RESUMEN
Objective: Using bioinformatics methods to analyze the core pathogenic genes and related pathways in elderly osteoporosis. Methods: From November 2020 and August 2021, eight elderly osteoporosis patients who received treatment and five healthy participants who underwent physical examinations in Beijing Jishuitan Hospital were selected as subjects. The expression level of RNA in the peripheral blood of eight elderly osteoporosis patients and five healthy participants was collected for high-throughput transcriptome sequencing and analysis. The gene ontology (GO) analysis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed for the differentially expressed genes (DEGs). The protein-protein interaction (PPI) network was constructed using the STRING website and Cytoscape software, and the most significant modules and hub genes were screened out. Results: Among the eight elderly osteoporosis patients, there were seven females and one male, with an average age of 72.4 years (SD=4.2). Among the five healthy participants, there were four females and one male, with an average age of 68.2 years (SD=5.7). A total of 1 635 DEGs (847 up-regulated and 788 down-regulated) were identified. GO analysis revealed that the molecular functions of DEGs were mainly enriched in structural constituents of the ribosome, protein dimerization activity, and cellular components were mainly enriched in the nucleosome, DNA packaging complex, cytosolic part, protein-DNA complex and the cytosolic ribosome. KEGG pathway analysis showed that DEGs were mainly enriched in systemic lupus erythematosus and ribosome. Gene UBA52, UBB, RPS27A, RPS15, RPS12, RPL13A, RPL23A, RPL10A, RPS25 and RPS6 were selected and seven of them could encode ribosome proteins. Conclusion: The pathogenesis of elderly osteoporosis may be associated with ribosome-related genes and pathways.
Asunto(s)
Perfilación de la Expresión Génica , Osteoporosis , Femenino , Humanos , Masculino , Anciano , Perfilación de la Expresión Génica/métodos , Transcriptoma , Mapas de Interacción de Proteínas/genética , Biología Computacional/métodos , Osteoporosis/genéticaRESUMEN
OBJECTIVE: To explore the mechanism of dexmedetomidine (DEX)-mediated miR-134 inhibition in hypoxia-induced damage in PC12 cells. METHODS: Hydrogen peroxide (H2O2)-stimulated PC12 cells were divided into control, H2O2, DEX + H2O2, miR-NC/inhibitor + H2O2, and miR-NC/ mimic + DEX + H2O2 groups. Cell viability and apoptosis were assessed by the 3-(4,5-dimethylthiazol(-2-y1)-2,5-diphenytetrazolium bromide (MTT) assay and Annexin V-FITC/PI staining, while gene and protein expression levels were detected by qRT-PCR and western blotting. Reactive oxygen species (ROS) levels were tested by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and malondialdehyde (MDA) content was determined with a detection kit. RESULTS: DEX treatment decreased H2O2-elevated miR-134 expression. H2O2-induced PC12 cell damage was improved by DEX and miR-134 inhibitor; additionally, cell viability was increased, while cell apoptosis was reduced. In addition, both DEX and miR-134 inhibitor reduced the upregulated expression of cleaved caspase-3 and increased the downregulated expression of Bcl-2 in H2O2-induced PC12 cells. However, compared to that in the DEX + H2O2 group, cell viability in the mimic + DEX + H2O2 group was decreased, and the apoptotic rate was elevated with increased cleaved caspase-3 and decreased Bcl-2 expression. Inflammation and oxidative stress were increased in H2O2-induced PC12 cells but improved with DEX or miR-134 inhibitor treatment. However, this improvement of H2O2-induced inflammation and oxidative stress induced by DEX in PC12 cells could be reversed by the miR-134 mimic. CONCLUSION: DEX exerts protective effects to promote viability and reduce cell apoptosis, inflammation, and oxidative stress in H2O2-induced PC12 cells by inhibiting the expression of miR-134.
