 1,2,3-triazole-linked sialic acid derivatives has been synthesized in 71-89% yield. A disaccharide mimic of sialic acid has also been prepared using the α-sialic acid azide 1 and a C-8 propargyl sialic acid acceptor in 68% yield. A model sialic acid coated dendrimer was also synthesized from a per-propargylated pentaerythritol acceptor. These novel sialic acid derivatives were then evaluated as potential neuraminidase inhibitors using a 96-well plate fluorescence assay; micromolar IC50 values were observed, comparable to the known sialidase inhibitor Neu5Ac2en.

11.
Gigascience ; 6(12): 1-5, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28327978

RESUMO

Background: Large-scale image sets acquired by automated microscopy of perturbed samples enable a detailed comparison of cell states induced by each perturbation, such as a small molecule from a diverse library. Highly multiplexed measurements of cellular morphology can be extracted from each image and subsequently mined for a number of applications. Findings: This microscopy dataset includes 919 265 five-channel fields of view, representing 30 616 tested compounds, available at "The Cell Image Library" (CIL) repository. It also includes data files containing morphological features derived from each cell in each image, both at the single-cell level and population-averaged (i.e., per-well) level; the image analysis workflows that generated the morphological features are also provided. Quality-control metrics are provided as metadata, indicating fields of view that are out-of-focus or containing highly fluorescent material or debris. Lastly, chemical annotations are supplied for the compound treatments applied. Conclusions: Because computational algorithms and methods for handling single-cell morphological measurements are not yet routine, the dataset serves as a useful resource for the wider scientific community applying morphological (image-based) profiling. The dataset can be mined for many purposes, including small-molecule library enrichment and chemical mechanism-of-action studies, such as target identification. Integration with genetically perturbed datasets could enable identification of small-molecule mimetics of particular disease- or gene-related phenotypes that could be useful as probes or potential starting points for development of future therapeutics.


Assuntos
Processamento de Imagem Assistida por Computador , Bibliotecas de Moléculas Pequenas , Linhagem Celular , Células/efeitos dos fármacos , Células/ultraestrutura , Humanos
12.
ACS Chem Biol ; 11(7): 1844-51, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27064299

RESUMO

Unbiased binding assays involving small-molecule microarrays were used to identify compounds that display unique patterns of selectivity among members of the zinc-dependent histone deacetylase family of enzymes. A novel, hydroxyquinoline-containing compound, BRD4354, was shown to preferentially inhibit activity of HDAC5 and HDAC9 in vitro. Inhibition of deacetylase activity appears to be time-dependent and reversible. Mechanistic studies suggest that the compound undergoes zinc-catalyzed decomposition to an ortho-quinone methide, which covalently modifies nucleophilic cysteines within the proteins. The covalent nature of the compound-enzyme interaction has been demonstrated in experiments with biotinylated probe compound and with electrospray ionization-mass spectrometry.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Animais , Linhagem Celular , Humanos
13.
PLoS One ; 8(12): e80999, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24312513

RESUMO

Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that "paints the cell" with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.


Assuntos
Corantes Fluorescentes/química , Processamento de Imagem Assistida por Computador , Linhagem Celular Tumoral , Humanos
14.
ACS Chem Biol ; 7(7): 1152-7, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22536950

RESUMO

Post-translational modifications of histones alter chromatin structure and play key roles in gene expression and specification of cell states. Small molecules that target chromatin-modifying enzymes selectively are useful as probes and have promise as therapeutics, although very few are currently available. G9a (also named euchromatin histone methyltransferase 2 (EHMT2)) catalyzes methylation of lysine 9 on histone H3 (H3K9), a modification linked to aberrant silencing of tumor-suppressor genes, among others. Here, we report the discovery of a novel histone methyltransferase inhibitor, BRD4770. This compound reduced cellular levels of di- and trimethylated H3K9 without inducing apoptosis, induced senescence, and inhibited both anchorage-dependent and -independent proliferation in the pancreatic cancer cell line PANC-1. ATM-pathway activation, caused by either genetic or small-molecule inhibition of G9a, may mediate BRD4770-induced cell senescence. BRD4770 may be a useful tool to study G9a and its role in senescence and cancer cell biology.


