RESUMEN
Radiation therapy is a mainstay in the therapeutic management of Head and Neck Squamous Cell Carcinoma (HNSCC). Despite significant progress in this field, radioresistance still accounts for most treatment failures. Gadolinium-based nanoparticles (GBNs) have shown great promises as radiosensitizers but the underlying sensitizing mechanism is still largely unknown with regards to the disparities obtained in in vitro studies. In this study, we show that a new formulation of GBNs, AGuIX®, can radiosensitize HNSCC after cell uptake and further accumulation in lysosomes. Although radiation alone triggered late apoptosis and mitochondrial impairment, the pre-treatment with GBNs led to complex DNA damage and a specific increase of autophagic cell death. In addition, a significant radio-enhancement effect was obtained after the pre-conditioning of cells with a glutathione inhibitor before GBNs treatment and radiation exposure. Overall, our results provide additional information on the radio-enhancing properties of GBNs in the management of radioresistant HNSCC.
Asunto(s)
Autofagia , Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Carcinoma de Células Escamosas de Cabeza y Cuello , Apoptosis , Línea Celular Tumoral , Gadolinio , Humanos , Nanopartículas del MetalAsunto(s)
Automonitorización de la Glucosa Sanguínea/instrumentación , Glucemia/análisis , Diabetes Mellitus Tipo 1/sangre , Sistemas de Infusión de Insulina , Educación del Paciente como Asunto , Adulto , Análisis de Varianza , Glucemia/metabolismo , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Femenino , Hemoglobina Glucada/análisis , Hemoglobina Glucada/metabolismo , Humanos , Insulina/administración & dosificación , Masculino , Persona de Mediana EdadRESUMEN
REASONS FOR PERFORMING STUDY: Rhodococcoccus equi is a significant cause of bronchopneumonia in foals worldwide. Infection of the lungs is believed to result from inhalation of virulent R. equi in dust from contaminated environments. A measure of infectious risk in an environment is the level of airborne contamination. OBJECTIVES: To assess and compare the level of airborne virulent R. equi in paddocks and stables. METHODS: Air samples were collected sequentially over the 2003 foaling season from the paddocks and stables on 3 Irish horse breeding farms affected by R. equi pneumonia. Colony blotting and DNA hybridisation techniques allowed quantitation of virulent R. equi. RESULTS: The odds of detecting airborne virulent R. equi in stables were 173 times greater than in paddocks. The median airborne concentration of virulent R. equi was significantly higher (P < 0.001) in stables than in paddocks on all farms. These observations suggested that stables were high-risk areas for infection. CONCLUSIONS AND POTENTIAL RELEVANCE: Our results indicate that contaminated stables are a significant risk factor in the epidemiology of R. equi pneumonia on horse-breeding farms in a temperate climate, such as in Ireland. Management strategies that improve the air hygiene of stables, through better ventilation, use of less fragile bedding material and the use of fogging agents to reduce the airborne concentration of virulent R. equi, may reduce the incidence and severity of R. equi pneumonia on farms.
Asunto(s)
Microbiología del Aire , Vivienda para Animales/normas , Rhodococcus equi/aislamiento & purificación , Infecciones por Actinomycetales/epidemiología , Infecciones por Actinomycetales/microbiología , Infecciones por Actinomycetales/veterinaria , Animales , Animales Recién Nacidos , Cruzamiento , Clima , Femenino , Enfermedades de los Caballos/epidemiología , Enfermedades de los Caballos/microbiología , Caballos , Irlanda , Masculino , Rhodococcus equi/patogenicidad , Estaciones del Año , VirulenciaRESUMEN
We describe the construction of plasmids which express fusion proteins representing various regions of Germiston virus M polyprotein. The fusion proteins were purified and inoculated into rabbits to produce antisera. The N- and C-terminal regions of the polyprotein induced specific antibodies which reacted with glycoproteins G2 and G1, respectively, and the intermediate region induced antibodies against the NSM polypeptide. This enabled us to determine the gene order: G2-NSM-G1. Glycoproteins G1 and G2 form the spikes on the surface of the virion. We attempted to determine the structural organization of the glycoproteins by using a membrane-permeable cross-linking reagent, dimethyl suberimidate, but were unable to demonstrate that G1 and/or G2 form oligomeric structures. We analysed the glycoproteins further and showed that, like peripheral membrane proteins, the G2 and NSM proteins are almost completely extracted into the aqueous phase of detergent Triton X114-treated cellular extracts, whereas glycoprotein G1 is distributed in almost equal proportions between the aqueous and the detergent fractions. This indicates that G1 is a membrane-associated protein, but its presence in the aqueous phase suggests that it is less hydrophobic than a typical membrane protein. We have also characterized the intracellular transport of the envelope glycoproteins from the endoplasmic reticulum to the Golgi complex. Pulse-chase labelling followed by immunoprecipitation and treatment with endoglycosidase H (endo H) showed that both G1 and G2 are transported from the endoplasmic reticulum to the Golgi complex. Conversion to the endo H-resistant form is a rather slow process which takes more than 2 h. The mature G1 and G2 proteins present in the virion particle contain almost completely endo-H-resistant glycans.
