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X-ray photoelectron diffraction (XPD) is a powerful technique that yields detailed structural information of solids and thin films that complements electronic structure measurements. Among the strongholds of XPD we can identify dopant sites, track structural phase transitions, and perform holographic reconstruction. High-resolution imaging of kll-distributions (momentum microscopy) presents a new approach to core-level photoemission. It yields full-field kx-ky XPD patterns with unprecedented acquisition speed and richness in details. Here, we show that beyond the pure diffraction information, XPD patterns exhibit pronounced circular dichroism in the angular distribution (CDAD) with asymmetries up to 80%, alongside with rapid variations on a small kll-scale (0.1 Å-1). Measurements with circularly-polarized hard X-rays (hν = 6 keV) for a number of core levels, including Si, Ge, Mo and W, prove that core-level CDAD is a general phenomenon that is independent of atomic number. The fine structure in CDAD is more pronounced compared to the corresponding intensity patterns. Additionally, they obey the same symmetry rules as found for atomic and molecular species, and valence bands. The CD is antisymmetric with respect to the mirror planes of the crystal, whose signatures are sharp zero lines. Calculations using both the Bloch-wave approach and one-step photoemission reveal the origin of the fine structure that represents the signature of Kikuchi diffraction. To disentangle the roles of photoexcitation and diffraction, XPD has been implemented into the Munich SPRKKR package to unify the one-step model of photoemission and multiple scattering theory.
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The performance of time-resolved photoemission experiments at fs-pulsed photon sources is ultimately limited by the e-e Coulomb interaction, downgrading energy and momentum resolution. Here, we present an approach to effectively suppress space-charge artifacts in momentum microscopes and photoemission microscopes. A retarding electrostatic field generated by a special objective lens repels slow electrons, retaining the k-image of the fast photoelectrons. The suppression of space-charge effects scales with the ratio of the photoelectron velocities of fast and slow electrons. Fields in the range from -20 to -1100 V/mm for Ekin = 100 eV to 4 keV direct secondaries and pump-induced slow electrons back to the sample surface. Ray tracing simulations reveal that this happens within the first 40 to 3 µm above the sample surface for Ekin = 100 eV to 4 keV. An optimized front-lens design allows switching between the conventional accelerating and the new retarding mode. Time-resolved experiments at Ekin = 107 eV using fs extreme ultraviolet probe pulses from the free-electron laser FLASH reveal that the width of the Fermi edge increases by just 30 meV at an incident pump fluence of 22 mJ/cm2 (retarding field -21 V/mm). For an accelerating field of +2 kV/mm and a pump fluence of only 5 mJ/cm2, it increases by 0.5 eV (pump wavelength 1030 nm). At the given conditions, the suppression mode permits increasing the slow-electron yield by three to four orders of magnitude. The feasibility of the method at high energies is demonstrated without a pump beam at Ekin = 3830 eV using hard x rays from the storage ring PETRA III. The approach opens up a previously inaccessible regime of pump fluences for photoemission experiments.
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PURPOSE: Multiple cell types of the tumour microenvironment, including macrophages, contribute to the response to cancer therapy. The anti-resorptive agent zoledronic acid (ZOL) has anti-tumour effects in vitro and in vivo, but it is not known to what extent macrophages are affected by this agent. We have therefore investigated the effects of ZOL on macrophages using a combination of in vitro and in vivo models. METHODS: J774 macrophages were treated with ZOL in vitro, alone and in combination with doxorubicin (DOX), and the levels of apoptosis and necrosis determined. Uptake of zoledronic acid was assessed by detection of unprenylated Rap1a in J774 macrophages in vitro, in peritoneal macrophages and in macrophage populations isolated from subcutaneously implanted breast cancer xenografts following ZOL treatment in vivo. RESULTS: Exposure of J774 macrophages to 5 µM ZOL for 24 h caused a significant increase in the levels of uRap1A, and higher doses/longer exposure induced apoptotic cell death. DOX (10 nM/24 h) and ZOL (10 µM/4 h) given in sequence induced significantly increased levels of apoptotic cell death compared to single agents. Peritoneal macrophages and macrophage populations isolated from breast tumour xenografts had detectable levels of uRap1A 24 h following a single, clinically achievable dose of 100 µg/kg ZOL in vivo. CONCLUSION: We demonstrate that macrophages are sensitive to sequential administration of DOX and ZOL, and that both peritoneal and breast tumour associated macrophages rapidly take up ZOL in vivo. Our data support that macrophages may contribute to the anti-tumour effect of ZOL.