Asunto(s)
Hipoxia de la Célula/efectos de los fármacos , Dexmedetomidina/farmacología , MicroARNs/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Interleukin-15 is a pleiotropic cytokine that is critical for the development and survival of multiple haematopoietic lineages. Mice lacking IL-15 have selective defects in populations of several pro-allergic immune cells including natural killer (NK) cells, NKT cells, and memory CD8+ T cells. We therefore hypothesized that IL-15-/- mice will have reduced inflammatory responses during the development of allergic airway disease (AAD). OBJECTIVE: To determine whether IL-15-/- mice have attenuated allergic responses in a mouse model of AAD. METHODS: C57BL/6 wild-type (WT) and IL-15-/- mice were sensitized and challenged with ovalbumin (OVA), and the development of AAD was ascertained by examining changes in airway inflammatory responses, Th2 responses, and lung histopathology. RESULTS: Here, we report that IL-15-/- mice developed enhanced allergic responses in an OVA-induced model of AAD. In the absence of IL-15, OVA-challenged mice exhibited enhanced bronchial eosinophilic inflammation, elevated IL-13 production, and severe lung histopathology in comparison with WT mice. In addition, increased numbers of CD4+ T and B cells in the spleens and bronchoalveolar lavage (BAL) were also observed. Examination of OVA-challenged IL-15Rα-/- animals revealed a similar phenotype resulting in enhanced airway eosinophilia compared to WT mice. Adoptive transfer of splenic CD8+ T cells from OVA-sensitized WT mice suppressed the enhancement of eosinophilia in IL-15-/- animals to levels observed in WT mice, but had no further effects. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that mice with an endogenous IL-15 deficiency are susceptible to the development of severe, enhanced Th2-mediated AAD, which can be regulated by CD8+ T cells. Furthermore, the development of disease as well as allergen-specific Th2 responses occurs despite deficiencies in several IL-15-dependent cell types including NK, NKT, and γδ T cells, suggesting that these cells or their subsets are dispensable for the induction of AAD in IL-15-deficient mice.
Asunto(s)
Alérgenos/toxicidad , Asma/inmunología , Linfocitos T CD8-positivos/inmunología , Interleucina-15/deficiencia , Células Th2/inmunología , Animales , Asma/inducido químicamente , Asma/genética , Asma/patología , Linfocitos T CD8-positivos/patología , Interleucina-15/inmunología , Ratones , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/patología , Células Th2/patologíaRESUMEN
OBJECTIVE: To explore the interaction between passive smoking and folic acid supplement during pregnancy on children autism spectrum disorder(ASD)behaviors. METHODS: Children aged about 3 years were enrolled at kindergarten entrance in Longhua district of Shenzhen in 2014. Self-administered questionnaires were completed by their primary caregivers and the information about children' s age, gender, history of preterm birth and low birth weight, parents' education level, parents' reproductive age and family income were collected. The children ASD behaviors were assessed with Autism Behavior Checklist(ABC). According to the cut point of ABC, the children were divided into normal group with score less than 31, sub-clinical group with score ranging from 31 to 61 and suspect clinical group with score no less than 62. After controlling for potential confounders, multivariate logistic regression analysis was performed to evaluate the main effects and the interaction between passive smoking and folic acid supplement during pregnancy on children ASD behaviors. RESULTS: Maternal passive smoking during pregnancy was significantly associated with children ASD behaviors(sub-clinical group: OR=1.48; suspect clinical group: OR= 2.85), and maternal folic acid supplement during pregnancy was not related to children ASD behaviors(sub-clinical group: OR=1.04; suspect clinical group: OR=0.75). Stratified analysis showed that folic acid supplement during pregnancy was negatively associated with children ASD behaviors(suspect clinical group: OR=0.30)among children without mothers' passive smoking during pregnancy, and that mothers' passive smoking during pregnancy was positively associated with children ASD behaviors(sub-clinical group: OR=1.52; suspect clinical group: OR=4.45)among the children whose mothers had folic acid supplement during pregnancy. Furthermore, an interaction effect on children ASD behaviors was found between passive smoking and folic acid supplement during pregnancy(suspect clinical group: OR=5.30). CONCLUSION: Passive smoking and folic acid supplement during pregnancy were related to children ASD behaviors and had an interaction on children ASD behaviors.