Assuntos
Adenocarcinoma/enzimologia , Senescência Celular/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Sondas Moleculares/farmacologia , Neoplasias Pancreáticas/enzimologia , Adenocarcinoma/patologia , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Benzimidazóis/química , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Senescência Celular/imunologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Neoplasias Pancreáticas/patologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-20049832

RESUMO

The combination of nanoparticles and biological molecules is of intense interest because of the synergistic properties offered by such newly synthesized composites. Heparin (HP), conjugated to nanomaterials, has recently been investigated for its chemical and biological properties. HP has a number of biological activities that can be enhanced when composited with nanoparticles. In addition, HP improves the biocompatibility of nanoparticles improving their performance in various biological applications. A variety of recent research combines HP and nanomaterials for a myriad of applications. HP has been conjugated to the surface of the nanoparticles, such as magnetic and metallic nanoparticles, or biodegradable and nondegradable synthetic polymers. HP has also been incorporated into the nanoparticles. There are numerous possibilities for material composites and chemistries that incorporate HP. This opens the door for novel applications ranging from improving anticoagulant activity, for anticancer and antitumor therapy, to tissue engineering and biosensors. This review examines the different possibilities of HP-based nanoparticle composites and their medicinal or biological applications.


Assuntos
Heparina/química , Nanocompostos/química , Animais , Tecnologia Biomédica/métodos , Sistemas de Liberação de Medicamentos/métodos , Sinergismo Farmacológico , Heparina/administração & dosagem , Heparina/farmacologia , Humanos , Nanocompostos/administração & dosagem
16.
Nanomedicine (Lond) ; 4(4): 421-9, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19505245

RESUMO

AIMS: Silver nanoparticles exhibit unique antibacterial properties that make these ideal candidates for biological and medical applications. We utilized a clean method involving a single synthetic step to prepare silver nanoparticles that exhibit antimicrobial activity. MATERIALS & METHODS: These nanoparticles were prepared by reducing silver nitrate with diaminopyridinylated heparin (DAPHP) and hyaluronan (HA) polysaccharides and tested for their efficacy in inhibiting microbial growth. RESULTS & DISCUSSION: The resulting silver nanoparticles exhibit potent antimicrobial activity against Staphylococcus aureus and modest activity against Escherichia coli. Silver-HA showed greater antimicrobial activity than silver-DAPHP, while silver-glucose nanoparticles exhibited very weak antimicrobial activity. Neither HA nor DAPHP showed activity against S. aureus or E. coli. CONCLUSION: These results suggest that DAPHP and HA silver nanoparticles have potential in antimicrobial therapeutic applications.


Assuntos
Anti-Infecciosos/química , Heparina/química , Ácido Hialurônico/química , Nanopartículas Metálicas/química , Nanopartículas/química , Prata/química , Anti-Infecciosos/uso terapêutico , Escherichia coli/efeitos dos fármacos , Nanopartículas Metálicas/uso terapêutico , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Prata/uso terapêutico , Staphylococcus aureus/efeitos dos fármacos
17.
J Med Chem ; 51(18): 5498-501, 2008 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-18754653

RESUMO

Heparin, a widely used anticoagulant, is being rapidly displaced by low-molecular-weight heparins. Recently, certain lots of heparin have been associated with anaphylactoid-type reactions resulting from contamination with oversulfated chondroitin sulfate. This impurity has also contaminated low-molecular-weight heparins obtained by chemical and enzymatic depolymerization of heparin. The sensitivity of oversulfated chondroitin sulfate to five different depolymerization processes similar to ones used in preparing low-molecular-weight heparins is reported.


