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Introducción: La variabilidad de la expresión y la evolución de la COVID no se explican completamente por los factores clínicos, atribuyéndose un importante papel a los genéticos. Además, se ha planteado si los mismos componentes genéticos que participan en la susceptibilidad y gravedad de la infección influyen en su evolución hacia long COVID. Como objetivo nos propusimos revisar la literatura a fin de conocer cuáles son los factores genéticos que intervienen en la génesis de la COVID persistente. Material y métodos: Revisión sistemática en PubMed y repositorios bioRxiv y medRxiv con base en los descriptores y términos Medical Subject Headings (MeSH) relacionados con COVID y factores genéticos. Fueron seleccionados 2.705 artículos. Un primer cribado, realizado de manera independiente por los autores, redujo la lista a 205 y finalmente, tras un análisis más detallado, se eligieron 85 trabajos para su lectura completa y revisión. Resultados: La enzima convertidora de angiotensina 2 (ACE2) y la proteasa transmembrana, serina 6 (TMPSS6) están implicadas en la susceptibilidad, sin embargo, no se ha encontrado su participación en long COVID. Sí se han hallado algunas asociaciones entre genes, que intervienen en la respuesta inflamatoria e inmune, con la gravedad de la enfermedad y el desarrollo de long COVID. La relación más importante se ha observado en el locus FOXP4. Conclusiones: Aunque actualmente la información sobre long COVID es limitada, parece claro que los factores genéticos identificados hasta ahora no justifican la progresión hacia una enfermedad persistente y se debe considerar la participación de otros componentes como la acción poligénica, de genes pleiotrópicos, de la microbiota y de los cambios epigenéticos.(AU)
Introduction: The variability in expression and evolution of COVID is not completely explained by clinical factors. In fact, genetic factors play an important role. Moreover, it is unknown whether the genetic factor that contribute to susceptibility and severity are also involved in the onset and evolution of long-COVID. The objective of this review is to gather information from literature to understand which genetic factors are involved in the onset of persistent COVID. Material and methods: Systematic review in PubMed and bioRxiv and medRxiv repositories based on MeSH-descriptors and MeSH-terms related to COVID and genetic factors. Using these terms 2715 articles were pooled. An initial screening performed by authors independently, selected 205 articles of interest. A final deeper screening a total of 85 articles were chosen for complete reading and summarized in this review. Results: Although ACE2 and TMPSS6 are involved in COVID susceptibility, their involvement in long-COVID has not been found. On the other hand, the severity of the disease and the onset of long-COVID has been associated with different genes involved in the inflammatory and immune response. Particularly interesting has been the association found with the FOXP4 locus. Conclusions: Although studies on long-COVID are insufficient to fully comprehend the cause, it is clear that the current identified genetic factors do not fully explain the progression and onset of long-COVID. Other factors such as polygenic action, pleiotropic genes, the microbiota and epigenetic changes must be considered and studied.(AU)
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Humanos , Masculino , Femenino , /diagnóstico , /genética , Variación Genética , Polimorfismo de Nucleótido Simple , Fenómenos Genéticos , /genéticaRESUMEN
INTRODUCTION: The variability in expression and evolution of COVID is not completely explained by clinical factors. In fact, genetic factors play an important role. Moreover, it is unknown whether the genetic factor that contribute to susceptibility and severity are also involved in the onset and evolution of long-COVID. The objective of this review is to gather information from literature to understand which genetic factors are involved in the onset of persistent COVID. MATERIAL AND METHODS: Systematic review in PubMed and bioRxiv and medRxiv repositories based on MeSH-descriptors and MeSH-terms related to COVID and genetic factors. Using these terms 2715 articles were pooled. An initial screening performed by authors independently, selected 205 articles of interest. A final deeper screening a total of 85 articles were chosen for complete reading and summarized in this review. RESULTS: Although ACE2 and TMPSS6 are involved in COVID susceptibility, their involvement in long-COVID has not been found. On the other hand, the severity of the disease and the onset of long-COVID has been associated with different genes involved in the inflammatory and immune response. Particularly interesting has been the association found with the FOXP4 locus. CONCLUSIONS: Although studies on long-COVID are insufficient to fully comprehend the cause, it is clear that the current identified genetic factors do not fully explain the progression and onset of long-COVID. Other factors such as polygenic action, pleiotropic genes, the microbiota and epigenetic changes must be considered and studied.