Asunto(s)
Trastorno del Espectro Autista/etiología , Desarrollo Fetal/efectos de los fármacos , Ácido Fólico/administración & dosificación , Complicaciones del Embarazo/fisiopatología , Fumar/efectos adversos , Contaminación por Humo de Tabaco , Trastorno del Espectro Autista/epidemiología , Preescolar , Suplementos Dietéticos , Femenino , Homocisteína/sangre , Humanos , Recién Nacido de Bajo Peso , Recién Nacido , Padres , Embarazo , Nacimiento Prematuro , Encuestas y CuestionariosRESUMEN
Antiviral defense of plants is usually enhanced by an elevated temperature under natural conditions. In order to better understand this phenomenon, we carried out temperature shift experiments with Nicotiana glutinosa plants that were infected with Potato virus X (PVX) or the necrotic strain of Potato virus Y (PVY(N)). The virus titer of the plants was found to be much lower when they were maintained at 30°C compared with 22°C, particularly in the upper leaves. PVX resistance at 30°C persisted for a short period even when temperature was shifted back to 22°C. In contrast, N. benthamiana lost the virus resistance immediately after the temperature dropped to 22°C. Expression analysis of two RNA-dependent RNA polymerases in N. glutinosa (NgRDR) showed that a 12-day treatment at 30°C increased the expression of NgRDR1, while NgRDR6 was not affected. In addition, the NgRDR6 mRNA level correlated with the PVX titer but was unaffected by PVY(N) infection. These observations indicate that PVX and PVY(N), although they are both RNA viruses, might trigger different defense responses at elevated temperatures. Our study provides valuable data for a better understanding of the temperature-regulated host virus interaction.
Asunto(s)
Calor , Nicotiana/genética , Enfermedades de las Plantas/virología , Potexvirus , Potyvirus , Enfermedades de las Plantas/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nicotiana/virologíaRESUMEN
BACKGROUND: Natural Killer (NK) cells have been implicated in the development of allergic airway inflammation. However, the in vivo role of NK cells has not been firmly established due to the lack of animal models with selective deficiencies in NK cells. OBJECTIVE: To determine the specific contribution of NK cells in a murine model of allergic airway disease (AAD). METHODS: The role of NK cells in AAD was studied using NK-deficient (NKD) mice, perforin(-/-) mice, and mice depleted of Ly49A/D/G(+) NK cell subsets in an ovalbumin-induced model of allergic airway disease (OVA-AAD). RESULTS: Induction of OVA-AAD in C57BL/6 wild-type (WT) mice resulted in the expansion of airway NK cells and the development of pronounced airway eosinophilia. In the absence of NK cells or specific subsets of NK cells, either in NKD mice, or after the depletion of Ly49A/D/G(+) NK cells, the development of OVA-AAD was significantly impaired as seen by decreased airway inflammation and eosinophilia, decreased secretion of the Th2 cytokines IL-4, IL-5 and IL-13 and diminished OVA-specific antibody production. Furthermore, while OVA-exposure induced a dramatic expansion of dendritic cells (DCs) in WT mice, their induction was significantly attenuated in NKD mice. Development of OVA-AAD in perforin(-/-) mice suggested that the proinflammatory role of NK cells is not dependent on perforin-mediated cytotoxicity. Lastly, induction of allergic disease by OVA-specific CD4 T cells from WT but not NK-depleted or NKD mice in RAG(-/-) recipients, demonstrates that NK cells are essential for T cell priming. CONCLUSIONS AND CLINICAL RELEVANCE: Our data demonstrate that conventional NK cells play an important and distinct role in the development of AAD. The presence of activated NK cells has been noted in patients with asthma. Understanding the mechanisms by which NK cells regulate allergic disease is therefore an important component of treatment approaches.
