RESUMO
INTRODUCTION AND AIMS: Percutaneous liver biopsy with histopathologic analysis is a valuable tool for the diagnosis, prognosis, and treatment evaluation of liver diseases. Its ultrasound-guided performance is useful, making the procedure safer and reducing the risk for complications and hospital stay. Our aim was to describe the indications, histopathologic study, and complications associated with the performance of ultrasound-guided percutaneous liver biopsy in pediatric patients. MATERIAL AND METHODS: The study included 102 ultrasound-guided percutaneous liver biopsies performed on patients <16 years of age, within the time frame of January 2014 and December 2019. The information was obtained from electronic files and histopathologic studies and the data were analyzed through descriptive statistics. RESULTS: A total of 102 procedures were carried out on 99 patients. Mean patient age was 72 months and 58.8% of the patients were female. Over 65% of the indications for liver biopsy included autoimmune hepatitis (23.5%), elevated liver enzymes (21.5%), and chronic liver disease (20.5%). Four patients presented with immediate complications (3.9%), three of which were major (2.9%), concurring with that reported in the international literature. CONCLUSIONS: Our study corroborates the importance of ultrasound-guided liver biopsy in the diagnosis and follow-up of pediatric patients. The procedure also had a low complication rate of only 3.9%.
Assuntos
Hepatopatias , Ultrassonografia de Intervenção , Criança , Feminino , Humanos , Biópsia Guiada por Imagem/efeitos adversos , Biópsia Guiada por Imagem/métodos , Hepatopatias/diagnóstico por imagem , Masculino , Estudos Retrospectivos , Centros de Atenção Terciária , Ultrassonografia de Intervenção/métodosRESUMO
BACKGROUND: Obesity promotes cellular immunometabolism changes that trigger the activation of macrophages and lymphocytes, leading to systemic inflammation. Activated leukocytes undergo metabolic reprogramming, increasing glycolytic activity. OBJECTIVE: To examine whether the reduction in the inflammatory state associated with bariatric surgery is associated with decreased glycolytic activity in leukocytes. Setting Single-center, prospective observational study. METHODS: This study involved 18 patients with obesity undergoing bariatric surgery. All measurements were performed preoperatively and six months postoperatively. Peripheral blood mononuclear cells and plasma were obtained to determine the glycolytic rate and mitochondrial membrane potential as surrogates of the metabolic switching and high-sensitivity C-reactive protein, adipokines, and CD69 expression as inflammatory and activation markers. RESULTS: Glycolytic activity engaged by CD3/CD28 activation was reduced six months after bariatric surgery, associated with decreased levels of T helper (Th) 1 and Th17 signature cytokines. An overall reduction in inflammatory markers was observed, which correlated with a higher adiponectin/leptin ratio. CONCLUSIONS: Metabolic and bariatric surgery-induced weight loss leads to reprogramming in T cells' metabolic machinery, resulting in reduced stimulation of glycolysis after activation, which may explain the decrease in systemic inflammation mediated by cytokines such as interferon-γ and interleukin-17A.
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Ativação Metabólica/imunologia , Cirurgia Bariátrica/métodos , Glicólise/imunologia , Leucócitos Mononucleares , Obesidade Mórbida , Células Th1 , Células Th17 , Adulto , Contagem de Células/métodos , Reprogramação Celular , Metabolismo Energético/imunologia , Feminino , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Obesidade Mórbida/metabolismo , Obesidade Mórbida/cirurgia , Período Pós-Operatório , Células Th1/metabolismo , Células Th1/patologia , Células Th17/metabolismo , Células Th17/patologiaRESUMO
INTRODUCTION AND AIMS: Percutaneous liver biopsy with histopathologic analysis is a valuable tool for the diagnosis, prognosis, and treatment evaluation of liver diseases. Its ultrasound-guided performance is useful, making the procedure safer and reducing the risk for complications and hospital stay. Our aim was to describe the indications, histopathologic study, and complications associated with the performance of ultrasound-guided percutaneous liver biopsy in pediatric patients. MATERIAL AND METHODS: The study included 102 ultrasound-guided percutaneous liver biopsies performed on patients <16 years of age, within the time frame of January 2014 and December 2019. The information was obtained from electronic files and histopathologic studies and the data were analyzed through descriptive statistics. RESULTS: A total of 102 procedures were carried out on 99 patients. Mean patient age was 72 months and 58.8% of the patients were female. Over 65% of the indications for liver biopsy included autoimmune hepatitis (23.5%), elevated liver enzymes (21.5%), and chronic liver disease (20.5%). Four patients presented with immediate complications (3.9%), three of which were major (2.9%), concurring with that reported in the international literature. CONCLUSIONS: Our study corroborates the importance of ultrasound-guided liver biopsy in the diagnosis and follow-up of pediatric patients. The procedure also had a low complication rate of only 3.9%.
RESUMO
Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.
Assuntos
Humanos , Condrócitos/metabolismo , Colágeno Tipo II/biossíntese , Glicosaminoglicanos/biossíntese , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Matrilinas/biossíntese , Fatores de Transcrição SOX9/metabolismo , Transfecção/métodos , Cartilagem Articular/lesões , Cartilagem Articular/metabolismo , Colágeno Tipo II/análise , Matriz Extracelular/química , Expressão Gênica , Glicosaminoglicanos/análise , Fator de Crescimento Insulin-Like I/genética , Proteínas Matrilinas/genética , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/genética , EspectrofotometriaRESUMO
The bimetallic nickel-tungsten catalysts were prepared via solvothermal method. The X-ray Diffractometer (XRD) analysis revealed that the corresponding peaks at 14°, 34°, and 58° were for tungsten disulfide (WS2 ) hexagonal phase. The catalysts displayed different crystalline phase with nickel addition, and as an effect the WS2 surface area decreased from 74.7 to 2.0 m(2) g(--1) . In this sense, high-resolution transmission electron microscopy (HRTEM) showed the layers set in direction (002) with an onion-like morphology, and in the center of the particles there is a large amount of nickel contained with 6-8 layers covering it. The catalytic dehydration of 2-propanol was selective to propene in 100% at 250 °C for the sample with 0.7 of atomic ratio of Ni/Ni + W.
RESUMO
BACKGROUND AND PURPOSE: Despite the importance of mitochondrial Ca(2+) to metabolic regulation and cell physiology, little is known about the mechanisms that regulate Ca(2+) entry into the mitochondria. Accordingly, we established a system to determine the role of the mitochondrial Ca(2+) uniporter in an isolated heart model, at baseline and during increased workload following ß-adrenoceptor stimulation. EXPERIMENTAL APPROACH: Cardiac contractility, oxygen consumption and intracellular Ca(2+) transients were measured in ex vivo perfused murine hearts. Ru360 and spermine were used to modify mitochondrial Ca(2+) uniporter activity. Changes in mitochondrial Ca(2+) content and energetic phosphate metabolite levels were determined. KEY RESULTS: The addition of Ru360 , a selective inhibitor of the mitochondrial Ca(2+) uniporter, induced progressively and sustained negative inotropic effects that were dose-dependent with an EC50 of 7 µM. Treatment with spermine, a uniporter agonist, showed a positive inotropic effect that was blocked by Ru360 . Inotropic stimulation with isoprenaline elevated oxygen consumption (2.7-fold), Ca(2+) -dependent activation of pyruvate dehydrogenase (5-fold) and mitochondrial Ca(2+) content (2.5-fold). However, in Ru360 -treated hearts, this parameter was attenuated. In addition, ß-adrenoceptor stimulation in the presence of Ru360 did not affect intracellular Ca(2+) handling, PKA or Ca(2+) /calmodulin-dependent PK signalling. CONCLUSIONS AND IMPLICATIONS: Inhibition of the mitochondrial Ca(2+) uniporter decreases ß-adrenoceptor response, uncoupling between workload and production of energetic metabolites. Our results support the hypothesis that the coupling of workload and energy supply is partly dependent on mitochondrial Ca(2+) uniporter activity.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/fisiologia , Coração/fisiologia , Mitocôndrias Cardíacas/fisiologia , Receptores Adrenérgicos beta/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glutationa/metabolismo , Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Masculino , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio , Ratos Wistar , Compostos de Rutênio/farmacologia , Espermina/farmacologiaAssuntos
Neoplasias do Córtex Suprarrenal/cirurgia , Adrenalectomia , Hemodinâmica , Monitorização Intraoperatória/métodos , Feocromocitoma/cirurgia , Análise de Onda de Pulso/métodos , Implante de Prótese Vascular , Humanos , Hipertensão/prevenção & controle , Complicações Intraoperatórias/prevenção & controle , Masculino , Pessoa de Meia-Idade , Monitorização Intraoperatória/instrumentação , Análise de Onda de Pulso/instrumentação , Choque/prevenção & controle , Veia Cava Inferior/cirurgiaRESUMO
Cell transplantation to regenerate injured tissues is a promising new treatment for patients suffering several diseases. Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Our goal was the isolation, characterization, and immortalization of porcine MSCs (pMSCs) to study their potential differentiation "in vitro" into cardiomyocytes. pMSCs were obtained from the aspirated bone marrow of Large-White pigs. After 4 weeks in culture, adherent cells were phenotypically characterized by flow cytometry and immunochemistry by using monoclonal antibodies. Primary pMSCs were transfected with the plasmid pRNS-1 to obtain continuous growing cloned cell lines. Fresh pMSCs and immortalized cells were treated with 5-azacytidine to differentiate them into cardiomyocytes. Flow cytometry analysis of isolated pMSCs demonstrated the following phenotype, CD90(pos), CD29(pos), CD44(pos), SLA-I(pos), CD106(pos), CD46(pos) and CD45(neg), CD14(neg), CD31(neg), and CD11b(neg), similar to that described for human MSC. We derived several stable immortalized MSC cell lines. One of these, called pBMC-2, was chosen for further characterization. After "in vitro" stimulation of both primary or immortalized cells with 5-azacytidine, we obtained different percentages (30%-50%) of cells with cardiomyocyte characteristics, namely, positive for alpha-Actin and T-Troponin. Thus, primary or immortalized pMSCs derived from bone marrow and cultured were able to differentiate "ex vivo" into cardiac-like muscle cells. These elements may be potentials tools to improve cardiac function in a swine myocardial infarct model.
Assuntos
Mesoderma/citologia , Células Musculares/citologia , Células Musculares/transplante , Miocárdio/citologia , Células-Tronco/citologia , Animais , Antígenos de Diferenciação/análise , Azacitidina/farmacologia , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Transplante de Células , Imuno-Histoquímica , Suínos , TransfecçãoRESUMO
La diabetes mellitus duplica la morbimortalidad cardiovascular posoperatoria, porque la neuropatía autonómica cardiovascular es uno de los principales factores de riesgo, con posible hipotensión perioperatoria y pérdida de variabilidad normal de la freuencia cardíaca Se diseño un estudio descriptivo, longitudinal, prospectivo en 28 pacientes diabéticos tipo 2 sometidos a cirugía con anestesia general inhalatoria. Se realizó test de Ewing preoperatorio, registro de tensión arterial y frecuencia cardíaca intraoperatorios, así como Holter de 24 horas en el posoperatorio. Se encuentra 32 por ciento de pacientes con neuropatía autonómica cardiovascular. Durante la ionducción anestésica, los pacientes con neuropatía autonómica cardiovascular presentaron tendencia al descenso de la tensión arterial; con la intubación, ambos grupos mostraron ascenso de sistólica, manteniendo los pacientes con neuropatía autonómica cardiovascular valores normales de tensión arterial diastólica. No hubo varación significativa de frecuencia cardíaca durante la cirugía; ni tensión arterial o frecuencia cardíaca a la extubación. La arritmias (92,9 por ciento) y depresión del ST (17,9 por ciento) en el posoperatorio no fueron estadísticamente significativas con respecto a presencia de neuropatía autonómica cardiovascular. Los diabéticos con neuropatía autonómica cardiovascular demostraron tendencia hacia mayor labilidad cardiovascular durante la inducción anestésica e intubación.
Assuntos
Masculino , Humanos , Feminino , Anestesia Geral , Doenças Cardiovasculares , Evolução Clínica , Neuropatias Diabéticas , Diabetes Mellitus/patologia , Medicina Interna , VenezuelaRESUMO
We present a six year-old patient suffering of adenoiditis and serous otitis. After surgical intervention, she developed septicemia with arthritis and acute renal failure. We discuss the need to carry out antibiotic prophylaxis in patients without previous risk factors.
Assuntos
Injúria Renal Aguda/etiologia , Adenoidectomia/métodos , Artrite/etiologia , Ventilação da Orelha Média/métodos , Complicações Pós-Operatórias , Sepse/etiologia , Criança , Feminino , Humanos , Otite Média/cirurgiaRESUMO
El consumo de agua potable envasada contaminada con microorganismos patógenos puede ocasionar enfermedades de origen entérico. El objetivo de este trabajo fue determinar y comparar, mediante dos métodos de análisis, la presencia de microorganismos indicadores de calidad sanitaria, tales como coliformes fecales y aerobios mesófilos, en dos marcas comerciales de agua potable, distribuidas en San Diego, estado Carabobo, Venezuela. Para ello se recolectaron 30 muestras de agua potable en presentación de bidones de 20 litros de capacidad, divididas en 15 muestras marca A y 15 marca B. Para la identificación microbiológica se emplearon el método rápido Petrifilm y el método tradicional de siembra en profundidad según las especificaciones del Método Estándar para el Análisis de Aguas Potables y las normas nacionales COVENIN. Los resultados no mostraron diferencias significativas para el recuento microbiológico de coliformes totales y de aerobios mesófilos, por el método tradicional de siembra en profundidad y el método rápido se siembra en placas Petrifilm (r=0,09), para las marcas de agua A y B (p=0,05). Tres muestras (13 por ciento) presentaron recuentos menores de 10 µfc/ml para coliformes totales para la marca de agua A, y siete (47 por ciento) para la marca B. Ninguna muestra permitió coliformes fecales. En conclusión, ambos métodos siguen siendo de elección para el análisis de agua potable envasada, y aunque el método de Petrifilm presente ciertas ventajas contra el método tradicional de siembra en profundidad, este último sigue siendo el más utilizado en la mayoría de los laboratorios de análisi microbiológicos fuera de especificaciones, según las recomendaciones de las normas respectivas, por lo que su consumo puede representar un riesgo para la salud del consumidor
Assuntos
Poluição da Água/análise , Água Potável , Poluentes da Água , Qualidade da Água , Microbiologia , VenezuelaRESUMO
Se estudiaron un total de 61 pacientes, con edades comprendidas entre 0 a 19 años, correspondiendo 27 (44,7 por ciento) al género femenino y 34 (55,7 por ciento) al masculino. El grupo etario donde hubo mayor número de pacientes con Tinea capitis fue el de 5 a 9 años, predominando en el género masculino. El tipo de dermatofito más frecuentemente aislado fue M. canis, 37/53 (69,8 por ciento), seguido de T. tonsurans, 12/53 (22,6 por ciento). No se logró demostrar ningún agente de Tinea capitis en 8/61 (13,1 por ciento). Al comparar el resultado del cultivo con el EMD, no hay discordancias entre los dos procedimientos utilizados
Assuntos
Humanos , Masculino , Adolescente , Feminino , Arthrodermataceae , Microsporum , Onicomicose , Micologia , VenezuelaRESUMO
El presente trabajo tiene como objetivo diseñar una metodología que permita a la Cátedra de Dentaduras Parciales Removibles obtener registros permanentes para el estudio y evaluación de posibles discontinuidades y/o defectos (forma, ubicación, tamaño, orientación y tipo) que puedan estar presentes en las estructuras metálicas de las prótesis parciales removibles (PPR). Se realizó una inspección visual y radiográfica para identificar la presencia de discontinuidades en las estructuras de PPR. Se evaluaron 108 estructuras seleccionadas aleatoriamente de la población de casos realizados por los estudiantes de pregrado de la Facultad de Odontología de la UCV en el período octubre 1998 - abril 1999. La inspección radiográfica permitió detectar una cantidad significativa de fallas en las estructuras evaluadas que no lograban determinarse con la inspección visual. Con relación a los elementos que constituyen una PPR, la mayor frecuencia de fallas se encontraron ubicadas en el conector mayor. Es de notar que sólo el 25,93 por ciento de las estructuras evaluadas no presentaron fallas. Entre las discontinuidades se detectaron; porosidades, inclusiones y fisuras en un porcentaje bastante importante (74,07 por ciento). Cuando contrastamos con la inspección visual, se demuestra la potencialidad de la inspección propuesta pues visualmente, sólo pudo detectarse un 7,41 por ciento de discontinuidades
Assuntos
Humanos , Masculino , Feminino , Prótese Parcial Removível , Falha de Prótese , Análise de Variância , Ligas de Cromo , Ligas Dentárias , Retenção em Prótese Dentária , Prótese Parcial Removível , Estudo de Avaliação , Porosidade , Controle de Qualidade , Radiografia Dentária/métodos , Interpretação Estatística de Dados , Técnica de Fundição Odontológica/efeitos adversos , VenezuelaRESUMO
Fluorescence spectroscopy, surface-enhanced Raman spectroscopy (SERS), and analytical centrifugation are applied in this work to study the interaction of the antitumor drug 9-aminoacridine (9AA) with a trypsin-like protease, guanidinobenzoatase (GB), extracted from an Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission can be detected at a certain drug and enzyme concentration. The 9AA exciplex emission was also studied for 9AA interacting with others serin proteases: alpha-chymotrypsin, trypsin, and penicillin G-acylase (PGA), as well as with bovine serum albumin (BSA) in order to obtain information about the active center of GB. We have found that the exciplex 9AA emission may be induced by a ring-stacking interaction between the monomeric drug, under the amino form, and an aromatic residue placed in the catalytic site of the protein. The results derived from Raman spectroscopy corroborate this interaction mechanism, as demonstrated by the existence of typical protonated amino 9AA marker bands as well as an important modification of the ring vibrations, thus indicating the existence of an interaction through ring stacking. The analytical centrifugation technique was applied to study the GB association in aqueous solution, demonstrating that the 9AA/GB interaction depends on the enzyme quaternary structure. An interaction of 9AA with an associate form of GB, which may be the actual enzyme active form, is suggested.
Assuntos
Aminacrina/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Endopeptidases/metabolismo , Aminacrina/química , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Biomarcadores Tumorais , Hidrolases de Éster Carboxílico/química , Carcinoma de Ehrlich/enzimologia , Domínio Catalítico , Bovinos , Endopeptidases/química , Humanos , Técnicas In Vitro , Camundongos , Sondas Moleculares , Estrutura Quaternária de Proteína , Espectrometria de Fluorescência , Células Tumorais CultivadasRESUMO
Se compararon las técnicas de inmunofluorescencia directa (IFD) y de Gomori-Grocott (G-G) para el diagnóstico de pneumocystis carinii en pacientes inmunocomprometidos con infección respiratoria baja. Se analizaron 30 muestras de esputo; a cada muestra se le practicó IFD y G-G. Se obtuvo el 12/30 (40 por ciento) de positividad, correspondiéndole 9 (30 por ciento) a la IFD, 2 (7 por ciento) al G-G y 1 (3 por ciento) para ambas. Este resultado fue estadísticamente significativo (p<0,05), concluyéndose que la IFD es la prueba más idónea para diagnósticar P. carinii en muestras de esputo
Assuntos
Humanos , Masculino , Feminino , Pneumocystis carinii , Coloração e Rotulagem , Técnica Direta de Fluorescência para Anticorpo , VenezuelaRESUMO
The affinity of mushroom polyphenol oxidase (PPO) towards gentisic acid (GA), a metabolite of acetyl salicylic acid (ASA), is demonstrated by spectrophotometry and by electrochemical techniques. The enzyme can selectively recognize GA even in the presence of large excess of ASA and its metabolic derivatives (salicylic acid (SA) and salicyluric acid (SUA)). At -0.150 V, the sensitivity is (6.1+/-0.1)x10(4) NAM(-1), the response is linear up to 2.0x10(-4) M and the detection limit is 5.0x10(-5) M. The kinetic parameters, obtained from Eadie-Hofstee plots, are I(max)=51.4 nA and K(m)(app)=6.7x10(-4) M.
RESUMO
Analizar la incidencia, características maternas, obstétricas, complicaciones más frecuentes y determinar la asociación entre desprendimiento prematuro de placenta y factores de riesgo como hipertensión asociada al embarazo. Estudio retrospectivo y descriptivo de 224 casos atendidos desde 1984 hasta 1993 en 38.747 nacimientos. Maternidad del Hospital "Dr. Adolfo Prince Lara", Puerto Cabello, Estado Carabobo, Venezuela. La incidencia fue de 0.57 por ciento, 1 caso por cada 175 partos. Predominó el grupo etáreo de 27 a 32 años (31,25 por ciento). El 62,06 por ciento fueron multíparas; 55,56 por ciento presentaron embarazos menores de 37 semanas. El 92,41 por ciento fue de cesárea y 7,59 por ciento parto espontáneo. En el 49,03 por ciento el grado de desprendimiento fue grave, 29,61 por ciento moderado y 21,36 por ciento leve. Las complicaciones maternas fueron anemia 57,72 por ciento, coagulación intravascular diseminada 17,45 por ciento, shock hemorrrágico 14,19 por ciento, útero couvelaire 8,72 por ciento y 2,01 por ciento falla renal. El bajo peso al nacer representó el 43,56 por ciento. La tasa de mortalidad fetal fue de 506x1000. Hubo 4 muertes maternas. El desprendimiento prematuro de placenta ha presentado un leve incremento en el período de estudio, amerita establecer un programa preventivo y de educación actualizada para el pronóstico y manejo adecuado
Assuntos
Humanos , Feminino , Parto/classificação , Descolamento Prematuro da Placenta/classificação , Descolamento Prematuro da Placenta/diagnóstico , Descolamento Prematuro da Placenta/prevenção & controle , Complicações do Trabalho de Parto/diagnóstico , Cesárea/métodos , HipertensãoRESUMO
The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.
Assuntos
Proteínas de Membrana , Vaccinia virus/química , Vaccinia virus/fisiologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/fisiologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/fisiologia , Proteínas Virais/química , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Dicroísmo Circular , Primers do DNA/genética , Escherichia coli/genética , Células HeLa , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Vaccinia virus/genética , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética , Proteínas Virais/genéticaRESUMO
The triphenylmethane derivative aurintricarboxylic acid has been used to inhibit angiogenesis, vascular smooth muscle cell proliferation and cell transformation, an effect that has been attributed to its relatively nonspecific inhibitory activity of protein-nucleic acid interactions. Here, we show that this compound binds to acidic fibroblast growth factor, a prototypic member of a family of protein mitogens activated by heparin, altering its physicochemical properties and decreasing its mitogenic activity. Counteraction of the effects of aurintricarboxylic acid by heparin shows that the two compounds have opposite and reversible effects on acidic fibroblast growth factor structure and biological activity. The studies reported here may contribute to a deeper understanding of the inhibition of fibroblast-growth-factor-dependent mitogenesis of relevance to future pharmacologic developments.