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Difosfonatos/farmacología , Imidazoles/farmacología , Macrófagos/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Difosfonatos/administración & dosificación , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Sinergismo Farmacológico , Femenino , Humanos , Imidazoles/administración & dosificación , Macrófagos/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Prenilación de Proteína/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Ácido Zoledrónico , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Genetic testing of an Irish kindred identified an exonic nucleotide substitution c.1664T>C (p.Leu555Pro) in the MLH1 mismatch repair (MMR) gene. This previously unreported variant is classified as a "variant of uncertain significance" (VUS). Immunohistochemical (IHC) analysis and microsatellite instability (MSI) studies, genetic testing, a literature and online MMR mutation database review, in silico phenotype prediction tools, and an in vitro MMR activity assay were used to study the clinical significance of this variant. The MLH1 c.1664T>C (p.Leu555Pro) VUS co-segregated with three cases of classic Lynch syndrome-associated malignancies over two generations, with consistent loss of MLH1 and PMS2 protein expression on IHC, and evidence of the MSI-High mutator phenotype. The leucine at position 555 is well conserved across a number of species, and this novel variant has not been reported as a normal polymorphism in the general population. In silico and in vitro analyses suggest that this variant may have a deleterious effect on the MLH1 protein and abrogate MMR activity. Evidence from clinical, histological, immunohistochemical, and molecular genetic data suggests that MLH1 c.1664T>C (p.Leu555Pro) is likely to be the pathogenic cause of Lynch syndrome in this family.
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Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Trifosfatasas/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Reparación de la Incompatibilidad de ADN/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Mutación/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Adulto , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Análisis Multivariante , Homólogo 1 de la Proteína MutL , Estadificación de Neoplasias , Proteínas Nucleares/metabolismo , Linaje , Fenotipo , Pronóstico , Adulto JovenRESUMEN
PURPOSE: Athletes are trained to choose the pace which is perceived to be correct during a specific effort, such as the 1500-m speed skating competition. The purpose of the present study was to "override" self-paced (SP) performance by instructing athletes to execute a theoretically optimal pacing profile. METHODS: Seven national-level speed-skaters performed a SP 1500-m which was analysed by obtaining velocity (every 100 m) and body position (every 200 m) with video to calculate total mechanical power output. Together with gross efficiency and aerobic kinetics, obtained in separate trials, data were used to calculate aerobic and anaerobic power output profiles. An energy flow model was applied to SP, simulating a range of pacing strategies, and a theoretically optimal pacing profile was imposed in a second race (IM). RESULTS: Final time for IM was â¼2 s slower than SP. Total power distribution per lap differed, with a higher power over the first 300 m for IM (637.0 (49.4) vs 612.5 (50.0) W). Anaerobic parameters did not differ. The faster first lap resulted in a higher aerodynamic drag coefficient and perhaps a less effective push-off. CONCLUSION: Experienced athletes have a well-developed performance template, and changing pacing strategy towards a theoretically optimal fast start protocol had negative consequences on speed-skating technique and did not result in better performance.
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Rendimiento Atlético/fisiología , Patinación/fisiología , Metabolismo Energético/fisiología , Fricción , Humanos , Modelos Biológicos , Consumo de Oxígeno/fisiología , Adulto JovenRESUMEN
The development of multidrug resistance (MDR) and subsequent relapse on therapy is a widespread problem in breast cancer, but our understanding of the underlying molecular mechanisms is incomplete. Numerous studies have aimed to establish the role of drug transporter pumps in MDR and to link their expression to response to chemotherapy. The ATP-binding cassette (ABC) transporters are central to breast cancer MDR, and increases in ABC expression levels have been shown to correlate with decreases in response to various chemotherapy drugs and a reduction in overall survival. But as there is a large degree of redundancy between different ABC transporters, this correlation has not been seen in all studies. This paper provides an introduction to the key molecules associated with breast cancer MDR and summarises evidence of their potential roles reported from model systems and clinical studies. We provide possible explanations for why despite several decades of research, the precise role of ABC transporters in breast cancer MDR remains elusive.
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Adenosina Trifosfatasas , Reparación del ADN , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células Eucariotas , Genoma Bacteriano , Humanos , Modelos Moleculares , Proteína MutS de Unión a los Apareamientos Incorrectos del ADNRESUMEN
We studied the morphogenesis of three pseudorabies virus mutants lacking parts of the gene homologous to the UL21 gene of the herpes simplex virus type 1. The mutants were examined in an SK-6 cell-line, in an SK-6 cell-line expressing the UL21 gene product, in porcine lung alveolar macrophages (PLAM) and in porcine nasal mucosa explants. Although on SK-6 cells and PLAM, the virus-assembly and egress of mutant virus M155, lacking almost the entire UL21 gene, was similar to that of the rescued PRV mutant, M155 producing virions containing little or no DNA (A-type particles). Virus mutants M133 and M134 (lacking 23 and 232 amino acids respectively) produced more C-type particles. In SK-6 cells stably expressing the UL21-encoded protein, all mutants produced C-type particles. All mutants produced C-type particles in nasal mucosa explants, indicating that the UL21-gene product is not essential for virus production in porcine tissue. These results support and extend previous work that indicated a role for the UL21 encoded protein in the packaging of newly replicated viral DNA.
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Proteínas de la Cápside , Cápside/genética , Cápside/fisiología , ADN Viral/genética , Herpesvirus Suido 1/genética , Animales , Cápside/ultraestructura , Células Cultivadas , ADN Viral/ultraestructura , Genes Virales , Herpesvirus Suido 1/ultraestructura , Microscopía Electrónica/veterinaria , Mutación , PorcinosRESUMEN
Mismatch repair (MMR) deficiency, which underlies hereditary nonpolyposis colorectal cancer, has recently been linked to a number of sporadic human cancers as well. Deficiency in this repair process renders cells resistant to many clinically active chemotherapy agents. As a result, it is of relevance to find an agent that selectively targets MMR-deficient cells. We have recently shown that the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) selectively target MutL homologue-1 (MLH1)-deficient human cancer cells for radiosensitization. The levels of IdUrd and BrdUrd in cellular DNA directly correlate with the ability of these analogues to increase the sensitivity of cells and tissues to ionizing radiation, and data from our laboratory have demonstrated that MLH1-mediated MMR status impacts dThd analogue DNA levels, and consequently, analogue-induced radiosensitization. Here, we have extended these studies and show that, both in human and murine cells, MutS homologue-2 (MSH2) is also involved in processing dThd analogues in DNA. Using both E1A-transformed Msh2+/+ and Msh2-/- murine embryonic stem (ES)-derived cells (throughout this report we use Msh2+/+ and Msh2-/- to refer to murine ES-derived cell lines that are wild type or mutant, respectively, for the murine Msh2 gene) and human endometrial cancer cells differing in MSH2 status, we see the classic cytotoxic response to 6-thioguanine (6-TG) in Msh2+/+ and human HEC59/2-4 (MSH2+) MMR-proficient cells, whereas Msh2-/- cells and human HEC59 (MSH2-/-) cells are tolerant (2-log difference) to this agent. In contrast, there is very little cytotoxicity in Msh2+/+ ES-derived and HEC59/2-4 cells to IdUrd, whereas Msh2-/- and HEC59 cells are more sensitive to IdUrd. High-performance liquid chromatography analysis of IdUrd and BrdUrd levels in DNA suggests that this differential cytotoxicity may be due to lower analogue levels in MSH2+ murine and human tumor cells. The DNA levels of IdUrd and BrdUrd continue to decrease over time in Msh2+/+ cells following incubation in drug-free medium, whereas they remain high in Msh2-/- cells. This trend was also found in MSH2-deficient human endometrial cancer cells (HEC59) when compared with HEC59/2-4 (hMsh2-corrected) cells. As a result of higher analogue levels in DNA, Msh2-/- cells are selectively targeted for radiosensitization by IdUrd. Fluorescence-activated cell-sorting analysis of Msh2+/+ and Msh2-/- cells shows that selective toxicity of the halogenated nucleotide analogues is not correlated with a G2-M cell cycle arrest and apoptosis, as is found for selective killing of Msh2+/+ cells by 6-TG. Together, these data demonstrate MSH2 involvement in the processing of IdUrd and BrdUrd in DNA, as well as the differential cytotoxicity and cell cycle effects of the halogenated dThd analogues compared with 6-TG. Therefore, IdUrd and BrdUrd may be used clinically to selectively target both MLH1- and MSH2-deficient, drug-resistant cells for radiosensitization.
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Antimetabolitos Antineoplásicos/farmacología , Bromodesoxiuridina/farmacología , Proteínas de Unión al ADN , ADN/metabolismo , Idoxuridina/farmacología , Proteínas Proto-Oncogénicas/fisiología , Fármacos Sensibilizantes a Radiaciones/farmacología , Tioguanina/farmacología , Proteínas E1A de Adenovirus/genética , Animales , Disparidad de Par Base , Bromodesoxiuridina/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Transformada , ADN/genética , Reparación del ADN , Nucleótidos de Desoxicitosina/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Idoxuridina/metabolismo , Cinética , Ratones , Ratones Noqueados , Proteína 2 Homóloga a MutS , Proteínas Proto-Oncogénicas/genética , Nucleótidos de Timina/metabolismoRESUMEN
DNA mismatch repair ensures genomic integrity on DNA replication. Recognition of a DNA mismatch by a dimeric MutS protein initiates a cascade of reactions and results in repair of the newly synthesized strand; however, details of the molecular mechanism remain controversial. Here we present the crystal structure at 2.2 A of MutS from Escherichia coli bound to a G x T mismatch. The two MutS monomers have different conformations and form a heterodimer at the structural level. Only one monomer recognizes the mismatch specifically and has ADP bound. Mismatch recognition occurs by extensive minor groove interactions causing unusual base pairing and kinking of the DNA. Nonspecific major groove DNA-binding domains from both monomers embrace the DNA in a clamp-like structure. The interleaved nucleotide-binding sites are located far from the DNA. Mutations in human MutS alpha (MSH2/MSH6) that lead to hereditary predisposition for cancer, such as hereditary non-polyposis colorectal cancer, can be mapped to this crystal structure.
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Proteínas Bacterianas/fisiología , Disparidad de Par Base , Reparación del ADN , ADN Bacteriano/química , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Cristalografía por Rayos X , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Dimerización , Escherichia coli/química , Escherichia coli/metabolismo , Guanina/metabolismo , Humanos , Hidrólisis , Modelos Moleculares , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Proteína 2 Homóloga a MutS , Mutación , Conformación de Ácido Nucleico , Conformación Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Timina/metabolismoRESUMEN
Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSalpha (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSbeta (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3 ) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6 -deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3. Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.
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Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Eliminación de Gen , Predisposición Genética a la Enfermedad/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Alelos , Animales , Disparidad de Par Base/genética , Muerte Celular/efectos de los fármacos , Neoplasias Colorrectales Hereditarias sin Poliposis/metabolismo , Neoplasias Colorrectales Hereditarias sin Poliposis/mortalidad , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Femenino , Incidencia , Masculino , Metilnitronitrosoguanidina/toxicidad , Ratones , Ratones Transgénicos , Proteína 3 Homóloga de MutS , Mutagénesis Insercional , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Tasa de Supervivencia , Factores de TiempoRESUMEN
To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vlambda1 light chain genes from naive and memory B cells in DNA repair-deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand-break repair, and AAG-mediated base excision repair.
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Linfocitos B/inmunología , Reparación del ADN/fisiología , Reordenamiento Génico de Linfocito B , Genes de Inmunoglobulinas , Memoria Inmunológica/inmunología , Mutación , Animales , Región Variable de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Ratones , Ratones Mutantes , Reacción en Cadena de la PolimerasaRESUMEN
Hemizygous germ-line defects in mismatch repair (MMR) genes underlie hereditary nonpolyposis colorectal cancer (HNPCC). Loss of the wild-type allele results in a mutator phenotype, accelerating tumorigenesis. Tumorigenesis specifically occurs in the gastrointestinal and genitourinary tracts; the cause of this tissue specificity is elusive. To understand the etiology and tissue distribution of tumors in HNPCC, we have developed mouse models carrying a deficiency in the MMR gene Msh2. Most of the completely Msh2-deficient mice succumbed to lymphomas at an early age; lymphomagenesis was synergistically enhanced by exposure to ethylnitrosourea. Lymphomas were absent in immunocompromised Tap1-/-;Msh2-/- mice; these mice generally succumbed to HNPCC-like tumors. Together, these data suggest that the HNPCC tumor spectrum is determined by exposure of MMR-deficient cells to exogenous mutagens, rather than by tissue-specific loss of the wild-type MMR allele or by immune surveillance. Msh2 hemizygous mice had an elevated tumor incidence that, surprisingly, was rarely correlated with loss of the Msh2+ allele. To develop a model for intestinal tumorigenesis in HNPCC, we introduced the Min allele of the Apc tumor suppressor gene. We observed loss of the wild-type Msh2 allele in a significant fraction of intestinal tumors in Apc+/Min;Msh2+/- mice. In some of the latter tumors, one area of the tumor displayed loss of the Msh2+ allele, but not of the Apc+ allele, whereas another area displayed the inverse genotype. This apparent biclonality might indicate a requirement for collaboration between independent tumor clones during intestinal tumorigenesis.
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Neoplasias Colorrectales Hereditarias sin Poliposis/etiología , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Modelos Animales de Enfermedad , Proteína de la Poliposis Adenomatosa del Colon , Animales , Células Clonales , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/genética , Etilnitrosourea/farmacología , Femenino , Eliminación de Gen , Huésped Inmunocomprometido , Pérdida de Heterocigocidad , Linfoma/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Proteína 2 Homóloga a MutS , Proteínas Proto-Oncogénicas/genética , Tasa de SupervivenciaRESUMEN
The study of genome structure and gene function is pivotal in understanding the mechanisms of replication, pathogenesis, and virulence of herpesviruses. In this respect, mutagenesis and sequence analysis of genes encoded by the virus are of great importance. However, the herpesvirus genomes are large, with sizes ranging between 120 and over 200 kbp and encoding between 70 and 200 genes (see ref. 1 for a review). This large size hampers handling and systematic mutagenesis of the virus genome using standard modern molecular biology techniques. Most current methods of mutagenesis therefore do not rely on direct modification of the viral genome in vitro but depend on exchange in vivo, by homologous recombination, of a viral gene by a copy of the latter gene that is truncated in vitro by insertion of a marker gene. Mutant virus progeny can be screened or selected for, depending on the marker gene that is used. Commonly used marker genes are thymidine kinase and lacZ. This procedure is generally used, reliable, and has yielded a wealth of information on the function of herpers simplex virus type 1 (HSV-1) encoded genes. However, it requires prior mapping and cloning of every gene to be mutagenized and is therefore less feasible if the virus is a novel or less-well-known herpesvirus.
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The glycoprotein E (gE) locus in the genome of pseudorabies virus (PRV) was used as an insertion site for the expression of glycoprotein E1 of classical swine fever virus (CSFV). Transcription of E1 in the recombinants M401, M402 or M403 was regulated by the gD promoter of PRV, the immediate early gene promoter of human cytomegalovirus, or the gE promoter of PRV, respectively. Groups of four pigs were vaccinated once intramuscularly with 10(6) plaque forming units (p.f.u.) of the recombinant viruses and challenged intranasally with 100 50% lethal doses of virulent CSFV and with 10(5) p.f.u. of virulent PRV. All pigs vaccinated with M402 were fully protected against both classical swine fever and pseudorabies.
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Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Regulación Viral de la Expresión Génica/inmunología , Herpesvirus Suido 1/inmunología , Regiones Promotoras Genéticas/inmunología , Seudorrabia/prevención & control , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Peste Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/patogenicidad , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/patogenicidad , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Seudorrabia/inmunología , Porcinos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , VirulenciaRESUMEN
We examined the influence of inactivation of various genes located in the unique short (U(S)) region of pseudorabies virus on virus replication and assembly in porcine nasal mucosa explant cultures. The following strains were used: the virulent wild-type strain NIA-3, and strains derived from NIA-3 containing a mutation inactivating the genes encoding either the US3-encoded protein kinase (PK), gG, gD, gI, gE, the 28 kDa ('28K') protein (single mutant), or the 28K and 11 kDa ('11K') proteins (double mutant). In addition a wild-type rescuant was used, which was generated by marker rescue from a PK- mutant. All virus strains infected nasal epithelium and had invaded the stroma after approximately 24 h. The morphogenesis in nasal epithelium cells of two PK- mutants showed the most striking differences compared to the parent NIA-3 strain and the other mutant strains. The changes could be ascribed to the US3-encoded PK because the rescue mutant showed a similar morphogenesis to wild-type NIA-3. The transmembrane transport of the PK- mutants was impaired at the outer nuclear membrane which resulted in an accumulation of virions in the perinuclear space. These results suggest that proteins, phosphorylated by the US3-encoded PK, are involved in debudding of virus particles at the outer nuclear membrane. This defect in the transport of the US3 mutant probably explains their reduced replication in vitro. The gG-, gD-, gI-, gE-, 28K- and 11K- mutant strains showed minor or no changes in viral assembly. Thus the reported decreased virulence of the gD-, gI- and gE- mutants was, in contrast to that of the PK- mutants, not associated with clear alterations in morphogenesis.