Asunto(s)
Células Asesinas Naturales/inmunología , Hipersensibilidad Respiratoria/inmunología , Traslado Adoptivo , Animales , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Eosinofilia/inmunología , Inflamación/inmunología , Células Asesinas Naturales/metabolismo , Pulmón/inmunología , Pulmón/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Ovalbúmina/inmunología , Hipersensibilidad Respiratoria/patología , Hipersensibilidad Respiratoria/terapia , Bazo/inmunologíaRESUMEN
Changes in chromosome number and structure are important contributors to adaptation, speciation and macroevolution. In flowering plants, polyploidy and subsequent reductions in chromosome number by fusion are major sources of chromosomal evolution, but chromosome number increase by fission has been relatively unexplored. Here, we use comparative linkage mapping with gene-based markers to reconstruct chromosomal synteny within the model flowering plant genus Mimulus (monkeyflowers). Two sections of the genus with haploid numbers ≥ 14 have been inferred to be relatively recent polyploids because they are phylogenetically nested within numerous taxa with low base numbers (n=8-10). We combined multiple data sets to build integrated genetic maps of the M. guttatus species complex (section Simiolus, n=14) and the M. lewisii group (section Erythranthe; n=8), and then aligned the two integrated maps using >100 shared markers. We observed strong segmental synteny between M. lewisii and M. guttatus maps, with essentially 1-to-1 correspondence across each of 16 chromosomal blocks. Assuming that the M. lewisii (and widespread) base number of 8 is ancestral, reconstruction of 14 M. guttatus chromosomes requires at least eight fission events (likely shared by Simiolus and sister section Paradanthus (n=16)), plus two fusion events. This apparent burst of fission in the yellow monkeyflower lineages raises new questions about mechanisms and consequences of chromosomal fission in plants. Our comparative maps also provide insight into the origins of a chromosome exhibiting centromere-associated female meiotic drive and create a framework for transferring M. guttatus genome resources across the entire genus.
Asunto(s)
Aneuploidia , Mapeo Cromosómico/métodos , Cromosomas de las Plantas/genética , Mimulus/genética , Poliploidía , Centrómero/genética , Evolución Molecular , Haploidia , Mimulus/clasificación , Especificidad de la Especie , SinteníaRESUMEN
Progesterone receptor membrane component-1 (PGRMC1) is present in both the cytoplasm and nucleus of spontaneously immortalized granulosa cells (SIGCs). PGRMC1 is detected as a monomer in the cytoplasm and a DTT-resistant PGRMC1 dimer in the nucleus. Transfected PGRMC1-GFP localizes mainly to the cytoplasm and does not form a DTT-resistant dimer. Moreover, forced expression of PGRMC1-GFP increases the sensitivity of the SIGCs to progesterone (P4)'s anti-apoptotic action, indicating that the PGRMC1 monomer is functional. However, when endogenous PGRMC1 is depleted by siRNA treatment and replaced with PGRMC1-GFP, P4 responsiveness is not enhanced, although overall levels of PGRMC1 are increased. P4's anti-apoptotic action is also attenuated by actinomycin D, an inhibitor of RNA synthesis, and P4 activation of PGRMC1 suppresses Bad and increases Bcl2a1d expression. Taken together, the present studies suggest a genomic component to PGRMC1's anti-apoptotic mechanism of action, which requires the presence of the PGRMC1 dimer.
Asunto(s)
Apoptosis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Progesterona/farmacología , Receptores de Progesterona/metabolismo , Animales , Western Blotting , Línea Celular Transformada , Dactinomicina/farmacología , Femenino , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The plant genus Mimulus is rapidly emerging as a model system for studies of evolutionary and ecological functional genomics. Mimulus contains a wide array of phenotypic, ecological and genomic diversity. Numerous studies have proven the experimental tractability of Mimulus in laboratory and field studies. Genomic resources currently under development are making Mimulus an excellent system for determining the genetic and genomic basis of adaptation and speciation. Here, we introduce some of the phenotypic and genetic diversity in the genus Mimulus and highlight how direct genetic studies with Mimulus can address a wide spectrum of ecological and evolutionary questions. In addition, we present the genomic resources currently available for Mimulus and discuss future directions for research. The integration of ecology and genetics with bioinformatics and genome technology offers great promise for exploring the mechanistic basis of adaptive evolution and the genetics of speciation.