Asunto(s)
Virus Bunyamwera/genética , Glicoproteínas/química , Proteínas del Envoltorio Viral/química , Proteínas de la Matriz Viral/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Virus Bunyamwera/crecimiento & desarrollo , Virus Bunyamwera/inmunología , Cricetinae , Escherichia coli/genética , Glicoproteínas/metabolismo , Glicoproteínas/ultraestructura , Glicósido Hidrolasas/metabolismo , Aparato de Golgi , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Procesamiento Proteico-Postraduccional , Proteínas/genética , Proteínas/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas de la Matriz Viral/inmunología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunologíaRESUMEN
Infection of Vero (monkey) cells by Germiston bunyavirus is highly cytopathic with cell lysis and virus production at a high titre, whereas infection of Aedes albopictus C6/36 (mosquito) cells leads, after an acute primary phase, to a persistent non-cytopathic infection with a loss in virus production. In this report we demonstrate that single-stranded RNA probes can be successfully used in an in situ hybridization assay to quantify viral expression during this persistent infection. The steady-state levels of viral S-RNA segment (genomic and messenger sense) during the acute phase were similar to those observed in lytically infected Vero cells, but appeared delayed. Both senses of S-RNA were detected throughout persistent infection but in lower amounts, in less than 10% of the cells and always in the cytoplasm of infected cells. The number of copies per cell of messenger sense S-RNAs remained low during persistent infection whereas a higher fluctuation was observed for genomic S-RNAs. In situ hybridization with specific stranded RNA probes provides both qualitative and quantitative informations, that can lead to a better understanding of virus-cell interactions.
Asunto(s)
Bunyaviridae/fisiología , Sondas ARN , ARN Viral/análisis , Aedes , Animales , Bunyaviridae/genética , Bunyaviridae/crecimiento & desarrollo , Línea Celular , Hibridación de Ácido Nucleico , ARN Viral/genética , Células Vero , Replicación Viral/genéticaRESUMEN
The 3' ends of the S and M messenger RNAs isolated from BHK21 cells infected with Germiston virus were analyzed by mapping with RNase T2 or nuclease S1. The transcription termination signal was found to be located approximately 115 and 80 nucleotides upstream from the 3' end of the S and M genomic RNA templates, respectively. Both mRNAs were found to possess several adenosine residues at their 3' ends, but were not polyadenylated. They have acquired at their 5' end a heterologous 12- to 18-nucleotide-long sequence, which is not coded for by the virus. Sequencing of the 5' terminal region from single molecules cloned into pBR327 revealed that these primers are rich in C and G residues and possess a U or a C adjacent to the viral sequence.
Asunto(s)
Bunyaviridae/genética , Genes Reguladores , ARN Mensajero/genética , ARN Viral/genética , Regiones Terminadoras Genéticas , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cricetinae , Sondas de ADN , ADN Viral/genética , Riñón , Datos de Secuencia Molecular , Mapeo Nucleótido , Plásmidos , ARN Mensajero/aislamiento & purificación , ARN Viral/aislamiento & purificación , Mapeo Restrictivo , Moldes Genéticos , Transcripción GenéticaRESUMEN
Using Germiston virus infected vertebrate (VERO) and invertebrate (Aedes albopictus C6/36) cells, paraformaldehyde-glutaraldehyde fixative allowed the best preservation of cellular morphology and the highest hybridization signals with cDNA and asymmetric RNA probes against the viral S segment. Asymmetric RNA probes always gave higher sensitivity and better specificity of in situ hybridization than the nick-translated symmetric DNA probe in both vertebrate and invertebrate cells. The study of Aedes albopictus C6/36 cells persistently infected with Germiston virus showed that only a small number of cells contained the S segment, and that the replication and transcription of the S segment took place in the cytoplasm of acutely and persistently infected cells.
Asunto(s)
Bunyaviridae/aislamiento & purificación , ARN Viral/análisis , Aedes/microbiología , Animales , Línea Celular , Sondas de ADN , Fijadores , Sondas ARN , Células VeroRESUMEN
The complete nucleotide sequence of the M RNA segment of Germiston bunyavirus was determined from plasmids containing overlapping M cDNA inserts. The M segment is 4534 nucleotides long and contains a 50-base-long inverted terminal repeat which can form a stable hydrogen-bonded secondary structure with a delta G of -45.8 kcal/mol. The RNA molecule complementary to viral RNA contains a single large open reading frame that encodes a 1437 amino acid-long protein with hydrophobic amino and carboxy terminal regions, which could represent signal and anchor sequences, respectively. It is presumed that this gene product is the polyprotein precursor to glycoproteins G1 and G2 and to the nonstructural polypeptide NSM. The nucleotide and amino acid sequences of the M RNA of Bunyamwera virus (prototype of the serogroup) and snowshow hare and La Crosse viruses (California serogroup) (Lees et al., 1986; Eshita and Bishop, 1984; Grady et al., 1987) were compared to those of Germiston virus. An overall amino acid sequence homology of 44% was found between Germiston and snowshoe hare viruses and of 61% between Germiston and Bunyamwera viruses. Most of the cysteines, three out of seven of the potential glycosylation sites, as well as the N and C terminal hydrophobic domains, are conserved between the four viruses.
Asunto(s)
Bunyaviridae/genética , ARN Viral/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Genes Virales , Datos de Secuencia Molecular , Especificidad de la Especie , Proteínas Virales/genéticaRESUMEN
The complete sequence of the S segment of Germiston bunyavirus has been determined from plasmids containing S cDNA inserts. The S segment is 980 nucleotides long with the first 15 bases at the 3' end complementary to the first 15 bases at the 5' end. Three overlapping open reading frames (ORF) were identified in the viral complementary RNA strand. The first ORF codes for a polypeptide of 233 amino acids (Mr 26,600) which is the nucleoprotein N. The second ORF codes for a polypeptide of 109 amino acids (Mr 11,800) which corresponds to the NSS protein, also called p12. Following this ORF, in the same frame, a third ORF which could encode a polypeptide of 75 amino acids was identified. Such a polypeptide has not yet been detected in infected cells. The N and NSS proteins of Germiston virus were compared with the corresponding proteins of La Crosse, snowshoe hare, and Aino viruses, and show a high extent of homology.
Asunto(s)
Virus Bunyamwera/genética , Bunyaviridae/genética , Genes Virales , ARN Viral/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Homología de Secuencia de Ácido NucleicoRESUMEN
We have isolated plasmid pIP112 (IncI1) from Salmonella panama and characterized by restriction endonucleases analysis and by recombinant DNA techniques a transposable element designated Tn1525. This 4.44 kilobase (kb) transposon confers resistance to kanamycin by synthesis of an aminoglycoside phosphotransferase (3') (5") type I and contains two copies of IS15 (1.5 kb) in direct orientation. The modular organisation of Tn1525 offers the possibility for intramolecular homologous recombination between the two terminal direct repeats and thus accounts for the in vivo structural lability of plasmid pIP112: instability of kanamycin resistance and tandem amplification of the kanamycin determinant. Other transposons mediating resistance to kanamycin by the same enzymatic mechanism were analysed by agarose and polyacrylamide gel electrophoresis, following digestion with restriction endonucleases, and by Southern hybridizations. These comparisons indicate that, although the structural genes for the phosphotransferases are homologous, Tn1525 differs from Tn903 and Tn2350 and is closely related but distinct from Tn6. Using the same techniques Tn1525 was detected on plasmids belonging to different incompatibility groups and originating from various species of Gram-negative clinical isolates. These results indicate that Tn1525 is representative of a new family of class I composite transposons already spread in diverse pathogenic bacterial genera.
Asunto(s)
Elementos Transponibles de ADN , Kanamicina/farmacología , Salmonella/genética , Mapeo Cromosómico , Enzimas de Restricción del ADN , ADN Bacteriano/genética , Farmacorresistencia Microbiana , Amplificación de Genes , Genes , Genes Bacterianos , Kanamicina Quinasa , Fosfotransferasas/genética , PlásmidosRESUMEN
A 10.8-kb transposable DNA sequence conferring resistance to tetracycline resides on the IncY Escherichia coli plasmid pIP231. This sequence, designated Tn1523, was shown to insert into different sites of the replicons of the IncY prophage P1Cm c1.100 and the IncI1 plasmid pIP112. This process is not dependent on the host recombination system recA. Genetic results indicate that Tn1523 transposition involves the formation of a cointegrate intermediate, either between pIP231 and P1Cm c1.100, or between pIP231 and pIP112. These intermediates were found to be resolved into donor and recipient plasmids, each harboring a copy of the Tn1523 transposon. A stable structure formed by fusion of the pIP231 plasmid with the pIP112 plasmid was also observed. This event occurs in the absence of the bacterial recA gene product and seems to involve a site-specific reciprocal recombination between "IS-like" elements.
Asunto(s)
Elementos Transponibles de ADN , Plásmidos , Recombinación Genética , Tetraciclina/farmacología , Colifagos/genética , Enzimas de Restricción del ADN , ADN Bacteriano , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Factores RRESUMEN
Curing of the P1 plasmid prophage in recA(P1) lysogens by superinfection with another P1 phage was specific and independent of immunity and incompatibility expression.