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1.
J Ultrasound Med ; 42(8): 1887-1892, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36896883

RESUMO

We present a 3-patient case series that support the use of ultrasound guided minimally invasive autopsy (MIA). This technique has a high diagnostic accuracy in specific clinical settings. It makes easier to diagnose pathologies once the patient has died, avoiding body deformation, with a notable reduction in sample processing time compared to the open autopsy study and, therefore, a shorter overall diagnostic response time. MIA shows some similarities with point of care ultrasound (POCUS), like examination protocols or that they can be performed at the bedside.


Assuntos
Testes Imediatos , Ultrassonografia de Intervenção , Humanos , Autopsia/métodos , Ultrassonografia/métodos , Sistemas Automatizados de Assistência Junto ao Leito
2.
Cardiovasc Diabetol ; 20(1): 155, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34320987

RESUMO

BACKGROUND: Metabolic syndrome (MetS) is a multimorbid long-term condition without consensual medical definition and a diagnostic based on compatible symptomatology. Here we have investigated the molecular signature of MetS in urine. METHODS: We used NMR-based metabolomics to investigate a European cohort including urine samples from 11,754 individuals (18-75 years old, 41% females), designed to populate all the intermediate conditions in MetS, from subjects without any risk factor up to individuals with developed MetS (4-5%, depending on the definition). A set of quantified metabolites were integrated from the urine spectra to obtain metabolic models (one for each definition), to discriminate between individuals with MetS. RESULTS: MetS progression produces a continuous and monotonic variation of the urine metabolome, characterized by up- or down-regulation of the pertinent metabolites (17 in total, including glucose, lipids, aromatic amino acids, salicyluric acid, maltitol, trimethylamine N-oxide, and p-cresol sulfate) with some of the metabolites associated to MetS for the first time. This metabolic signature, based solely on information extracted from the urine spectrum, adds a molecular dimension to MetS definition and it was used to generate models that can identify subjects with MetS (AUROC values between 0.83 and 0.87). This signature is particularly suitable to add meaning to the conditions that are in the interface between healthy subjects and MetS patients. Aging and non-alcoholic fatty liver disease are also risk factors that may enhance MetS probability, but they do not directly interfere with the metabolic discrimination of the syndrome. CONCLUSIONS: Urine metabolomics, studied by NMR spectroscopy, unravelled a set of metabolites that concomitantly evolve with MetS progression, that were used to derive and validate a molecular definition of MetS and to discriminate the conditions that are in the interface between healthy individuals and the metabolic syndrome.


Assuntos
Síndrome Metabólica/urina , Metaboloma , Metabolômica , Espectroscopia de Prótons por Ressonância Magnética , Adolescente , Adulto , Idoso , Biomarcadores/urina , Estudos de Casos e Controles , Progressão da Doença , Europa (Continente) , Feminino , Humanos , Masculino , Síndrome Metabólica/diagnóstico , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Urinálise , Adulto Jovem
3.
Immunity ; 29(6): 863-75, 2008 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-19100700

RESUMO

Differentiation of memory cells involves DNA-sequence changes in B lymphocytes but is less clearly defined in T cells. RNA rearrangement is identified here as a key event in memory T cell differentiation by analysis of a mouse mutation that altered the proportions of naive and memory T cells and crippled the process of Ptprc exon silencing needed to generate CD45RO in memory T cells. A single substitution in a memory-induced RNA-binding protein, hnRNPLL, destabilized an RNA-recognition domain that bound with micromolar affinity to RNA containing the Ptprc exon-silencing sequence. Hnrpll mutation selectively diminished T cell accumulation in peripheral lymphoid tissues but not proliferation. Exon-array analysis of Hnrpll mutant naive and memory T cells revealed an extensive program of alternative mRNA splicing in memory T cells, coordinated by hnRNPLL. A remarkable overlap with alternative splicing in neural tissues may reflect a co-opted strategy for diversifying memory T cells.


Assuntos
Processamento Alternativo/genética , Éxons/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Memória Imunológica/genética , RNA/genética , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/imunologia , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , RNA/imunologia , Subpopulações de Linfócitos T/metabolismo
4.
Rev Argent Microbiol ; 48(2): 119-21, 2016.
Artigo em Espanhol | MEDLINE | ID: mdl-27086257

RESUMO

Staphylococcus lugdunensis is a coagulase-negative staphylococcus of growing importance and atypical behavior. The infections caused by this microorganism are becoming more frequent, having a broader spectrum. Psoas abscesses caused by this germ are rare, with few cases reported in the literature. In this work, we present a case of a psoas abscess caused by S. lugdunensis in a patient suffering from diabetes mellitus and rheumatoid arthritis, which was treated with intravenous cloxacillin with a good outcome.


Assuntos
Abscesso do Psoas/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus lugdunensis/isolamento & purificação , Antibacterianos/uso terapêutico , Artrite Reumatoide/complicações , Técnicas de Tipagem Bacteriana , Cloxacilina/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Abscesso Epidural/tratamento farmacológico , Abscesso Epidural/microbiologia , Feminino , Humanos , Hospedeiro Imunocomprometido , Pessoa de Meia-Idade , Abscesso do Psoas/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus lugdunensis/patogenicidade
5.
Med Oral Patol Oral Cir Bucal ; 19(4): e335-9, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24608210

RESUMO

OBJECTIVES: to evaluate the ability of the diagnostic criteria proposed by Cerero et al in 2010 to perform an early diagnose in patients with proliferative verrucous leukoplakia. STUDY DESIGN: retrospective study with patients diagnosed with leukoplakia at Oral Medicine Service at Oral Medicine and Surgery Department at Dentistry Faculty at Universidad Complutense of Madrid. RESULTS: the criteria were applied in 116 patients, turning positive in 40 cases. Out of these, 24 (60%) had been previously diagnosed with PVL. Most frequent criteria were major criteria A and E, concerning lesion's site and histopathology, and minor criteria b and c, concerning sex and smoking habit. CONCLUSIONS: diagnostic criteria developed by Cerero et al can be a useful tool for an early diagnose of PVL, as in 60% of the cases, the criteria would have allowed to make an early diagnose of the disease.


Assuntos
Leucoplasia Oral/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
6.
Food Chem ; 461: 140831, 2024 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-39226795

RESUMO

Eight sample digestion procedures were compared to determine 41 elements in rice samples by ICP-MS and CV-AFS. Analytical methods were evaluated using certified rice flour reference material (NIST 1568b) and recovery experiments. The microwave-assisted digestion of 0.5 g rice sample and reagent mixture of 2 mL HNO3, 0.5 mL H2O2, and 0.5 mL deionized water yielded the best recovery for all elements ranging from 90 to 120% at three different levels, bias% within 10%, and precision (coefficient of variation percent, CV% intra- and inter-day) below 15%. The best analytical method was applied to the elemental determination in nine types of rice available on the Italian market. Daily or weekly rice consumption meets the nutritional and safety requirements of EFSA and WHO. The present study allows extensive and detailed knowledge of the content of essential and non-essential/toxic elements in different types of rice produced or packaged in Italy.


Assuntos
Espectrometria de Massas , Oryza , Oryza/química , Itália , Oligoelementos/análise , Micro-Ondas , Humanos
7.
Data Brief ; 42: 108253, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35599817

RESUMO

Noticeable within the Mexican Caribbean is the Arrecife de Puerto Morelos National Park (APMNP), a marine protected area established as an essential component for managing and protecting coral reefs. In June 2019, we conducted a survey in eight shallow reef sites of the APMNP with the purpose of applying a coral reef assessment method, based on biological indicators of the condition of both benthos and fish communities. In this paper we present tables with data of biological and ecological variables such as: benthos coverage, species composition and abundance of corals, abundance of urchins and coral recruits, bleaching, coral diseases and coral mortality percent, reef relief, and composition and abundance of key commercial and herbivorous fish species. The research article related to these databases was published in the journal Diversity with the title: Puerto Morelos coral reefs, current state and their classification by a scoring system.

8.
J Am Chem Soc ; 133(47): 19205-15, 2011 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-22007671

RESUMO

The two-component dengue virus NS2B-NS3 protease (DEN NS2B-NS3pro) is an established drug target, but inhibitor design is hampered by the lack of a crystal structure of the protease in its fully active form. In solution and without inhibitors, the functionally important C-terminal segment of the NS2B cofactor is dissociated from DEN NS3pro ("open state"), necessitating a large structural change to produce the "closed state" thought to underpin activity. We analyzed the fold of DEN NS2B-NS3pro in solution with and without bound inhibitor by nuclear magnetic resonance (NMR) spectroscopy. Multiple paramagnetic lanthanide tags were attached to different sites to generate pseudocontact shifts (PCS). In the face of severe spectral overlap and broadening of many signals by conformational exchange, methods for assignment of (15)N-HSQC cross-peaks included selective mutation, combinatorial isotope labeling, and comparison of experimental PCSs and PCSs back-calculated for a structural model of the closed conformation built by using the structure of the related West Nile virus (WNV) protease as a template. The PCSs show that, in the presence of a positively charged low-molecular weight inhibitor, the enzyme assumes a closed state that is very similar to the closed state previously observed for the WNV protease. Therefore, a model of the protease built on the closed conformation of the WNV protease is a better template for rational drug design than available crystal structures, at least for positively charged inhibitors. To assess the open state, we created a binding site for a Gd(3+) complex and measured paramagnetic relaxation enhancements. The results show that the specific open conformation displayed in the crystal of DEN NS2B-NS3pro is barely populated in solution. The techniques used open an avenue to the fold analysis of proteins that yield poor NMR spectra, as PCSs from multiple sites in combination with model building generate powerful information even from incompletely assigned (15)N-HSQC spectra.


Assuntos
Vírus da Dengue/enzimologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sítios de Ligação/efeitos dos fármacos , Modelos Moleculares , Conformação Molecular , Peso Molecular , Inibidores de Proteases/química , Dobramento de Proteína/efeitos dos fármacos , Serina Endopeptidases/química , Proteínas não Estruturais Virais/química
9.
Am J Med Genet A ; 155A(2): 301-6, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21271646

RESUMO

We present two unrelated male infants with strikingly similar clinical features which have not previously been reported together. The most unusual feature was the presence of multiple small capillary malformations (port-wine stains) on the skin from birth. Both infants had intractable seizures, microcephaly with progressive cortical atrophy, severe developmental delay, dysmorphic facial features, and hypoplasia of the distal phalanges. To our knowledge, no other person with this unique constellation of features has been described.


Assuntos
Anormalidades Múltiplas , Capilares/anormalidades , Deformidades Congênitas dos Membros/patologia , Microcefalia/patologia , Mancha Vinho do Porto/patologia , Convulsões/patologia , Pré-Escolar , Humanos , Recém-Nascido , Cariotipagem , Masculino , Análise em Microsséries , Síndrome
10.
Pathogens ; 9(9)2020 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-32825097

RESUMO

Mycoplasma gallisepticum (MG) is among the most significant problems in the poultry industry worldwide, representing a serious threat to international trade. Despite the fact that the mgc2 gene has been widely used for diagnostic and molecular characterization purposes, there is a lack of evidence supporting the reliability of this gene as a marker for molecular epidemiology approaches. Therefore, the current study aimed to assess the accuracy of the mgc2 gene for phylogenetic, phylodynamic, and phylogeographic evaluations. Furthermore, the global phylodynamic expansion of MG is described, and the origin and extension of the outbreak caused by MG in Ecuador were tracked and characterized. The results obtained strongly supported the use of the mgc2 gene as a reliable phylogenetic marker and accurate estimator for the temporal and phylogeographic structure reconstruction of MG. The phylodynamic analysis denoted the failures in the current policies to control MG and highlighted the imperative need to implement more sensitive methodologies of diagnosis and more efficient vaccines. Framed in Ecuador, the present study provides the first piece of evidence of the circulation of virulent field MG strains in Ecuadorian commercial poultry. The findings derived from the current study provide novel and significant insights into the origin, diversification, and evolutionary process of MG globally.

11.
Drug Target Insights ; 13: 1177392819861987, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31320796

RESUMO

A 43-year-old man with a history of severe extrinsic allergic asthma treated with once-monthly omalizumab (600 mg) for the last 15 months. He presented to the emergency room with a 2-week history of right lower limb pain and chest pleuritic pain. Computed tomography pulmonary angiography showed bilateral pulmonary embolism with right-sided pulmonary infarction and ultrasound of right lower limb confirmed distal deep vein thrombosis. No other known risk factors were identified. Treatment with omalizumab was stopped during hospitalization. The Naranjo Adverse Drug Reaction (ADR) Probability Scale classifies this as a probable ADR (score of 6). Omalizumab is a humanized monoclonal anti-IgE antibody indicated for the treatment of persistent moderate-to-severe asthma and certain chronic refractory urticaria. The EXCELS study (The Epidemiologic Study of Xolair (omalizumab): Evaluating Clinical Effectiveness and Long-term Safety in Patients with Moderate-to-Severe Asthma), a postmarketing observational cohort study to assess clinical safety profile of omalizumab, showed a significant increase in venous thromboembolism. In conclusion, omalizumab has been associated with arterial and venous thromboembolic events, although the evidence is not definitive.

13.
Rev. colomb. ciencias quim. farm ; 51(1)ene.-abr. 2022.
Artigo em Espanhol | LILACS-Express | LILACS | ID: biblio-1535832

RESUMO

Introducción: el estradiol es una hormona esteroide sexual femenina usada ampliamente como terapia hormonal que presenta una baja biodisponibilidad, debido a su baja solubilidad acuosa y a su alta hidrofobicidad, perteneciendo a la clase II del sistema de clasificación de biofarmacéutica. Objetivos: diseñar y caracterizar un sistema de entrega de fármacos autoemulsificable (SEDDS) para el fármaco estradiol por pruebas fisicoquímicas con el fin de obtener la relación óptima que permitiera mejorar su solubilidad acuosa, velocidad de disolución y potencialmente su biodisponibilidad. Método: estudios de solubilidad en diferentes solventes, diagramas de fases pseudoternarios constituidos por aceites, tensioactivos, cotensioactivos y agua permitieron reconocer las diferentes regiones de formación de SEDDS e identificar los porcentajes de excipientes que conducen a la formación de soluciones isotrópicas; las formulaciones resultantes fueron caracterizadas en tiempo de autoemulsificación, robustez a la dilución, punto de nube y perfil de disolución en capsula dura. Resultados: las formulaciones que contenían Capmul MCM®, Kolliphor® RH40 y Transcutol®, tuvieron un tiempo de autoemulsificación de aproximadamente 1 min; fueron estables en tres distintos pH (1,2; 4,5 y 7,2), en diferentes volúmenes de dilución, exhibiendo una apariencia transparente, ligeramente azulada, sin precipitados, o separación de fases, puntos de nube mayores en comparación de las formulaciones que contenían Gelucire® 44/14. Conclusiones: las estrategias de caracterización empleadas en el desarrollo de esta investigación demostraron ser eficientes para la selección adecuada de excipientes y su proporción óptima para el diseño eficaz de un sistema de entrega de fármaco autoemulsificable (SEDDS).


SUMMARY Introduction: Estradiol is a female sex steroid hormone widely used as hormonal therapy that has low bioavailability, due to its low aqueous solubility and high hydro-phobicity, belonging to class II of the Biopharmaceutical Classification System. Aim: To design and characterize a self-emulsifying drug delivery system (SEDDS) for the drug estradiol by physicochemical tests to obtain the most optimal ratio that would improve its aqueous solubility, dissolution rate, and potentially its bioavailability. Method: Solubility studies in different solvents; pseudo ternary phase diagrams made up of oils, surfactants, co-surfactants, and water, allowed to recognize the different regions of SEDDS formation and identify the percentages of excipients that lead to the formation of isotropic solutions; The resulting formulations were characterized in autoemulsification time, robustness to dilution, cloud point and dissolution profile in a hard capsule. Results: The formulations containing Capmul MCM®, Kolliphor® RH40, and Transcutol®, had an autoemulsification time of approximately 1 minute; were stable at three different pHs (1.2, 4.5 and 7.2), at different dilution volumes, exhibiting a transparent, slightly bluish appearance, without precipitates, or phase separation, higher cloud points compared to the formulations containing Gelucire® 44/14. Conclusions: The characterization strategies used in the development of this research proved to be efficient for the adequate selection of excipients and their optimal ratio for the effective design of a self-emulsifying drug delivery system (SEDDS).


Introdução: o estradiol é um hormônio esteroide sexual feminino amplamente utilizado como terapia hormonal que apresenta baixa biodisponibilidade devido à sua baixa solubilidade aquosa e alta hidrofobicidade, pertencente à classe II do sistema de classificação biofarmacêutica. Objetivos: projetar e caracterizar um sistema de liberação de drogas autoemulsificante (SEDDS) para o fármaco estradiol por meio de testes físico-químicos a fim de obter a proporção ideal que melhore sua solubilidade aquosa, taxa de dissolução e potencialmente sua biodisponibilidade. Método: estudos de solubilidade em diferentes solventes, diagramas de fases pseudoternários compostos por óleos, tensoativos, cotensoativos e água permitiram reconhecer as diferentes regiões de formação de SEDDS e identificar as porcentagens de excipientes que levam à formação de soluções isotrópicas; as formulações resultantes foram caracterizadas quanto ao tempo de autoemulsificação, robustez à diluição, ponto de turvação e perfil de dissolução da cápsula dura. Resultados: as formulações contendo Capmul MCM®, Kolliphor® RH40 e Transcutol®, tiveram um tempo de autoemulsificação de aproximadamente 1 min; foram estáveis em três diferentes pH's (1,2; 4,5 e 7,2), em diferentes volumes de diluição, apresentando aspecto transparente, levemente azulado, sem precipitados ou separação de fases, pontos de turvação mais elevados em relação às formulações contendo Gelucire® 44/14. Conclusões: as estratégias de caracterização utilizadas no desenvolvimento desta pesquisa mostraram-se eficientes para a seleção adequada de excipientes e sua proporção ideal para o desenho eficaz de um sistema de liberação de fármacos autoemulsificante (SEDDS).

14.
J Dent Educ ; 79(8): 959-64, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26246535

RESUMO

The aim of this study was to assess the ability of students at the School of Dentistry, Complutense University of Madrid, Spain, to diagnose oral cancer and other potentially malignant disorders, as well as to compare their ability at different stages of the learning process and evaluate their knowledge retention. Students were surveyed after they had studied oral medicine and oral pathology at two time points: midway through and near the end of their studies. The survey consisted of questions about 40 photographs of benign oral lesions, malignant oral lesions, and potentially malignant disorders. The response rate for all groups was greater than 70%. The results showed that these students' overall success rate in differentiating benign from malignant lesions averaged 73.9%. When the distinction for potentially malignant disorders was included, their average overall success rate decreased to 42.8% (p<0.001). Furthermore, the students' average success rate was at its lowest at the end of the dental program (p<0.001). Results from this study suggest that, given these students' difficulties in identifying potentially malignant disorders, an increased emphasis on cancer education in the dental curriculum may be needed for future practitioners to master this ability.


Assuntos
Educação em Odontologia , Neoplasias Bucais/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Estudantes de Odontologia , Competência Clínica , Estudos de Coortes , Registros Odontológicos , Avaliação Educacional/métodos , Humanos , Aprendizagem , Doenças da Boca/diagnóstico , Medicina Bucal/educação , Patologia Bucal/educação , Espanha , Ensino/métodos
15.
FEBS J ; 281(6): 1517-33, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24472363

RESUMO

The two-component dengue virus NS2B-NS3 protease (NS2B-NS3pro) is an established drug target but inhibitor design is hampered by uncertainties about its 3D structure in solution. Crystal structures reported very different conformations for the functionally important C-terminal segment of the NS2B cofactor (NS2Bc), indicating open and closed conformations in the absence and presence of inhibitors, respectively. An earlier NMR study in solution indicated that a closed state is the preferred conformation in the absence of an artificial linker engineered between NS2B and NS3pro. To obtain direct structural information on the fold of unlinked NS2B-NS3pro in solution, we tagged NS3pro with paramagnetic tags and measured pseudocontact shifts by NMR to position NS2Bc relative to NS3pro. NS2Bc was found to bind to NS3pro in the same way as reported in a previously published model and crystal structure of the closed state. The structure is destabilized, however, by high ionic strength and basic pH, showing the importance of electrostatic forces to tie NS2Bc to NS3pro. Narrow NMR signals previously thought to represent the open state are associated with protein degradation. In conclusion, the closed conformation of the NS2B-NS3 protease is the best model for structure-guided drug design.


Assuntos
Vírus da Dengue/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sítios de Ligação , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
16.
Am J Clin Pathol ; 135(3): 386-90, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21350092

RESUMO

The erythrocyte sedimentation rate (ESR) is a nonspecific indicator of disease activity and is often used by clinicians in assisting diagnosis and follow-up of many inflammatory disorders. The Westergren method is considered the standard method for measuring ESR. Recently, many automated instruments have become available to address laboratory safety and time efficiency. We validated the Streck ESR-Auto Plus instrument (Streck, Omaha, NE) using the Sediplast (Polymedco, Cortlandt Manor, NY) Westergren method as the reference method. Blood samples collected in 113 EDTA tubes were transferred into Sediplast vials and Streck high-altitude vacuum tubes for measuring the ESR. There was good correlation between the Sediplast and Streck methods (0.95) using Pearson correlation. The y-intercept was at 6.5 using linear regression, indicating systematic bias with a mean difference of 7.13 using the t test (P < .0001). We modified our reference ranges to rectify the systematic bias found during validation.


Assuntos
Sedimentação Sanguínea , Testes Diagnósticos de Rotina/métodos , Testes Hematológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Adulto Jovem
17.
Assay Drug Dev Technol ; 9(1): 50-7, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21050031

RESUMO

Development of enzyme inhibitors requires an activity assay for the identification of hits and lead compounds. To determine dissociation constants in a straightforward manner, we explored the use of a genetically encoded fluorescent amino acid for site-specific tagging of the target protein. The unnatural amino acid 7-(hydroxy-coumarin-4-yl) ethylglycine (Hco) was site-specifically incorporated in the target protein by cell-free protein synthesis using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair. Using the West Nile virus nonstructural protein 2B-nonstructural protein 3 protease as the target protein, the fluorescence of Hco-tagged samples proved to be exquisitely sensitive to the presence of inhibitors and small ligand molecules if they bind in the vicinity of the Hco residue. No significant change in fluorescence was observed when the ligand-binding site was far from the Hco residue. Hco-tagged proteins thus combine outstanding sensitivity with accurate information on the site of binding, making Hco labeling an attractive tool in drug discovery.


Assuntos
Aminoácidos/análise , Aminoácidos/genética , Corantes Fluorescentes , Técnicas de Sonda Molecular , Mapeamento de Interação de Proteínas/métodos , Espectrometria de Fluorescência/métodos , Proteínas Virais/química , Engenharia Genética/métodos , Peso Molecular
18.
Protein Sci ; 19(11): 2045-54, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20718036

RESUMO

Proteins targeted to the mitochondrial matrix are translocated through the outer and the inner mitochondrial membranes by two protein complexes, the translocase of the outer membrane (TOM) and one of the translocases of the inner membrane (TIM23). The protein Tim23, the core component of TIM23, consists of an N-terminal, soluble domain in the intermembrane space (IMS) and a C-terminal domain that forms the import pore across the inner membrane. Before translocation proceeds, precursor proteins are recognized by the N-terminal domain of Tim23, Tim23N (residues 1-96). By using NMR spectroscopy, we show that Tim23N is a monomeric protein belonging to the family of intrinsically disordered proteins. Titrations of Tim23N with two presequences revealed a distinct binding region of Tim23N formed by residues 71-84. In a charge-hydropathy plot containing all soluble domains of TOM and TIM23, Tim23N was found to be the only domain with more than 40 residues in the IMS that is predicted to be intrinsically disordered, suggesting that Tim23N might function as hub in the mitochondrial import machinery protein network.


Assuntos
Proteínas de Membrana Transportadoras/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Dicroísmo Circular , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Moleculares , Anotação de Sequência Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Dev Biol ; 272(2): 286-309, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15282149

RESUMO

Receptor-mediated cell-extracellular matrix (ECM) interactions are critical regulators of cell survival, and perturbing these signaling pathways can disrupt cellular differentiation and function in a variety of tissues, including the mammary gland. One such receptor is the cell surface-associated, long isoform of beta1,4-galactosyltransferase I (GalT I). Deletion of long GalT I leads to increased mammary ductal branching morphogenesis [Dev. Biol., 244 (2002) 114]. Here, we show that this expansion in the mammary epithelial (ME) cell compartment is accomplished through decreased apoptosis during pregnancy and involution. Decreased apoptosis during involution is concomitant with delayed alveolar collapse, persistent expression of the milk protein gene alpha-lactalbumin and delayed expression of genes associated with the tissue-remodeling phase of involution. Using 3-dimensional in vitro cultures, we show that the decrease in apoptosis is dependent on laminin 1, a ligand for surface GalT I, suggesting that surface GalT I negatively influences ECM-dependent cell survival, a novel function for an ECM receptor. In the best-studied examples, ECM promotes survival through integrin receptor-mediated activation of focal adhesion kinase (FAK). Aggregation of surface GalT I also activates FAK, therefore, we asked if FAK activation was altered in ME from long GalT I null mice. Activated FAK was appropriately localized to focal adhesions in long GalT I null ME. However, FAK activation was constitutively reduced 4.5-fold in long GalT I nulls relative to wild type. Expression of the integrin beta1 subunit was not affected by loss of long GalT I. Collectively, these results suggest that surface GalT I might negatively regulate ME cell survival by linking integrin-independent FAK activation to apoptotic rather than survival signaling events.


Assuntos
Apoptose/fisiologia , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Glândulas Mamárias Animais/patologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Feminino , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Integrina beta1/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lactalbumina/genética , Lactação , Laminina/metabolismo , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Mutantes , Gravidez , Prenhez , Proteínas Tirosina Quinases/metabolismo , Vinculina/metabolismo
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