Asunto(s)
Mimulus/genética , Evolución Biológica , Ecología , Ecosistema , Genoma , Mimulus/anatomía & histología , Mimulus/fisiologíaRESUMEN
Progesterone (P4) inhibits apoptosis of rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs), which were derived from rat granulosa cells. Defining the mechanism through which P4 mediates its action has been difficult because these cells do not express the classic nuclear P4 receptor. Previous studies have shown that a P4 receptor antibody, C-262, detects a 60-kDa protein that is involved in regulating P4's antiapoptotic action. Using a C-262 affinity column, this 60-kDa protein was isolated and sequenced by mass spectrometry. This analysis revealed that the C-262-detectable protein is an unnamed protein referred to as RDA288. This protein has several putative hyaluronic acid binding sites. Further hyaluronic acid antagonizes (3)H-P4 binding to SIGCs and mimics P4's action, whereas exogenous hyaluronic acid binding protein attenuates P4's actions. RT-PCR demonstrated that RDA288 mRNA was present in SIGCs, immature rat ovary, lung, and skeletal muscle but was not present in several other organs. Forced expression of RDA288 increased the capacity of SIGCs to bind and respond to P4. An antibody was also developed against RDA288. Using this antibody in a Western blot protocol, RDA288 expression was confirmed in both SIGCs and granulosa cells. An immunohistochemical study detected RDA288 in the cytoplasm and plasma membrane components of granulosa cells of antral follicles. Immunocytochemical studies on living nonpermeabilized SIGCs revealed that RDA288 was present on the extracellular surface of the plasma membrane. Finally, pretreatment with the RDA288 antibody blocked P4's antiapoptotic actions. Taken together, these data suggest that RDA288 plays a significant role in mediating P4's antiapoptotic action in granulosa cells.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/fisiología , Células de la Granulosa/fisiología , Progesterona/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Espacio Extracelular , Femenino , Inmunohistoquímica , Datos de Secuencia Molecular , Proteínas de Unión al ARN , Ratas , Ratas Wistar , Distribución TisularRESUMEN
One cause of reproductive isolation is gamete competition, in which conspecific pollen has an advantage over heterospecific pollen in siring seeds, thereby decreasing the formation of F1 hybrids. Analogous pollen interactions between hybrid pollen and conspecific pollen can contribute to post-zygotic isolation. The herbaceous plants Ipomopsis aggregata and I. tenuituba frequently hybridize in nature. Hand-pollination of I. aggregata with pollen from F1 or F2 hybrids produced as many seeds as hand-pollination with conspecific pollen, suggesting equal pollen viability. However, when mixed pollen loads with 50% conspecific pollen and 50% hybrid pollen were applied to I. aggregata stigmas, fewer than half of the seeds had hybrid sires. Such pollen mixtures are frequently received if plants of the two species and F1 and F2 hybrids are intermixed, suggesting that this advantage of conspecific over hybrid pollen reduces backcrossing and contributes to reproductive isolation.
Asunto(s)
Hibridación Genética/fisiología , Magnoliopsida/fisiología , Polen/fisiología , Semillas/fisiología , Colorado , Cruzamientos Genéticos , Fertilidad/genética , Magnoliopsida/genética , Reproducción/genéticaRESUMEN
Concomitant infection of murine CMV (MCMV), an opportunistic respiratory pathogen, altered Th1/Th2 cytokine expression, decreased bronchoalveolar lavage (BAL) fluid eosinophilia, and increased mucus production in a murine model of OVA-induced allergic airway disease. Although no change in the total number of leukocytes infiltrating the lung was observed between challenged and MCMV/challenged mice, the cellular profile differed dramatically. After 10 days of OVA-aerosol challenge, eosinophils comprised 64% of the total leukocyte population in BAL fluid from challenged mice compared with 11% in MCMV/challenged mice. Lymphocytes increased from 11% in challenged mice to 30% in MCMV/challenged mice, and this increase corresponded with an increase in the ratio of CD8(+) to CD4(+)TCRalphabeta lymphocytes. The decline in BAL fluid eosinophilia was associated with a change in local Th1/Th2 cytokine profiles. Enhanced levels of IL-4, IL-5, IL-10, and IL-13 were detected in lung tissue from challenged mice by RNase protection assays. In contrast, MCMV/challenged mice transiently expressed elevated levels of IFN-gamma and IL-10 mRNAs, as well as decreased levels of IL-4, IL-5, and IL-13 mRNAs. Elevated levels of IFN-gamma and reduced levels of IL-5 were also demonstrated in BAL fluid from MCMV/challenged mice. Histological evaluation of lung sections revealed extensive mucus plugging and epithelial cell hypertrophy/hyperplasia only in MCMV/challenged mice. Interestingly, the development of airway hyperresponsiveness was observed in challenged mice, not MCMV/challenged mice. Thus, MCMV infection can modulate allergic airway inflammation, and these findings suggest that enhanced mucus production may occur independently of BAL fluid eosinophilia.
