RESUMEN
Objetivos: Las enfermedades minoritarias constituyen un grupo heterogéneo de patologías de baja prevalencia, con un origen genético en la mayoría de los casos y frecuentemente asociadas a un retraso en su diagnóstico y notable morbi-mortalidad. Conocer las características clínicas y el grado de complejidad asistencial de los pacientes con enfermedades minoritarias que acuden a las consultas de medicina interna en Galicia podría facilitar una atención más eficaz y eficiente. Material y métodos: Estudio epidemiológico, transversal y multicéntrico en pacientes con enfermedad de baja prevalencia ≥ 18 años atendidos en las consultas y unidades específicas de medicina interna en Galicia hasta el 31 de Diciembre de 2020. Se obtuvieron de cada centro el número de pacientes atendidos según la patología, la media en años desde la aparición de los síntomas, número de comorbilidades, número de visitas anuales al centro y a otros especialistas, número de tratamientos (incluidos aquellos de alto impacto económico), grado de deterioro cognitivo y estimación del nivel de dependencia. Resultados: Se analizaron los indicadores de seis centros correspondientes a seis áreas sanitarias de Galicia, representando a un total de 324 pacientes, con una edad media de 41,3 ±15,8 años. Los tres principales grupos de patologías atendidas fueron por este orden las enfermedades genéticas raras, los errores innatos del metabolismo y las enfermedades neurológicas raras. El retraso medio en el diagnóstico fue de 4,8 ±7,9 años y un 34,17% de los pacientes tardaron 5 o más años en tener una confirmación diagnóstica. Este grupo de mayor retraso diagnóstico presenta menor puntuación en la escala de dependencia de Barthel y 1,75 veces mayor utilización de recursos sanitarios (consultas a medicina interna y otras especialidades). El 11,75% de los pacientes presentan un nivel de dependencia severa o total y el 9,9%, un bajo coeficiente intelectual. Conclusiones: ... (AU)
Objectives: Rare diseases (RD) constitute a heterogeneous group of low prevalence conditions, with genetic origin in most cases and frequently associated with a delay in their diagnosis and notable morbidity and mortality. Knowing the clinical profile and the complexity degree of patientswith RD who attend internal medicine units in Galicia could facilitate more effective and efficient care settings. Methods: Epidemiological, cross-sectional and multicenter study in patients with low prevalence diseases ≥ 18 years attending the outpatients departments and specific units of internal medicine in Galicia up to December 31, 2020. Data were collected on the number of patients treatedregarding their condition at each center, the average age at disease onset, number of comorbidities, number of annual visits to the center and other specialists, number of treatments (including those with a high economicimpact), degree of cognitive impairment and assessment of the degree of autonomy. Results: We analyzed data from six participating centers (from six healthareas of Galicia), representing a total of 324 patients, with a mean age of 41.3 ±15.8 years. The three main groups of pathologies treated were, in this order, rare genetic diseases, innate errors of metabolism and rareneurological diseases. The mean delay in diagnosis was 4.8 ±7.9 years and 34.17% of patients took 5 or more years to have a diagnostic confirmation. This group with the longest diagnostic delay has a lower scoreon the Barthel dependency scale and 1.75 times greater use of health resources (consultations with internal medicine and other specialties). 11.75% of the patients present a level of severe or total dependence and 9.9%, a low IQ. Conclusions: Despite their low average age, patients with minority diseases treated in internal medicine services present a high complexity of care derived from the number of comorbidities, visits to consultations, the need for hospital admissions and polypharmacy... (AU)
Asunto(s)
Humanos , Adolescente , Adulto Joven , Adulto , Enfermedades Raras , Diagnóstico Tardío , Manejo de Atención al Paciente , Repertorio de Barthel , Comorbilidad , España , Estudios Transversales , Estudios Multicéntricos como AsuntoRESUMEN
OBJETIVO: analizar las variables demográficas y diagnósticas que condicionan la demanda asistencial ambulatoria de la consulta neurológica privada por primera vez. MÉTODOS: estudio observacional, descriptivo, retrospectivo, de la demanda de consultas ambulatorias de primera vez en un centro neurológico privado, durante 24 meses (enero-2016 hasta diciembre-2017). La información médica fue obtenida a partir de registros clínicos computarizados registrándose: edad, género y diagnóstico según la Clasificación Internacional de Enfermedades, 10 edición (CIE-10). RESULTADOS: el total de pacientes del estudio fue de 2 372, (60%) fueron mujeres. La edad media fue de 42,6 años ± 18,8 años, (42,20%) son mayores de 65 años. El diagnóstico más frecuente fue cefalea primaria (33,4%); seguido por epilepsia (14%); cervicalgia, dorsalgia y lumbalgia (13,2%), trastornos psiquiátricos (6,5%). En mayores de 65 años el deterioro cognitivo fue el diagnóstico más frecuente (14,68%), seguido de movimientos anormales. CONCLUSIÓN: los pacientes neurológicos son predominantemente menores de 65 años y de sexo femenino. La patología más frecuente fue la cefalea primaria.(AU)
OBJECTIVES: to analyze the demographic and diagnostic variables that condition the ambulatory care demand of the private neurological consultation for the first time. METHODS: observational, descriptive, retrospective study of the demand for first-time outpatient consultations in a private neurological center, for 24 months (January-2016 to December-2017). The medical information was obtained from computerized clinical records by registering: age, gender and diagnosis according to the International Classification of Diseases, 10 edition (ICD-10). RESULTS: the total number of patients in the study was 2372, (60%) were women. The mean age was 42.6 years ± 18.8 years, (42.20%) are over 65 years. The most frequent diagnosis was primary headache (33.4%); followed by epilepsy (14%); cervicalgia, dorsalgia and low back pain (13.2%), psychiatric disorders (6.5%). In older than 65 years cognitive impairment was the most frequent diagnosis (14.68%), followed by abnormal movements. CONCLUSIONS: neurological patients are predominantly under 65 years of age and female. The most frequent pathology was primary headache.(AU)
Asunto(s)
Humanos , Masculino , Femenino , Cefalea , Neurología , Demografía , Sistemas de Registros Médicos ComputarizadosRESUMEN
In plants, adverse conditions often induce an increase in reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). H2O2 is reduced to water, and thus becomes detoxified by enzymes such as Cytisus multiflorus peroxidase (CMP). Here, the steady-state kinetics of the H2O2-supported oxidation of different organic substrates by CMP was investigated. Analysis of the initial rates vs. H2O2 and reducing substrate concentrations proved to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters [Formula: see text] , [Formula: see text] , kcat, [Formula: see text] , [Formula: see text] and of the microscopic rate constants, k1 and k3, of the shared three-step catalytic cycle of peroxidases.
Asunto(s)
Cytisus/enzimología , Peroxidasa/metabolismo , Biocatálisis , Guayacol/metabolismo , Peróxido de Hidrógeno/metabolismo , Cinética , Modelos Moleculares , Oxidación-Reducción , Peroxidasa/antagonistas & inhibidores , Especificidad por SustratoRESUMEN
Programmed cell death is essential to survival of multicellular organisms. Previously restricted to apoptosis, the concept of programmed cell death is now extended to other mechanisms, as programmed necrosis or necroptosis, autophagic cell death, pyroptosis and parthanatos, among others. Viruses have evolved to manipulate and take control over the programmed cell death response, and the infected cell attempts to neutralize viral infections displaying different stress signals and defensive pathways before taking the critical decision of self-destruction. Learning from viruses and their interplay with the host may help us to better understand the complexity of the self-defense death response that when altered might cause disorders as important as cancer. In addition, as the fields of immunotherapy and oncolytic viruses advance as promising novel cancer therapies, the programmed cell death response reemerges as a key point for the success of both therapeutic approaches. In this review we summarize the research of the multimodal cell death response induced by Newcastle disease viruses (NDV), considered nowadays a promising viral oncolytic therapeutic, and how the manipulation of the host programmed cell death response can enhance the NDV antitumor capacity.
Asunto(s)
Virus de la Enfermedad de Newcastle/fisiología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Animales , Apoptosis , Autofagia , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Humanos , Virus de la Enfermedad de Newcastle/crecimiento & desarrollo , Virus Oncolíticos/crecimiento & desarrolloRESUMEN
Newcastle disease virus (NDV) is considered a promising agent for cancer therapy due to its oncolytic properties. These include preferential replication in transformed cells, induction of innate and adaptive immune responses within tumors, and cytopathic effects in infected tumor cells due to the activation of apoptosis. To enhance the latter and thus possibly enhance the overall oncolytic activity of NDV, we generated a recombinant NDV encoding the human TNF receptor Fas (rNDV-B1/Fas). rNDV-B1/Fas replicates to similar titers as its wild-type (rNDV-B1) counterpart; however, overexpression of Fas in infected cells leads to higher levels of cytotoxicity correlated with faster and increased apoptosis responses, in which both the intrinsic and extrinsic pathways are activated earlier. Furthermore, in vivo studies in syngeneic murine melanoma models show an enhancement of the oncolytic properties of rNDV-B1/Fas, with major improvements in survival and tumor remission. Altogether, our data suggest that upregulation of the proapoptotic function of NDV is a viable approach to enhance its antitumor properties and adds to the currently known, rationally based strategies to design optimized therapeutic viral vectors for the treatment of cancer.
Asunto(s)
Melanoma Experimental/terapia , Virus de la Enfermedad de Newcastle/fisiología , Neoplasias Cutáneas/terapia , Receptor fas/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Células HeLa , Humanos , Melanoma Experimental/inmunología , Ratones , Células 3T3 NIH , Virus de la Enfermedad de Newcastle/genética , Viroterapia Oncolítica , Virus Oncolíticos/genética , Virus Oncolíticos/fisiología , Neoplasias Cutáneas/inmunología , Células Vero , Receptor fas/genéticaRESUMEN
New plant peroxidase has been isolated to homogeneity from the white Spanish broom Cytisus multiflorus. The enzyme purification steps included homogenization, NH(4)SO(4) precipitation, extraction of broom colored compounds and consecutive chromatography on Phenyl-Sepharose, HiTrap™ SP HP and Superdex-75 and 200. The novel peroxidase was characterized as having a molecular weight of 50 ± 3 kDa. Steady-state tryptophan fluorescence and far-UV circular dichroism (CD) studies, together with enzymatic assays, were carried out to monitor the structural stability of C. multiflorus peroxidase (CMP) at pH 7.0. Thus changes in far-UV CD corresponded to changes in the overall secondary structure of enzyme, while changes in intrinsic tryptophan fluorescence emission corresponded to changes in the tertiary structure of the enzyme. It is shown that the process of CMP denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, N ⶠkD, where k is a first-order kinetic constant that changes with temperature following the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.
Asunto(s)
Cytisus/enzimología , Estabilidad de Enzimas , Peroxidasa/aislamiento & purificación , Dicroismo Circular , Peroxidasa/química , Desnaturalización Proteica , Estructura Secundaria de Proteína , TemperaturaRESUMEN
Although it is well documented that the initial attachment receptors for Newcastle Disease Virus (NDV) and Respiratory Syncytial Virus (RSV) are sialic acid-containing molecules and glycosaminoglycans respectively, the exact nature of the receptors for both viruses remains to be deciphered. Moreover, additional molecules at the host cell surface might be involved in the entry mechanism. With the aim of identifying the cellular proteins that interact with NDV and RSV at the cell surface, we performed a virus overlay protein binding assay (VOPBA). Cell membrane lysates were separated by two dimensional (2D) gel electrophoresis and electrotransferred to PVDF membranes, after which they were probed with high viral concentrations. NDV interacted with a Protein Disulfide Isomerase from chicken fibroblasts. In the case of RSV, we detected 15 reactive spots, which were identified as six different proteins, of which nucleolin was outstanding. We discuss the possible role of PDI and nucleolin in NDV and RSV entry, respectively.
Asunto(s)
Enfermedad de Newcastle/metabolismo , Virus de la Enfermedad de Newcastle/metabolismo , Enfermedades de las Aves de Corral/metabolismo , Proteínas/metabolismo , Infecciones por Virus Sincitial Respiratorio/metabolismo , Virus Sincitial Respiratorio Humano/metabolismo , Animales , Línea Celular , Pollos , Electroforesis en Gel Bidimensional , Humanos , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/genética , Enfermedades de las Aves de Corral/virología , Unión Proteica , Proteínas/química , Proteínas/genética , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/genéticaRESUMEN
Most paramyxoviruses enter the cell by direct fusion of the viral envelope with the plasma membrane. Our previous studies have shown the colocalization of Newcastle Disease Virus (NDV) with the early endosome marker EEA1 and the inhibition of NDV fusion by the caveolin-phosphorylating drug phorbol 12-myristate 13-acetate (PMA) prompted us to propose that NDV enters the cells via endocytosis. Here we show that the virus-cell fusion and cell-cell fusion promoted by NDV-F are increased by about 30% after brief exposure to low pH in HeLa and ELL-0 cells but not in NDV receptor- deficient cell lines such as GM95 or Lec1. After a brief low-pH exposure, the percentage of NDV fusion at 29 °C was similar to that at 37 °C without acid-pH stimulation, meaning that acid pH would decrease the energetic barrier to enhance fusion. Furthermore, preincubation of cells with the protein kinase C inhibitor bisindolylmaleimide led to the inhibition of about 30% of NDV infectivity, suggesting that a population of virus enters cells through receptor-mediated endocytosis. Moreover, the involvement of the GTPase dynamin in NDV entry is shown as its specific inhibitor, dynasore, also impaired NDV fusion and infectivity. Optimal infection of the host cells was significantly affected by drugs that inhibit endosomal acidification such as concanamycin A, monensin and chloroquine. These results support our hypothesis that entry of NDV into ELL-0 and HeLa cells occurs through the plasma membrane as well as by dynamin- low pH- and receptor- dependent endocytosis.
Asunto(s)
Membrana Celular/metabolismo , Dinaminas/metabolismo , Endocitosis , Virus de la Enfermedad de Newcastle/fisiología , Receptores Virales/metabolismo , Virión/fisiología , Animales , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/virología , Cloroquina/farmacología , Cricetulus , Dinaminas/antagonistas & inhibidores , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Hidrazonas/farmacología , Concentración de Iones de Hidrógeno , Indoles/farmacología , Macrólidos/farmacología , Maleimidas/farmacología , Fusión de Membrana/efectos de los fármacos , Monensina/farmacología , Virus de la Enfermedad de Newcastle/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Receptores Virales/antagonistas & inhibidores , Termodinámica , Virión/efectos de los fármacos , Internalización del Virus/efectos de los fármacosRESUMEN
The thermal stability of the matrix protein (M protein) of Newcastle disease virus (NDV) has been investigated using high-sensitivity differential scanning calorimetry (DSC) at pH 7.4. The thermal folding/unfolding of M protein at this pH value is a reversible process involving a highly cooperative transition between folded and unfolded monomers with a transition temperature (Tm) of 63 °C, an unfolding enthalpy, ΔH(Tm), of 340 kcal mol(-1), and the difference in heat capacity between the native and denatured states of the protein, ΔCp, of 5.1 kcal K(-1) mol(-1). The heat capacity of the native state of the protein is in good agreement with the values calculated using a structure-based parameterization, whereas the calculated values for the hypothetical fully-unfolded state of the protein is higher than those determined experimentally. This difference between the heat capacity of denatured M protein and the heat capacity expected for an unstructured polypeptide of the same sequence, together with the data derived from the heat-induced changes in the steady-state fluorescence of the protein, indicates that the polypeptide chain maintains a significant amount of residual structure after thermal denaturation.
Asunto(s)
Virus de la Enfermedad de Newcastle/química , Proteínas de la Matriz Viral/química , Rastreo Diferencial de Calorimetría , Concentración de Iones de Hidrógeno , Estabilidad Proteica , Termodinámica , Proteínas de la Matriz Viral/aislamiento & purificaciónRESUMEN
Receptor recognition and binding is the first step in the viral cycle. It has been established that Newcastle Disease Virus (NDV) interacts with sialylated molecules such as gangliosides and glycoproteins at the cell surface. Nevertheless, the specific receptor(s) that mediate virus entry are not well known. We have analysed the role of the sialic acid linkage in the early steps of the viral infection cycle. Pretreatment of ELL-0 cells with both α2,3 and α2,6 specific sialidases led to the inhibition of NDV binding, fusion and infectivity, which were restored after α2,3(N)- and α2,6(N)-sialyltransferase incubation. Moreover, α2,6(N)-sialyltransferases also restored NDV activities in α2-6-linked sialic acid deficient cells. Competition with α2-6 sialic acid-binding lectins led to a reduction in the three NDV activities (binding, fusion and infectivity) suggesting a role for α2-6- linked sialic acid in NDV entry. We conclude that both α2-3- and α2-6- linked sialic acid containing glycoconjugates may be used for NDV infection. NDV was able to efficiently bind, fuse and infect the ganglioside-deficient cell line GM95 to a similar extent to that of its parental MEB4, suggesting that gangliosides are not essential for NDV binding, fusion and infectivity. Nevertheless, the fact that the interaction of NDV with cells deficient in N-glycoprotein expression such as Lec1 was less efficient prompted us to conclude that NDV requires N-linked glycoproteins for efficient attachment and entry into the host cell.
Asunto(s)
Glicoproteínas/metabolismo , Enfermedad de Newcastle/metabolismo , Virus de la Enfermedad de Newcastle/fisiología , Ácidos Siálicos/metabolismo , Acoplamiento Viral , Internalización del Virus , Animales , Células CHO , Pollos , Cricetinae , Cricetulus , Células HEK293 , Humanos , Sialiltransferasas/metabolismoRESUMEN
Endoglin is a TGF-ß co-receptor expressed in endothelial cells, where it plays a crucial role in angiogenesis, cardiovascular development and vascular remodeling. In humans, mutations in the endoglin gene give rise to Hereditary Hemorrhagic Telangiectasia type 1 (HHT1), an autosomal dominant disorder associated with vascular lesions in skin, mucosa and internal organs. So far, endoglin cDNA has been sequenced in several species from mammals, amphibians and birds. While in mammals the characterization of endoglin protein expression and function is well documented, little is known about the protein homologue in birds. In silico analysis by multiple sequences alignment showed a low homology score of 30-33 between the full length chicken endoglin protein and several mammalian homologues. However, a high homology score (80-85) was observed with the cytoplasmic and transmembrane regions and the overall structure of the zona pellucida (ZP) and orphan domains of the extracellular region appear to be conserved. Transient expression of chicken endoglin allowed the identification of a 180-kDa disulfide linked homodimer similar to the mammalian homologues. To further characterize its tissue expression, the novel specific monoclonal antibody (mAb) 7H5A8 was generated against chicken endoglin transfectant cells. The mAb 7H5A8 specifically recognized chicken endoglin by western blot, immunoprecipitation, immunofluorescence flow cytometry as well as immunofluorescence microscopy assays and displayed a positive staining of the endothelium in veins and arteries from frozen tissue sections of lung and bursa of Fabricius. These results may help to further understand the endoglin expression in vertebrates.
Asunto(s)
Pollos/genética , Animales , Antígenos CD , Distribución Tisular , Zona Pelúcida/metabolismoRESUMEN
Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-ß-cyclodextrin (MßCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MßCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4°C and then shifted to detergent-soluble fractions at 37°C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.
Asunto(s)
Colesterol/metabolismo , Microdominios de Membrana/metabolismo , Enfermedad de Newcastle/metabolismo , Virus de la Enfermedad de Newcastle/fisiología , Acoplamiento Viral , Internalización del Virus , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Receptores Virales/biosíntesis , beta-Ciclodextrinas/farmacologíaRESUMEN
The entry of enveloped viruses into host cells is preceded by membrane fusion, which in paramyxoviruses is triggered by the fusion (F) protein. Refolding of the F protein from a metastable conformation to a highly stable postfusion form is critical for the promotion of fusion, although the mechanism is still not well understood. Here we examined the effects of mutations of individual residues of the F protein of Newcastle disease virus, located at critical regions of the protein, such as the C terminus of the N-terminal heptad repeat (HRA) and the N terminus of the C-terminal heptad repeat (HRB). Seven of the mutants were expressed at the cell surface, showing differences in antibody reactivity in comparison with the F wild type. The N211A, L461A, I463A, and I463F mutants showed a hyperfusogenic phenotype both in syncytium and in dye transfer assays. The four mutants promoted fusion more efficiently at lower temperatures than the wild type did, meaning they probably had lower energy requirements for activation. Moreover, the N211A, I463A, and I463F mutants exhibited hemagglutinin-neuraminidase (HN)-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.
Asunto(s)
Proteína HN/metabolismo , Mutación , Virus de la Enfermedad de Newcastle/metabolismo , Proteínas Virales de Fusión/genética , Animales , Línea Celular , Cricetinae , Células Gigantes/metabolismo , Proteína HN/genética , Células HeLa , Humanos , Fusión de Membrana , Mutagénesis Sitio-Dirigida , Virus de la Enfermedad de Newcastle/genética , Fenotipo , Conformación Proteica , Transfección , Proteínas Virales de Fusión/metabolismoRESUMEN
The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N-->kD, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization.
Asunto(s)
Arecaceae/enzimología , Peroxidasa/química , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Pliegue de Proteína , Multimerización de Proteína , TermodinámicaRESUMEN
The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.
Asunto(s)
Expresión Génica , Neuraminidasa/genética , Virus de la Enfermedad de Newcastle/fisiología , Internalización del Virus , Replicación Viral , Animales , Western Blotting , Células COS , Fusión Celular , Membrana Celular/virología , Chlorocebus aethiops , Cromatografía en Capa Delgada , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/metabolismo , Células Gigantes/virología , Proteína HN/metabolismo , Neuraminidasa/metabolismo , Enfermedad de Newcastle/virologíaRESUMEN
The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins purified from Newcastle disease virus adsorb on a phospholipid bilayer and condense into fluidlike domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.
Asunto(s)
Glicoproteínas/metabolismo , Liposomas Unilamelares/metabolismo , Proteínas de la Matriz Viral/biosíntesis , Colesterol/química , Etanolaminas/química , Colorantes Fluorescentes , Glicoproteínas/química , Glicoproteínas/ultraestructura , Membrana Dobles de Lípidos/química , Microscopía Electrónica , Virus de la Enfermedad de Newcastle/química , Virus de la Enfermedad de Newcastle/fisiología , Técnicas de Placa-Clamp , Fosfolípidos/química , Fosfolípidos/metabolismo , Fosforilcolina/química , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Temperatura , Termodinámica , Proteínas del Envoltorio Viral/metabolismo , Proteínas de la Matriz Viral/químicaRESUMEN
The entry into cells of Newcastle disease virus (NDV), a prototype member of the paramyxoviruses, is believed to occur by direct fusion at the plasma membrane through a pH-independent mechanism. In addition, NDV may enter host cells by an endocytic pathway. Treatment of cells with drugs that block caveolae-dependent endocytosis reduced NDV fusion and infectivity, the degree of inhibition being dependent on virus concentration. The inhibitory effect was reduced greatly when drugs were added after virus adsorption. Cells treated with methyl beta-cyclodextrin, a drug that sequesters cholesterol from membranes, reduced the extent of fusion, infectivity and virus-cell binding; this indicates that cholesterol plays a role in NDV entry. Double-labelling immunofluorescence assays performed with anti-NDV monoclonal antibodies and antibodies against the early endosome marker EEA1 revealed the localization of the virus in these intracellular structures. Using fluorescence microscopy, it was found that cell-cell fusion was enhanced at low pH. It is concluded that NDV may infect cells through a caveolae-dependent endocytic pathway, suggesting that this pathway could be an alternative route for virus entry into cells.
Asunto(s)
Caveolas/virología , Endocitosis/fisiología , Virus de la Enfermedad de Newcastle/patogenicidad , Internalización del Virus , Animales , Células COS , Fusión Celular , Chlorocebus aethiops , Endocitosis/efectos de los fármacos , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Fusión de Membrana , Microscopía Fluorescente , Virus de la Enfermedad de Newcastle/fisiología , Células Vero , beta-Ciclodextrinas/farmacologíaRESUMEN
Sialic acid-containing compounds play a key role in the initial steps of the paramyxovirus life cycle. As enveloped viruses, their entry into the host cell consists of two main events: binding to the host cell and membrane fusion. Virus adsorption occurs at the surface of the host cell with the recognition of specific receptor molecules located at the cell membrane by specific viral attachment proteins. The viral attachment protein present in some paramyxoviruses (Respirovirus, Rubulavirus and Avulavirus) is the HN glycoprotein, which binds to cellular sialic acid-containing molecules and exhibits sialidase and fusion promotion activities. Gangliosides of the gangliotetraose series bearing the sialic acid N-acetylneuraminic (Neu5Ac) on the terminal galactose attached in alpha2-3 linkage, such as GD1a, GT1b, and GQ1b, and neolacto-series gangliosides are the major receptors for Sendai virus. Much less is known about the receptors for other paramyxoviruses than for Sendai virus. Human parainfluenza viruses 1 and 3 preferentially recognize oligosaccharides containing N-acetyllactosaminoglycan branches with terminal Neu5Acalpha2-3Gal. In the case of Newcastle disease virus, has been reported the absence of a specific pattern of the gangliosides that interact with the virus. Additionally, several works have described the use of sialylated glycoproteins as paramyxovirus receptors. Accordingly, the design of specific sialic acid analogs to inhibit the sialidase and/or receptor binding activity of viral attachment proteins is an important antiviral strategy. In spite of all these data, the exact nature of paramyxovirus receptors, apart from their sialylated nature, and the mechanism(s) of viral attachment to the cell surface are poorly understood.