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COVID-19 , Síndrome Post Agudo de COVID-19 , Humanos , COVID-19/genética , Factores de Transcripción ForkheadRESUMEN
The main of this study was to evaluate the effect of supplementation of tropical tree foliage in ruminant diets on the in vitro fermentation, bacterial population, volatile fatty acids (VFAs), and enteric CH4 production. Seven experimental diets were evaluated: a control treatment of Pennisetum purpureum (T7) and six treatments of P. purpureum supplemented (30%) with the foliage of Neomillspaughia emargiata (T1), Tabernaemontana amygdalifolia (T2), Caesalpinia gaumeri (T3), Piscidia piscipula (T4), Leucaena leucocephala (T5), and Havardia albicans (T6). The T2, T7, and T5 treatments had the highest (p < 0.05) digestibility of dry matter. Overall, supplementation increased (p < 0.05) the concentrations of propionic and butyric acid and decreased acetic acid. Methanogenic bacteria decreased (p < 0.05) in T1, T2, T5, and T6. Ruminococcus albus decreased in T1, T2, T3, and T5 and Selenomonas ruminiantum increased in T3. Fibrobacter succinogenes increased, except in T5. Methane production decreased (p < 0.05) in T1, T4, T5, and T6. The supplementation with Leucaena leucocephala, Tabernaemontana amygdalifolia, Neomillspaughia emargiata, Piscidia piscipula, Havardia albicans, and Caesalpinia gaumeri is a potential alternative nutritional strategy for ruminants that results in positive changes in VFAs profile, a decrease on CH4 production and methanogenic bacteria, and changes on fibrolytic and non-fibrolytic bacteria composition.HIGHLIGHTSTropical tree foliage supplementation increased propionic and butyric acid and decreased acetic acid concentrations.Fibrolytic, non-fibrolytic, and Methanogenic bacteria were selectively modulated with the supplementation of tropical tree foliage.The enteric methane (CH4) production decreased with the supplementation of tree foliage.The supplementation of Tabernaemontana amygdalifolia and Leucaena leucocephala had the highest digestibility and is a potential alternative nutritional strategy for ruminants.
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Fabaceae , Árboles , Animales , Fermentación , Rumen/metabolismo , Dieta , Suplementos Dietéticos , Rumiantes , Ácidos Grasos Volátiles , Ácido Acético/metabolismo , Ácido Butírico , Metano/metabolismo , Alimentación Animal/análisisRESUMEN
El genograma y el árbol genealógico son herramientas de representación gráfica de la familia que, aunque comparten similitudes, tienen una funcionalidad y significados diferentes. Si bien el objetivo básico del primero es representar la estructura familiar y las relaciones entre sus miembros, el segundo pretende la identificación del patrón de herencia y de rasgos heredables, aunque su uso y aplicaciones van mucho más allá. Por otra parte, la representación gráfica en ambas herramientas se encuentra normalizada con símbolos similares, aunque algunos de ellos con significado distinto. Tanto una como otra, son utilizadas por el médico de familia, y éste debe tener las competencias necesarias para su manejo efectivo. En este trabajo se pretende mejorar dichas competencias al definir las principales diferencias de una y otra herramienta, establecer los elementos básicos para la construcción e interpretación del árbol genealógico, además de señalar su utilidad clínica y sus usos más frecuentes (AU)
A genogram and a genealogical tree are graphic representations of families that share similarities but have different functionality and meanings. While the first basic objective is to represent the family structure and the relationships between its members, the second seeks to identify the inheritance pattern and inheritable traits, although its use and applications go much further. Graphic representation is normalized with similar symbols, although some of them have different meanings. Both are used by family doctors, and they must have the necessary skills for their effective management. This work aims to improve these skills by defining the main differences between the two tools, establishing the basic elements for the construction and interpretation of a genealogical tree, and pointing out its clinical utility and its most frequent uses (AU)
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Humanos , Linaje , Enfermedades Genéticas Congénitas , Predisposición Genética a la EnfermedadRESUMEN
The first licensed dengue vaccine, CYD-TDV (Dengvaxia) is efficacious in seropositive individuals, but increases the risk for severe dengue in seronegative persons about two years after administration of the first dose. For countries considering the introduction of Dengvaxia, WHO recommends a pre-vaccination screening strategy whereby only persons with evidence of a past dengue infection would be vaccinated. Policy-makers need to consider the risk-benefit of vaccination strategies based on such screening tests, the optimal age to introduce the vaccine, communication and implementation strategies. To address these questions, the Global Dengue and Aedes-transmitted diseases Consortium (GDAC) organized a 3-day workshop in January 2019 with country representatives from Asia and Latin America. The meeting discussions highlighted many challenges in introducing Dengvaxia, in terms of screening test characteristics, costs of such tests combined with a 3-dose schedule, logistics, achieving high coverage rates, vaccine confidence and communication; more challenges than for any other vaccine introduction programme. A screening test would require a high specificity to minimize individual risk, and at the same time high sensitivity to maximize individual and population benefit. The underlying seroprevalence dependent positive predictive value is the best indicator for an acceptable safety profile of a pre-vaccination screening strategy. The working groups discussed many possible implementation strategies. Addressing the bottlenecks in school-based vaccine introduction for Dengvaxia will also benefit other vaccines such as HPV and booster doses for tetanus and pertussis. Levels of public trust are highly variable and context specific, and understanding of population perceptions and concerns is essential to tailor interventions, monitor and mitigate risks.
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Vacunas contra el Dengue/uso terapéutico , Adolescente , Adulto , Anticuerpos Antivirales/inmunología , Niño , Dengue/inmunología , Dengue/microbiología , Dengue/prevención & control , Vacunas contra el Dengue/inmunología , Virus del Dengue , Humanos , Programas de Inmunización/métodos , Salud Pública , Estudios Seroepidemiológicos , Vacunas Atenuadas/uso terapéutico , Organización Mundial de la Salud , Adulto JovenRESUMEN
BK virus-(BKV) associated nephropathy (BKVN) is a major cause of allograft injury in kidney transplant recipients. In such patients, subclinical reactivation of latent BKV infection can occur in the pre-transplant period. The purpose of this study was to determine whether urinary BKV shedding in the immediate pre-transplant period is associated with a higher incidence of viruria and viremia during the first year after kidney transplantation. We examined urine samples from 34 kidney transplant recipients, using real-time quantitative polymerase chain reaction to detect BKV. Urine samples were obtained in the immediate pre-transplant period and during the first year after transplant on a monthly basis. If BKV viruria was detected, blood samples were collected and screened for BKV viremia. In the immediate pre-transplant period, we detected BKV viruria in 11 (32.3%) of the 34 recipients. During the first year after transplantation, we detected BKV viruria in all 34 patients and viremia in eight (23.5%). We found no correlation between pre-transplant viruria and post-transplant viruria or viremia (p = 0.2). Although reactivation of latent BKV infection in the pre-transplant period is fairly common among kidney transplant recipients, it is not a risk factor for post-transplant BKV viruria or viremia.
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Virus BK/genética , ADN Viral/biosíntesis , ADN Viral/orina , Trasplante de Riñón/efectos adversos , Infecciones por Polyomavirus/metabolismo , Infecciones Tumorales por Virus/metabolismo , Viremia/metabolismo , Adolescente , Adulto , Brasil/epidemiología , Femenino , Estudios de Seguimiento , Supervivencia de Injerto , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Infecciones por Polyomavirus/epidemiología , Infecciones por Polyomavirus/virología , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Riesgo , Receptores de Trasplantes , Infecciones Tumorales por Virus/epidemiología , Infecciones Tumorales por Virus/virología , Urinálisis , Viremia/epidemiología , Viremia/virología , Esparcimiento de Virus , Adulto JovenRESUMEN
Schistosomiasis constitutes a major public health problem, with an estimated 200 million people infected worldwide. Many areas of Brazil show low endemicity of schistosomiasis, and the current standard parasitological techniques are not sufficiently sensitive to detect the low-level helminth infections common in areas of low endemicity (ALEs). This study compared the Kato-Katz (KK); Hoffman, Pons, and Janer (HH); enzyme-linked immunosorbent assay- (ELISA-) IgG and ELISA-IgM; indirect immunofluorescence technique (IFT-IgM); and qPCR techniques for schistosomiasis detection in serum and fecal samples, using the circumoval precipitin test (COPT) as reference. An epidemiological survey was conducted in a randomized sample of residents from five neighborhoods of Barra Mansa, RJ, with 610 fecal and 612 serum samples. ELISA-IgM (21.4%) showed the highest positivity and HH and KK techniques were the least sensitive (0.8%). All techniques except qPCR-serum showed high accuracy (8295.5%), differed significantly from COPT in positivity , and showed poor agreement with COPT. Medium agreement was seen with ELISA-IgG (Kappa = 0.377) and IFA (Kappa = 0.347). Parasitological techniques showed much lower positivity rates than those by other techniques. We suggest the possibility of using a combination of laboratory tools for the diagnosis of schistosomiasis in ALEs.
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Esquistosomiasis mansoni/diagnóstico , Esquistosomiasis mansoni/parasitología , Esquistosomiasis mansoni/epidemiología , Anciano de 80 o más Años , Brasil/epidemiología , Anciano , Humanos , Inmunoensayo/métodos , Inmunoensayo/estadística & datos numéricos , Pruebas de Precipitina/métodos , Niño , Preescolar , Vigilancia de la Población/métodos , Prevalencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Medición de Riesgo/métodos , Adulto , Técnicas de Laboratorio Clínico/métodos , Técnicas de Laboratorio Clínico/estadística & datos numéricos , Enfermedades Endémicas/estadística & datos numéricos , Adulto Joven , Lactante , Persona de Mediana EdadRESUMEN
Schistosomiasis is a major public health concern, with 200 million people infected worldwide. In Brazil, this disease has been reported in 19 states, and its prevalence in the city of Barra Mansa in Rio de Janeiro State is 1 %. The parasitological diagnostic methods currently available in these areas lack sensitivity; however, enzyme-linked immunosorbent assays (ELISAs) have been employed successfully for the diagnosis of schistosomiasis by using antibodies against antigens of Schistosoma mansoni adult worms and eggs, and for the detection of circulating antigens. The objective of this study was to determine systematically the prevalence of S. mansoni infection in the peripheral areas of Barra Mansa. A cross-sectional study was conducted from April to December 2011 by using probabilistic sampling that collected 610 fecal samples and 612 serum samples. ELISA-IgG with total extracts and ELISA-IgM with trichloroacetic acid-soluble fractions were employed to detect antibodies against S. mansoni and were compared with the Kato-Katz and Hoffman parasitological techniques. Among the individuals studied, anti-S. mansoni antibodies were detected in 11.16 % (n = 71) by ELISA-IgG and in 20.75 % (n = 132) by ELISA-IgM, while the parasitological techniques showed 0.82 % (n = 5) positivity. The agreement between the two ELISA tests was 85.38 % (n = 543), and 8.65 % (n = 55) of the serum samples showed positive results in both tests. The higher positivity of the ELISA-IgM test corroborates the results of previous reports and indicates that the test may be a useful tool in epidemiological studies, particularly in areas of low endemicity for S. mansoni.
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Anticuerpos Antihelmínticos/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Schistosoma mansoni/aislamiento & purificación , Esquistosomiasis mansoni/diagnóstico , Adolescente , Adulto , Animales , Brasil/epidemiología , Niño , Preescolar , Cricetinae , Estudios Transversales , Heces , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Prevalencia , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/sangre , Esquistosomiasis mansoni/epidemiología , Esquistosomiasis mansoni/inmunología , Adulto JovenRESUMEN
To explore the association of the Newcastle disease virus (NDV) fusion (F) protein with cholesterol-rich membrane domains, its localization in detergent-resistant membranes (DRMs) in transfected cells was characterized. After solubilization of cells expressing the F protein with 1% Triton X-100 at 4 degrees C, ca. 40% of total, cell-associated F protein fractionated with classical DRMs with densities of 1.07 to l.14 as defined by flotation into sucrose density gradients. Association of the F protein with this cell fraction was unaffected by the cleavage of F(0) to F(1) and F(2) or by coexpression of the NDV attachment protein, the hemagglutinin-neuraminidase protein (HN). Furthermore, elimination by mutation, of potential palmitate addition sites in and near the F-protein transmembrane domain had no effect on F-protein association with DRMs. Rather, specific deletions of the cytoplasmic domain of the F protein eliminated association with classical DRMs. Comparisons of deletions that affected fusion activity of the protein and deletions that affected DRM association suggested that there is no direct link between the cell-cell fusion activity of the F protein and DRM association. Furthermore, depletion of cholesterol from cells expressing F and HN protein, while eliminating DRM association, had no effect on the ability of these cells to fuse with avian red blood cells. These results suggest that specific localization of the F protein in cholesterol-rich membrane domains is not required for cell-to-cell fusion. Paramyxovirus F-protein cytoplasmic domains have been implicated in virus assembly. The results presented here raise the possibility that the cytoplasmic domain is important in virus assembly at least in part because it directs the protein to cholesterol-rich membrane domains.
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Citoplasma/química , Fusión de Membrana , Microdominios de Membrana/metabolismo , Virus de la Enfermedad de Newcastle/metabolismo , Proteínas Virales de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Fusión Celular , Chlorocebus aethiops , Eritrocitos , Datos de Secuencia Molecular , Virus de la Enfermedad de Newcastle/genética , Eliminación de Secuencia , Transfección , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genéticaRESUMEN
The villin-type "headpiece" domain is a modular motif found at the extreme C-terminus of larger "core" domains in over 25 cytoskeletal proteins in plants and animals. Although headpiece is classified as an F-actin-binding domain, it has been suggested that some expressed fusion-proteins containing headpiece may lack F-actin-binding in vivo. To determine the intrinsic F-actin affinity of headpiece domains, we quantified the F-actin affinity of seven headpiece domains and three N-terminal truncations, under identical in vitro conditions. The constructs are folded and adopt the native headpiece structure. However, they show a wide range of affinities that can be grouped into high, low, and nonspecific-binding categories. Computer models of the structure and charged surface potential of these headpiece domains suggest features important for high F-actin affinity. We conclude that not all headpiece domains are intrinsically F-actin-binding motifs, and suggest that the surface charge distribution may be an important element for F-actin recognition.
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Actinas/química , Proteínas Portadoras/química , Proteínas de Microfilamentos/química , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Pollos , Cromatografía en Gel , Dicroismo Circular , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Programas Informáticos , Temperatura , Rayos UltravioletaRESUMEN
Objetivo. Conocer la variabilidad en el patrón de uso de los fármacos antihipertensivos (ATH) entre las diferentes Zonas Básicas de Salud que componen un Área de Gestión Sanitaria, así como qué ATH son los más usados y qué coste farmacológico directo suponen. Material y métodos. Diseño: estudio descriptivo transversal. Ámbito de estudio: Atención Primaria, Área rural. Sujetos: 145.498 habitantes pertenecientes a las 7 Zonas Básicas de Salud que componen el Área de Gestión Sanitaria de Osuna. Marco temporal: 1 de septiembre de 1999 a 31 de agosto de 2000. Variables: dosis definida por habitante (DHD).Dosis diaria definida (DDD). Precio de venta al público. Fuente de datos: fichero de facturación mensual de recetas remitido desde el Servicio de Asistencia Farmacéutica del Servicio Andaluz de Salud. Resultados. El coste total de ATH fue de 587.710.110 pesetas, lo que representa un coste de 27.694 ptas/año por DDD. El análisis por grupos terapéuticos mostró gran variabilidad y no pudo establecerse un patrón uniforme de comparación. El mayor rango de variación sobre la media de la dosis definida por habitante fue para los inhibidores de la enzima de conversión de la angiotensina (IECA) (17,5 DHD), seguido de calcioantagonistas (11,5 DHD) y diuréticos (10,5 DHD). El grupo más usado fue el de los IECA (34,6 por ciento), seguido de calcioantagonistas (20,2 por ciento) y diuréticos no asociados (19,1 por ciento). Enalapril (18,1 por ciento), amlodipino (11,1 por ciento) y furosemida (5,3 por ciento) explican más de la tercera parte de las prescripciones. Conclusiones. Los grupos más recomendados por los Consensos Internacionales tienen una menor representación de la que cabría esperar. Se debería profundizar no sólo en el establecimiento de consensos, sino en la difusión efectiva de las conclusiones de los mismos (AU)
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Humanos , Antihipertensivos/economía , Atención a la Salud/economía , Atención Primaria de Salud/economía , Antihipertensivos/administración & dosificación , Costos y Análisis de Costo , 34003 , Estudios TransversalesRESUMEN
BACKGROUND: The yellow fever vaccine is regarded as one of the safest attenuated virus vaccines, with few side-effects or adverse events. We report the occurrence of two fatal cases of haemorrhagic fever associated with yellow fever 17DD substrain vaccine in Brazil. METHODS: We obtained epidemiological, serological, virological, pathological, immunocytochemical, and molecular biological data on the two cases to determine the cause of the illnesses. FINDINGS: The first case, in a 5-year-old white girl, was characterised by sudden onset of fever accompanied by headache, malaise, and vomiting 3 days after receiving yellow fever and measles-mumps-rubella vaccines. Afterwards she decompensated with icterus and haemorrhagic signs and died after a 5-day illness. The second patient-a 22-year-old black woman-developed a sore throat and fever accompanied by headache, myalgia, nausea, and vomiting 4 days after yellow fever vaccination. She then developed icterus, renal failure, and haemorrhagic diathesis, and died after 6 days of illness. Yellow fever virus was recovered in suckling mice and C6/36 cells from blood in both cases, as well as from fragments of liver, spleen, skin, and heart from the first case and from these and other viscera fragments in case 2. RNA of yellow fever virus was identical to that previously described for 17D genomic sequences. IgM ELISA tests for yellow fever virus were negative in case 1 and positive in case 2; similar tests for dengue, hantaviruses, arenaviruses, Leptospira, and hepatitis viruses A-D were negative. Tissue injuries from both patients were typical of wild-type yellow fever. INTERPRETATION: These serious and hitherto unknown complications of yellow fever vaccination are extremely rare, but the safety of yellow fever 17DD vaccine needs to be reviewed. Host factors, probably idiosyncratic reactions, might have had a substantial contributed to the unexpected outcome.
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Lesión Renal Aguda/etiología , Fiebre/etiología , Cefalea/etiología , Hemorragia/etiología , Ictericia/etiología , Faringitis/etiología , Vómitos/etiología , Vacuna contra la Fiebre Amarilla/efectos adversos , Lesión Renal Aguda/epidemiología , Lesión Renal Aguda/patología , Adulto , Sistemas de Registro de Reacción Adversa a Medicamentos , Autopsia , Brasil/epidemiología , Preescolar , ADN Viral/análisis , Ensayo de Inmunoadsorción Enzimática , Resultado Fatal , Femenino , Fiebre/epidemiología , Fiebre/patología , Cefalea/epidemiología , Cefalea/patología , Hemorragia/epidemiología , Hemorragia/patología , Humanos , Inmunohistoquímica , Ictericia/epidemiología , Ictericia/patología , Faringitis/epidemiología , Faringitis/patología , Alineación de Secuencia , Vacunas Atenuadas/efectos adversos , Vómitos/epidemiología , Vómitos/patología , Virus de la Fiebre Amarilla/genéticaRESUMEN
This unit contains three protocols for biotinylation of isolated proteins: attaching biotin to primary amines (e.g., amino groups of lysyl residues); attaching biotin to sulfhydryls (i.e., thiol groups of cysteinyl residues); and attaching biotin to carbohydrate residues on proteins. As biotinylation of lysyl and cysteinyl residues may alter protein function, modification of protein carbohydrates, which is usually innocuous, may be preferable if intact protein function is required (e.g., for activity assays or affinity purification). Biotinylation of cysteinyl thiols requires that disulfide bonds in isolated proteins be reduced before labeling. Biotinylation of surface proteins on living cells is also described using mild reaction conditions. Finally, this unit includes a brief description of methods for detecting biotinylated proteins.
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Biotinilación/métodos , Proteínas de la Membrana/química , Proteínas/química , Membrana Celular/metabolismo , Disulfuros/química , Lisina/química , Solubilidad , Compuestos de Sulfhidrilo/químicaRESUMEN
Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.
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Actinas/metabolismo , Citoesqueleto/fisiología , Dictyostelium/fisiología , Endocitosis , Neuropéptidos/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Actinas/inmunología , Animales , Adhesión Celular , División Celular , Membrana Celular/metabolismo , Movimiento Celular , Células Cultivadas , Quimiotaxis , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Humanos , Mutagénesis Sitio-Dirigida , Neuropéptidos/genética , Proteínas Recombinantes de Fusión , Homología de Secuencia , Transducción de Señal , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1RESUMEN
A recent application of optical tweezers has shown that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels control adhesion of the membrane bilayer to the underlying cytoskeleton, by regulated direct binding of PIP(2) to cytoskeletal proteins and/or indirect effects on cytoskeleton structure.
Asunto(s)
Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Isoenzimas/metabolismo , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/fisiología , Fosfolipasa C gamma , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Activation of human platelets with thrombin transiently increases phosphorylation at (558)threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or 125I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with alpha- or beta/gamma-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, approximately 1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin's high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane.
Asunto(s)
Actinas/metabolismo , Plaquetas/metabolismo , Proteínas de Microfilamentos/metabolismo , Secuencia de Aminoácidos , Bioquímica/métodos , Citoesqueleto/química , Citoesqueleto/metabolismo , Detergentes/química , Humanos , Proteínas de Microfilamentos/aislamiento & purificación , Datos de Secuencia Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Fosforilación , Compuestos de Amonio Cuaternario , Treonina/metabolismoRESUMEN
A growing number of actin-associated membrane proteins have been implicated in motile processes, adhesive interactions, and signal transduction to the cell nucleus. We report here that supervillin, an F-actin binding protein originally isolated from bovine neutrophil plasma membranes, contains functional nuclear targeting signals and localizes at or near vinculin-containing focal adhesion plaques in COS7-2 and CV1 cells. Overexpression of full-length supervillin in these cells disrupts the integrity of focal adhesion plaques and results in increased levels of F-actin and vinculin. Localization studies of chimeric proteins containing supervillin sequences fused with the enhanced green fluorescent protein indicate that: (1) the amino terminus promotes F-actin binding, targeting to focal adhesions, and limited nuclear localization; (2) the dominant nuclear targeting signal is in the center of the protein; and (3) the carboxy-terminal villin/gelsolin homology domain of supervillin does not, by itself, bind tightly to the actin cytoskeleton in vivo. Overexpression of chimeras containing both the amino-terminal F-actin binding site(s) and the dominant nuclear targeting signal results in the formation of large nuclear bundles containing F-actin, supervillin, and lamin. These results suggest that supervillin may contribute to cytoarchitecture in the nucleus, as well as at the plasma membrane.
Asunto(s)
Actinas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Bovinos , Adhesión Celular , Línea Celular , Citoesqueleto/metabolismo , Cartilla de ADN/genética , Expresión Génica , Proteínas Fluorescentes Verdes , Laminas , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Señales de Localización Nuclear , Proteínas Nucleares/metabolismo , Fenotipo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vinculina/metabolismoRESUMEN
Supervillin is a 205-kDa F-actin binding protein originally isolated from bovine neutrophils. This protein is tightly associated with both actin filaments and plasma membranes, suggesting that it forms a high-affinity link between the actin cytoskeleton and the membrane. Human supervillin cDNAs cloned from normal human kidney and from the cervical carcinoma HeLa S3 predict a bipartite structure with three potential nuclear localization signals in the NH2-terminus and three potential actin-binding sequences in the COOH-terminus. In fact, throughout its length, the COOH-terminal half of supervillin is similar to segments 2-6 plus the COOH-terminal "headpiece" of villin, an actin-binding protein in intestinal microvilli. A comparison of the bovine and human sequences indicates that supervillin is highly conserved at the amino acid level, with 79.2% identity of the NH2-terminus and conservation of three of the four nuclear localization signals found in bovine supervillin. The COOH-terminus is even more conserved, with 95.1% amino acid identity overall and 100% conservation of the villin-like headpiece. Supervillin mRNAs are expressed in all human tissue tested, bu are most abundant in muscle, bone marrow, thyroid gland, and salivary gland; comparatively little message is found in brain. Human supervillin mRNA is approximately 7.5 kb; this message is especially abundant in HeLa S3 cervical carcinoma, SW480 adenocarcinoma, and A549 lung carcinoma cell lines. The human supervillin gene (SVIL) is localized to a single chromosomal locus at 10p11.2, a region that is deleted in some prostate tumors.