Assuntos
Anticoagulantes/química , Sulfatos de Condroitina/efeitos adversos , Sulfatos de Condroitina/isolamento & purificação , Heparina de Baixo Peso Molecular/química , Anticoagulantes/efeitos adversos , Heparina de Baixo Peso Molecular/efeitos adversos , Espectroscopia de Ressonância Magnética , Polímeros
18.
J Biol Chem ; 282(35): 25376-84, 2007 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-17597060

RESUMO

Heparan sulfate has been isolated for the first time from the mosquito Anopheles stephensi, a known vector for Plasmodium parasites, the causative agents of malaria. Chondroitin sulfate, but not dermatan sulfate or hyaluronan, was also present in the mosquito. The glycosaminoglycans were isolated, from salivary glands and midguts of the mosquito in quantities sufficient for disaccharide microanalysis. Both of these organs are invaded at different stages of the Plasmodium life cycle. Mosquito heparan sulfate was found to contain the critical trisulfated disaccharide sequence, -->4)beta-D-GlcNS6S(1-->4)-alpha-L-IdoA2S(1-->, that is commonly found in human liver heparan sulfate, which serves as the receptor for apolipoprotein E and is also believed to be responsible for binding to the circumsporozoite protein found on the surface of the Plasmodium sporozoite. The heparan sulfate isolated from the whole mosquito binds to circumsporozoite protein, suggesting a role within the mosquito for infection and transmission of the Plasmodium parasite.


Assuntos
Anopheles/metabolismo , Heparitina Sulfato/metabolismo , Fígado/metabolismo , Malária Falciparum/transmissão , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Anopheles/química , Anopheles/parasitologia , Sequência de Carboidratos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/química , Dermatan Sulfato/metabolismo , Dissacarídeos/química , Dissacarídeos/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/genética , Humanos , Fígado/química , Fígado/parasitologia , Malária Falciparum/metabolismo , Plasmodium falciparum/química , Ligação Proteica , Proteínas de Protozoários/química , Glândulas Salivares/química , Glândulas Salivares/metabolismo , Glândulas Salivares/parasitologia
19.
Biochemistry ; 46(10): 2697-706, 2007 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-17305366

RESUMO

Listeria monocytogenes, a food-borne pathogen that infects immunocompromised patients, enters and proliferates within mammalian cells by taking advantage of host cell machinery. While entry into macrophages and other phagocytic cells occurs constitutively, intracellular invasion of nonphagocytic cells, such as epithelial and endothelial cells, occurs through induced phagocytosis. Invasion of these nonphagocytic cell types is under the control of the secreted L. monocytogenes protein internalin B (InlB), which directly associates with and activates the receptor tyrosine kinase Met. Activation of Met by InlB has previously been shown to be potentiated by binding of glycosaminoglycans to the GW domains of this protein. We studied the interaction between heparin and full-length InlB as well as a truncated, functional form of InlB to understand the mode of interaction between these two molecules. InlB preferred long-chain (>or=dp14) heparin oligosaccharides, and the interaction with heparin fit a complicated binding model with a dissociation constant in the nanomolar range. While there are various explanations for this complicated binding model, one supported by our data involves binding and rebinding of InlB to multiple binding sites on heparin in a positive and weakly cooperative manner. This mode is consistent with enhancement of interaction of InlB with glycosaminoglycans for activation of Met.


Assuntos
Proteínas de Bactérias/metabolismo , Ligação Competitiva/fisiologia , Heparina/metabolismo , Proteínas de Membrana/metabolismo , Ligação Proteica/fisiologia , Ressonância de Plasmônio de Superfície/métodos , Sítios de Ligação , Humanos , Cinética
20.
J Biol Chem ; 281(42): 31689-95, 2006 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-16882661

RESUMO

Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.


Assuntos
Anexina A2/química , Cálcio/metabolismo , Heparina/química , Sítios de Ligação , Cálcio/química , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Modelos Moleculares , Oligossacarídeos/química , Oxigênio/química , Ligação Proteica , Conformação Proteica , Ressonância de Plasmônio de Superfície
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA