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1.
Lasers Surg Med ; 2021 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-34940986

RESUMO

OBJECTIVES: Bacteremia is a serious and potentially lethal condition. Staphylococcus aureus is a leading cause of bacteremia and methicillin-resistant S. aureus (MRSA) accounts for more than a third of the cases. Compared to methicillin-sensitive S. aureus, MRSA is more than twice as likely to be fatal. Furthermore, subpopulations of seemingly isogenic bacteria may exhibit a range of susceptibilities, often called heterogenous resistance. These heterogeneous antibiotic-resistant infections are often misdiagnosed as hospital-acquired secondary infections because there are no clinically used tests that can differentiate between homogeneous and heterogeneous antibiotic resistance. We describe the development and proof of concept of rapid bacterial identification using photoacoustic flow cytometry and labeled bacteriophages with the characterization and differentiation of heterogeneous antibiotic-resistant bacterial infections. METHODS: In photoacoustic flow cytometry, pulsed laser light is delivered to a sample flowing past a focused transducer and particles that absorb laser light create an acoustic response. Optically labeled bacteriophage are added to a bacterial mixture that flows through the photoacoustic chamber. The presence of target bacteria is determined by bound labeled phage which are detected photoacoustically. Incubation of bacterial samples in the presence and absence of the antibiotic daptomycin creates a difference in bacterial cell numbers that is quantified using photoacoustic flow cytometry. RESULTS: Four clinical isolates were tested in the presence and absence of daptomycin. Photoacoustic events for each isolate were recorded and compared to growth curves. Samples treated with daptomycin fell into three categories: resistant, susceptible, and heterogeneous resistant. CONCLUSIONS: Here we show a method to determine the presence of bacteria as a marker for bloodstream infection level and antibiotic sensitivity in less than 4 hours. Additionally, these results show an ability to identify heterogeneous resistant strains that are often misidentified.

2.
Nucleic Acids Res ; 2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34788843

RESUMO

The Reactome Knowledgebase (https://reactome.org), an Elixir core resource, provides manually curated molecular details across a broad range of physiological and pathological biological processes in humans, including both hereditary and acquired disease processes. The processes are annotated as an ordered network of molecular transformations in a single consistent data model. Reactome thus functions both as a digital archive of manually curated human biological processes and as a tool for discovering functional relationships in data such as gene expression profiles or somatic mutation catalogs from tumor cells. Recent curation work has expanded our annotations of normal and disease-associated signaling processes and of the drugs that target them, in particular infections caused by the SARS-CoV-1 and SARS-CoV-2 coronaviruses and the host response to infection. New tools support better simultaneous analysis of high-throughput data from multiple sources and the placement of understudied ('dark') proteins from analyzed datasets in the context of Reactome's manually curated pathways.

3.
Methods Mol Biol ; 2265: 203-212, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704716

RESUMO

Early detection of cancer has been a goal of cancer research in general and melanoma research in particular (Birnbaum et al., Lancet Glob Health 6:e885-e893, 2018; Alendar et al., Bosnian J Basic Med Sci 9:77-80, 2009). Early detection of metastasis has been targeted as pivotal to increasing survival rates (Menezes et al., Adv Cancer Res 132:1-44, 2016). Melanoma, though curable in its early stages, has a dramatic decrease in survival rates once metastasis has occurred (Sharma et al., Biotechnol Adv 36:1063-1078, 2018). The transition to metastasis is not well understood and is an area of increasing interest. Metastasis is always premeditated by the shedding of circulating tumor cells (CTCs) from the primary tumor. The ability to isolate rare CTCs from the bloodstream has led to a host of new targets and therapies for cancer (Micalizzi et al., Genes Dev 31:1827-1840, 2017). Detection of CTCs also allows for disease progression to be tracked in real time while eliminating the need to wait for additional tumors to grow. Using a photoacoustic flowmeter, in which we induce ultrasonic responses from circulating melanoma cells (CMCs), we identify and quantify these cells in order to track disease progression. Additionally, these CMCs are captured and isolated allowing for future analysis such as RNA-Seq or microarray analysis.


Assuntos
Citometria de Fluxo/métodos , Melanoma/diagnóstico , Células Neoplásicas Circulantes , Técnicas Fotoacústicas/instrumentação , Técnicas Fotoacústicas/métodos , Reologia/instrumentação , Reologia/métodos , Neoplasias Cutâneas/diagnóstico , Progressão da Doença , Detecção Precoce de Câncer/métodos , Citometria de Fluxo/instrumentação , Biblioteca Gênica , Humanos , Imuno-Histoquímica/métodos , Melanoma/sangue , Melanoma/genética , Melanoma/patologia , Metástase Neoplásica/diagnóstico , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes/patologia , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Ultrassonografia/métodos
4.
Am J Obstet Gynecol ; 224(1): 78.e1-78.e7, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32707267

RESUMO

BACKGROUND: Stress urinary incontinence carries a significant healthcare burden for women worldwide. Single incision slings are minimally invasive mesh devices designed to treat stress urinary incontinence. For prolapse repair, meshes with higher porosity and lower structural stiffness have been associated with improved outcomes. OBJECTIVE: In this study, we compared the higher stiffness, lower porosity Altis sling with the lower stiffness, higher porosity Solyx sling in an ovine model. We hypothesized that SIS-B would have a negative impact on the host response. STUDY DESIGN: A total of Altis and Solyx single incision slings were implanted suburethrally into sheep according to the manufacturer's instructions on minimal tension. The mesh-urethral-vaginal complex and adjacent ungrafted vagina (no mesh control) were harvested en bloc at 3 months. Masson's trichrome and picrosirius red staining of 6 µm thin sections was performed to measure interfiber distance and tissue integration. Smooth muscle contractility to a 120 mM KCl stimulus was performed in an organ bath to measure myofiber-driven contractions. Standard biochemical assays were used to quantify glycosaminoglycan, total collagen, and elastin content, and collagen subtypes. Bending stiffness was performed in response to a uniaxial force to define susceptibility to folding/buckling. Statistical analysis was performed using Mann-Whitney, Gabriel's pairwise post hoc, Wilcoxon matched-pairs, and chi-square tests. RESULTS: The animals had similar ages (3-5 years), parity (multiparous), and weights (45-72 kg). Trichrome cross sections showed that the Altis sling buckled in a "C" or "S" shape in most samples (8 of 11), whereas buckling after Solyx sling implantation was observed in only a single sample (1 of 13; P=.004). Tissue integration, as measured by the presence of collagen or smooth muscle between the mesh fibers on trichrome 4× imaging, was increased in samples implanted with the Solyx sling compared with the Altis sling (P<.05). Total collagen content decreased significantly with both products when compared with the ungrafted vagina consistent with stress shielding. There was no difference in the 2 groups with regard to glycosaminoglycan or elastin content. The Altis sling mesh tissue complex demonstrated significantly higher amounts of both collagen types I and III than the Solyx sling-implanted tissue and the ungrafted control. Smooth muscle contractility in response to 120 mM KCl was decreased after implantation of both slings compared with the sham (P=.011 and P<.01), with no difference between mesh types (P=.099). Bending stiffness in the Altis sling was more than 4 times lower than in the Solyx, indicating an increased propensity to buckle (0.0186 vs 0.0883). CONCLUSION: The structurally stiffer Altis sling had decreased tissue integration and increased propensity to buckle after implantation. Increased collagen types I and III after the implantation of this device suggests that these changes may be associated with a fibrotic response. In contrast, the Solyx sling largely maintained a flat configuration and had improved tissue integration. The deformation of the Altis sling is not an intended effect and is likely caused by its lower bending stiffness. Both meshes induced a decrease in collagen content and smooth muscle contractility similar to previous findings for prolapse meshes and consistent with stress shielding. The long-term impact of buckling warrants further investigation.


Assuntos
Modelos Animais , Slings Suburetrais , Incontinência Urinária por Estresse/cirurgia , Vagina/cirurgia , Animais , Feminino , Ovinos
5.
Nucleic Acids Res ; 49(D1): D1452-D1463, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33170273

RESUMO

Gramene (http://www.gramene.org), a knowledgebase founded on comparative functional analyses of genomic and pathway data for model plants and major crops, supports agricultural researchers worldwide. The resource is committed to open access and reproducible science based on the FAIR data principles. Since the last NAR update, we made nine releases; doubled the genome portal's content; expanded curated genes, pathways and expression sets; and implemented the Domain Informational Vocabulary Extraction (DIVE) algorithm for extracting gene function information from publications. The current release, #63 (October 2020), hosts 93 reference genomes-over 3.9 million genes in 122 947 families with orthologous and paralogous classifications. Plant Reactome portrays pathway networks using a combination of manual biocuration in rice (320 reference pathways) and orthology-based projections to 106 species. The Reactome platform facilitates comparison between reference and projected pathways, gene expression analyses and overlays of gene-gene interactions. Gramene integrates ontology-based protein structure-function annotation; information on genetic, epigenetic, expression, and phenotypic diversity; and gene functional annotations extracted from plant-focused journals using DIVE. We train plant researchers in biocuration of genes and pathways; host curated maize gene structures as tracks in the maize genome browser; and integrate curated rice genes and pathways in the Plant Reactome.


Assuntos
Bases de Dados Genéticas , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Genômica/métodos , Proteínas de Plantas/genética , Plantas/genética , Produtos Agrícolas , Elementos de DNA Transponíveis , Duplicação Gênica , Ontologia Genética , Redes Reguladoras de Genes , Internet , Bases de Conhecimento , Redes e Vias Metabólicas , Anotação de Sequência Molecular , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plantas/classificação , Plantas/metabolismo , Poliploidia , Mapeamento de Interação de Proteínas , Software , Zea mays/genética , Zea mays/metabolismo
6.
Lasers Surg Med ; 53(4): 578-586, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32557708

RESUMO

BACKGROUND AND OBJECTIVES: Enumerating circulating tumor cells has been used as a method of monitoring progression of various cancers. Various methods for detecting circulating melanoma cells (CMCs) have been reported, but none has had sufficient sensitivity to determine if the presence of rare CMCs in the blood of Stage I-III melanoma patients predicts if those patients eventually develop metastatic disease. STUDY DESIGN: We quantified CMCs in serial blood samples from 38 early stage melanoma patients to determine if CMC numbers predict development of metastatic melanoma. CMCs were enumerated using a photoacoustic flow cytometric detection system that uses a laser to induce high frequency acoustic signals in pigmented CMCs. RESULTS: We observed that detection of greater than 2 CMCs/ml of blood from patients with Stage I-III melanoma predicts metastatic disease. Of the 11 patients we studied who had two or fewer CMCs detected at all time points tested, none progressed to metastatic disease over a mean follow-up of 1288 days. In contrast, 18 of the 27 patients (67%) having more than 2 CMCs/ml at one or more time points progressed to metastatic disease over a mean follow-up of 850 days. CONCLUSIONS: Photoacoustic flow cytometry can detect rare CMCs in the blood of Stage I-III melanoma patients and detectionof these cells is predictive of subsequent development of metastatic disease. Lasers Surg. Med.


Assuntos
Melanoma , Células Neoplásicas Circulantes , Neoplasias Cutâneas , Citometria de Fluxo , Humanos , Melanoma/diagnóstico por imagem , Neoplasias Cutâneas/diagnóstico por imagem
7.
Nucleic Acids Res ; 48(D1): D498-D503, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31691815

RESUMO

The Reactome Knowledgebase (https://reactome.org) provides molecular details of signal transduction, transport, DNA replication, metabolism and other cellular processes as an ordered network of molecular transformations in a single consistent data model, an extended version of a classic metabolic map. Reactome functions both as an archive of biological processes and as a tool for discovering functional relationships in data such as gene expression profiles or somatic mutation catalogs from tumor cells. To extend our ability to annotate human disease processes, we have implemented a new drug class and have used it initially to annotate drugs relevant to cardiovascular disease. Our annotation model depends on external domain experts to identify new areas for annotation and to review new content. New web pages facilitate recruitment of community experts and allow those who have contributed to Reactome to identify their contributions and link them to their ORCID records. To improve visualization of our content, we have implemented a new tool to automatically lay out the components of individual reactions with multiple options for downloading the reaction diagrams and associated data, and a new display of our event hierarchy that will facilitate visual interpretation of pathway analysis results.


Assuntos
Bases de Dados de Compostos Químicos , Bases de Dados de Produtos Farmacêuticos , Bases de Conhecimento , Software , Genoma Humano , Humanos , Redes e Vias Metabólicas , Mapas de Interação de Proteínas , Transdução de Sinais
8.
Nucleic Acids Res ; 48(D1): D1093-D1103, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31680153

RESUMO

Plant Reactome (https://plantreactome.gramene.org) is an open-source, comparative plant pathway knowledgebase of the Gramene project. It uses Oryza sativa (rice) as a reference species for manual curation of pathways and extends pathway knowledge to another 82 plant species via gene-orthology projection using the Reactome data model and framework. It currently hosts 298 reference pathways, including metabolic and transport pathways, transcriptional networks, hormone signaling pathways, and plant developmental processes. In addition to browsing plant pathways, users can upload and analyze their omics data, such as the gene-expression data, and overlay curated or experimental gene-gene interaction data to extend pathway knowledge. The curation team actively engages researchers and students on gene and pathway curation by offering workshops and online tutorials. The Plant Reactome supports, implements and collaborates with the wider community to make data and tools related to genes, genomes, and pathways Findable, Accessible, Interoperable and Re-usable (FAIR).


Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Genômica , Metabolômica , Plantas/genética , Plantas/metabolismo , Proteômica , Redes Reguladoras de Genes , Genômica/métodos , Humanos , Redes e Vias Metabólicas , Metabolômica/métodos , Proteômica/métodos , Transdução de Sinais , Navegador
9.
Database (Oxford) ; 20192019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31802127

RESUMO

Reactome is a manually curated, open-source, open-data knowledge base of biomolecular pathways. Reactome has always provided clear credit attribution for authors, curators and reviewers through fine-grained annotation of all three roles at the reaction and pathway level. These data are visible in the web interface and provided through the various data download formats. To enhance visibility and credit attribution for the work of authors, curators and reviewers, and to provide additional opportunities for Reactome community engagement, we have implemented key changes to Reactome: contributor names are now fully searchable in the web interface, and contributors can 'claim' their contributions to their ORCID profile with a few clicks. In addition, we are reaching out to domain experts to request their help in reviewing and editing Reactome pathways through a new 'Contribution' section, highlighting pathways which are awaiting community review. Database URL: https://reactome.org.


Assuntos
Curadoria de Dados , Transdução de Sinais , Interface Usuário-Computador
10.
Opt Express ; 27(21): 30653, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31684309

RESUMO

The authors wish to acknowledge an additional funding source which was absent from our manuscript.

11.
Opt Express ; 27(20): 27682-27690, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31684531

RESUMO

We report efficient coupling of a quantum cascade laser (QCL) into step-index chalcogenide fibers (As 2S 3). Mechanically robust and low-loss chalcogenide fibers were fabricated using a hybrid, multi-material thermal drawing process. With suitable free-space optics, more than 160 mW of optical power was coupled into the fiber with predominantly single-mode excitation. Antireflection coatings on the fiber facets enabled 88.9% transmission with strong core confinement. By accurately tailoring the core diameter and antireflection-coating thickness, these fibers offer a versatile platform for high-power and low-loss transmission across the infrared spectrum. This work introduces an attractive alternative to the use of hollow-core fibers or multimode solid-core fibers for diffraction-limited infrared beam delivery.

12.
J Biomed Opt ; 24(11): 1-7, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31758676

RESUMO

Infection with resistant bacteria has become an ever increasing problem in modern medical practice. Currently, broad spectrum antibiotics are prescribed until bacteria can be identified through blood cultures, a process that can take two to three days and is unable to provide quantitative information. To detect and quantify bacteria rapidly in blood samples, we designed a method using labeled bacteriophage in conjunction with photoacoustic flow cytometry (PAFC). PAFC is the generation of ultrasonic waves created by the absorption of laser light in particles under flow. Bacteriophage is a virus that infects bacteria and possesses the ability to discriminate bacterial surface antigens, allowing the bacteriophage to bind only to their target bacteria. Bacteria can be tagged with dyed phage and processed through a photoacoustic flow cytometer where they are detected by the acoustic response. We demonstrate that E. coli; can be detected and discriminated from Salmonella; using this method. Our goal is to develop a method to determine bacterial content in blood samples. We hope to develop this technology into future clinical use and decrease the time required to identify bacterial species from 3 to 4 days to less than 1 hour.


Assuntos
Bacteriófagos/fisiologia , Escherichia coli/citologia , Citometria de Fluxo/métodos , Técnicas Fotoacústicas/métodos , Salmonella/citologia , Corantes , Epitopos , Escherichia coli/virologia , Lasers , Salmonella/virologia , Processamento de Sinais Assistido por Computador , Ultrassom
13.
Adv Mater ; 30(39): e1803628, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30101495

RESUMO

A novel photothermal process to spatially modulate the concentration of sub-wavelength, high-index nanocrystals in a multicomponent Ge-As-Pb-Se chalcogenide glass thin film resulting in an optically functional infrared grating is demonstrated. The process results in the formation of an optical nanocomposite possessing ultralow dispersion over unprecedented bandwidth. The spatially tailored index and dispersion modification enables creation of arbitrary refractive index gradients. Sub-bandgap laser exposure generates a Pb-rich amorphous phase transforming on heat treatment to high-index crystal phases. Spatially varying nanocrystal density is controlled by laser dose and is correlated to index change, yielding local index modification to ≈+0.1 in the mid-infrared.

15.
Emerg Med Clin North Am ; 34(1): 151-7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26614246

RESUMO

Pulmonary ultrasound continues to develop and is ideally suited for the evaluation and treatment of respiratory emergencies. It is portable, can be performed rapidly, has no ionizing radiation, and is highly sensitive and specific for the diagnosis of pneumothorax, pneumonia, pulmonary edema, and free fluid in the chest.


Assuntos
Tratamento de Emergência/métodos , Pneumopatias/diagnóstico por imagem , Sistemas Automatizados de Assistência Junto ao Leito , Tórax/diagnóstico por imagem , Doença Aguda , Hemotórax/diagnóstico por imagem , Humanos , Derrame Pleural/diagnóstico por imagem , Pneumonia/diagnóstico por imagem , Pneumotórax/diagnóstico por imagem , Edema Pulmonar/diagnóstico por imagem , Ultrassonografia
16.
Trials ; 15: 34, 2014 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-24456998

RESUMO

BACKGROUND: Diabetes, a highly prevalent, chronic disease, is associated with increasing frailty and functional decline in older people, with concomitant personal, social, and public health implications. We describe the rationale and methods of the multi-modal intervention in diabetes in frailty (MID-Frail) study. METHODS/DESIGN: The MID-Frail study is an open, randomised, multicentre study, with random allocation by clusters (each trial site) to a usual care group or an intervention group. A total of 1,718 subjects will be randomised with each site enrolling on average 14 or 15 subjects. The primary objective of the study is to evaluate, in comparison with usual clinical practice, the effectiveness of a multi-modal intervention (specific clinical targets, education, diet, and resistance training exercise) in frail and pre-frail subjects aged ≥70 years with type 2 diabetes in terms of the difference in function 2 years post-randomisation. Difference in function will be measured by changes in a summary ordinal score on the short physical performance battery (SPPB) of at least one point. Secondary outcomes include daily activities, economic evaluation, and quality of life. DISCUSSION: The MID-Frail study will provide evidence on the clinical, functional, social, and economic impact of a multi-modal approach in frail and pre-frail older people with type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01654341.


Assuntos
Diabetes Mellitus Tipo 2/terapia , Dieta , Conhecimentos, Atitudes e Prática em Saúde , Educação de Pacientes como Assunto , Projetos de Pesquisa , Treinamento de Força , Atividades Cotidianas , Fatores Etários , Idoso , Protocolos Clínicos , Terapia Combinada , Análise Custo-Benefício , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/economia , Dieta/efeitos adversos , Dieta/economia , Europa (Continente) , Idoso Fragilizado , Custos de Cuidados de Saúde , Humanos , Educação de Pacientes como Assunto/economia , Qualidade de Vida , Treinamento de Força/economia , Fatores de Tempo , Resultado do Tratamento
18.
J Phys Chem A ; 116(20): 4934-46, 2012 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-22515263

RESUMO

Mechanisms for the reaction of thiophene and 2-methylthiophene with molecular oxygen on both the triplet and singlet potential energy surfaces (PESs) have been investigated using ab initio methods. Geometries of various stationary points involved in the complex reaction routes are optimized at the MP2/6-311++G(d, p) level. The barriers and energies of reaction for all product channels were refined using single-point calculations at the G4MP2 level of theory. For thiophene, CCSD(T) single point energies were also determined for comparison with the G4MP2 energies. Thiophene and 2-methylthiophene were shown to react with O(2) via two types of mechanisms, namely, direct hydrogen abstraction and addition/elimination. The barriers for reaction with triplet oxygen are all significantly large (i.e., >30 kcal mol(-1)), indicating that the direct oxidation of thiophene by ground state oxygen might be important only in high temperature processes. Reaction of thiophene with singlet oxygen via a 2 + 4 cycloaddition leading to endoperoxides is the most favorable channel. Moreover, it was found that alkylation of the thiophene ring (i.e., methyl-substituted thiophene) is capable of lowering the barrier height for the addition pathway. The implication of the current theoretical results may shed new light on the initiation mechanisms for combustion of asphaltenes.


Assuntos
Oxigênio/química , Teoria Quântica , Tiofenos/química
19.
J Phys Chem B ; 115(26): 8597-608, 2011 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-21661730

RESUMO

(rac)-1,1'-Binaphthyl-based simple receptors 1 and 2 have been designed, synthesized and studied theoretically. The receptors utilize naphthyridine as the binding motifs for complexation of dicarboxylic acids in CHCl(3). The emission of the BINOL moiety was monitored experimentally to ascertain the selectivity and sensitivity of the receptors. Receptor 1 distinguishes maleic acid from isomeric fumaric acid by exhibiting different fluorescence behavior and demonstrates stronger binding in the excited state. Modulation of the binding sites of 1 leads to a new receptor structure 2, which was found to be less efficient in distinguishing maleic from fumaric acid, fluorometrically. Both 1 and 2 also recognize other hydroxy di- and tricarboxylic acids. The binding interactions were monitored by (1)H NMR, fluorescence and UV-vis spectroscopic methods. Structures of apo-hosts, guests and host-guest complexes were determined using force-field based conformational searching. Low energy ensembles were grouped into geometrically similar families, and low energy structures from each family were verified using B3LYP/6-31G*/PB-SCRF(CHCl(3)) calculations. The atomistic calculations provide insight into the differential dicarboxylic acid binding behavior of receptors 1 and 2.


Assuntos
Fluorometria/métodos , Fumaratos/análise , Maleatos/análise , Naftalenos/química , Simulação por Computador , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Naftalenos/síntese química
20.
J Immunol ; 168(4): 1787-95, 2002 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-11823511

RESUMO

Soluble fragments of the alpha-chain of FcepsilonRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcepsilonRIalpha is used in this study, in conjunction with the crystal structure of the FcepsilonRIalpha/IgE complex, to define the relative importance of specific residues within human FcepsilonRIalpha for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcepsilonRIalpha and attenuate allergic disease. Three residues within the C-C' region of the FcepsilonRIalpha2 domain and two residues within the alpha2 proximal loops of the alpha1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three alpha2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in alpha1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the alpha1/alpha2 interface. Our results demonstrate that the effects of mutations in human FcepsilonRIalpha on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.


Assuntos
Imunoglobulina E/metabolismo , Receptores de IgE/genética , Receptores de IgE/metabolismo , Animais , Basófilos/imunologia , Degranulação Celular , Dicroísmo Circular , Liberação de Histamina , Humanos , Imunoglobulina E/imunologia , Cinética , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pichia/genética , Estrutura Terciária de Proteína , Ratos , Receptores de IgE/química , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Ressonância de Plasmônio de Superfície , Células Tumorais Cultivadas
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