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Núcleo Celular/genética , Núcleo Celular/virología , Herpesvirus Suido 1/enzimología , Herpesvirus Suido 1/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Núcleo Celular/enzimología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Herpesvirus Suido 1/ultraestructura , Mutagénesis Insercional , Mucosa Nasal/ultraestructura , Mucosa Nasal/virología , Proteínas Serina-Treonina Quinasas/fisiología , Porcinos , Proteínas Virales , Virión/efectos de los fármacosRESUMEN
To investigate the role of the presumed DNA mismatch repair (MMR) gene Msh2 in genome stability and tumorigenesis, we have generated cells and mice that are deficient for the gene. Msh2-deficient cells have lost mismatch binding and have acquired microsatellite instability, a mutator phenotype, and tolerance to methylating agents. Moreover, in these cells, homologous recombination has lost dependence on complete identity between interacting DNA sequences, suggesting that Msh2 is involved in safeguarding the genome from promiscuous recombination. Msh2-deficient mice display no major abnormalities, but a significant fraction develops lymphomas at an early age. Thus, Msh2 is involved in MMR, controlling several aspects of genome stability; loss of MMR-controlled genome stability predisposes to cancer.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/genética , ADN/genética , Proteínas Fúngicas , Linfoma/genética , Recombinación Genética , Animales , Composición de Base , Secuencia de Bases , Quimera , ADN/metabolismo , Predisposición Genética a la Enfermedad , Metilación , Metilnitronitrosoguanidina/farmacología , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Proteína 2 Homóloga a MutS , Mutagénesis , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Células Madre/citología , Células Madre/fisiologíaRESUMEN
To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.
Asunto(s)
Antígenos Virales/inmunología , Herpesvirus Suido 1/inmunología , Animales , Antígenos Virales/genética , Línea Celular , Herpesvirus Suido 1/enzimología , Herpesvirus Suido 1/crecimiento & desarrollo , Mutación , Pruebas de Neutralización , Proteínas Quinasas/inmunología , Seudorrabia/inmunología , Porcinos , Enfermedades de los Porcinos/inmunología , Timidina Quinasa/inmunología , Proteínas del Envoltorio Viral/inmunología , Cultivo de Virus , Replicación Viral/inmunología , Esparcimiento de Virus/inmunologíaRESUMEN
Bacterial MutS protein and its yeast and human homologs MSH2 trigger the mismatch repair process by their initial binding to mispaired and unpaired bases in DNA. We describe the cloning and sequencing of genes from Xenopus laevis and Mus musculus encoding the homolog of the Saccharomyces cerevisiae MSH2 (the major DNA mismatch binding protein). Mutations in the human homolog of this gene have recently been implicated in microsatellite instability and DNA mismatch repair deficiency in tumour cells from patients with the most common hereditary predisposition to cancer (Lynch syndrome, or hereditary non-polyposis colorectal cancer, HNPCC), as well as in a significant percentage of sporadic tumours. Expression of the amphibian and murine Msh2 gene in different tissues appears to be ubiquitous. The Xenopus gene is highly expressed in eggs, a model system for the biochemistry of DNA mismatch repair. Expression of the murine gene is low in all tissues examined, and is relatively high in a rapidly dividing cell line. These data are suggestive of a role for MSH2 during DNA replication.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/química , Expresión Génica , Genes , Secuencias Hélice-Asa-Hélice , Ratones , Datos de Secuencia Molecular , Proteína 2 Homóloga a MutS , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Distribución Tisular , Xenopus laevisRESUMEN
We mutagenized and characterized a 41-kilobase-pair subgenomic cloned fragment from the unique long (UL) region of pseudorabies virus (PRV). Forty mutant clones, each carrying a single inserted oligonucleotide, were used for cotransfection with overlapping cloned viral DNA fragments. More than half of these transfections yielded viable virus mutants. Short viral DNA fragments, flanking the oligonucleotide insertions, were cloned and used as probes on Northern blots with RNA isolated from cells infected with wild-type PRV. In this way we were able to construct a partial transcriptional map of this region of the PRV genome. In addition, we used these probes in cross-hybridization studies with cloned genomic fragments from the prototype alphaherpesvirus herpes simplex virus type 1 (HSV-1). This allowed us to define homology between the corresponding regions of these viruses. Most viable PRV mutants were assayed for virulence in mice. Mutagenesis of the identified homologs of HSV-1 genes UL39 (encoding the large subunit of ribonucleotide reductase), UL40 (small subunit of ribonucleotide reductase), UL42 (DNA polymerase accessory factor), UL23 (thymidine kinase), UL21 (a capsid-associated protein), and UL12 (alkaline nuclease) completely abrogated or strongly reduced virulence.