Asunto(s)
Asma/complicaciones , Asma/inmunología , Infecciones por Citomegalovirus/complicaciones , Infecciones por Citomegalovirus/inmunología , Células TH1/inmunología , Células Th2/inmunología , Animales , Asma/patología , Asma/fisiopatología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/biosíntesis , Citocinas/genética , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/fisiopatología , Modelos Animales de Enfermedad , Eosinofilia/patología , Femenino , Humanos , Pulmón/inmunología , Pulmón/patología , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Moco/fisiología , Ovalbúmina/sangre , Ovalbúmina/genética , Ovalbúmina/inmunología , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
The murine cytomegalovirus (MCMV) monoclonal antibody 5C7:6 was used in Western analysis to probe MCMV infected murine embryo cells (MEC). This antibody recognizes three virus specific polypeptides of 130, 105, and 95 kDa and pulse-chase experiments demonstrated that these three proteins, although antigenically related, are distinct. The 105- and 95-kDa species were expressed with early kinetics, whereas the 130-kDa protein was synthesized as a true late. By screening a lambdagt11 MCMV cDNA library, the gene encoding these proteins was identified as the M25 open reading frame previously reported by Dallas et al. (Dallas, P. B., Lyons, P. A., Hudson, J. B., Scalzo, A. A., and Shellam, G. R., 1994, Virology 200, 643-650). Immunofluorescent studies monitored the location of pM25, present in the nucleus at 15 h after infection, condensing around the periphery of the nucleus at 18 h, before finally accumulating in the cytoplasm. Immunoelectron microscopy detected gold particles associated with the viral tegument of enveloped virions located in the cytoplasm and extracellular space but not with naked nucleocapsids. Western analysis of MCMV purified virions depicted the presence of the 130-kDa protein, the predominant M25 species, in mature virus particles. Together these findings provide compelling evidence that the 130-kDa M25 polypeptide is a component of the viral tegument.
Asunto(s)
Muromegalovirus/química , Muromegalovirus/genética , Sistemas de Lectura Abierta/genética , Proteínas del Envoltorio Viral/genética , Animales , Anticuerpos Monoclonales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Núcleo Celular/virología , Células Cultivadas , Clonación Molecular , Citoplasma/metabolismo , Citoplasma/ultraestructura , Citoplasma/virología , ADN Complementario/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/virología , Femenino , Regulación Viral de la Expresión Génica , Genes Virales/genética , Ratones , Ratones Endogámicos , Microscopía Inmunoelectrónica , Peso Molecular , Muromegalovirus/aislamiento & purificación , Muromegalovirus/ultraestructura , Nucleocápside/metabolismo , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Ensamble de VirusRESUMEN
Human herpesvirus-6 (HHV-6) exhibits a predominant tropism for CD4+ T-lymphocytes, but can infect other components of the blood as well as surrounding tissue and organs. To understand the role of the endothelium in the transmission and haematogenous spread of this virus, human umbilical vein endothelial cells (HUVEC) were infected with HHV-6 and monitored for viral gene expression. The presence of both early and late viral antigens was demonstrated by indirect immunofluorescence in 37.6 and 6.5%, respectively, of HUVEC. However, attempts to detect the release of infectious virus were not successful, indicating infection is semipermissive in nature. Upon continued passage of infected HUVEC monolayers, HHV-6 antigen-positive cells persisted up to 27 days post-infection. Furthermore, the virus could be recovered from HUVEC monolayers that contained fewer than 1% antigen-positive cells by co-cultivation with peripheral blood mononuclear cells. Together, these findings suggest that endothelial cells may serve as a reservoir for harbouring HHV-6.
Asunto(s)
Endotelio Vascular/virología , Herpesvirus Humano 6/fisiología , Antivirales/farmacología , Línea Celular , Replicación del ADN , Herpesvirus Humano 6/metabolismo , Humanos , Interferón-alfa/farmacología , Interferón gamma/farmacología , Fenotipo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales , Proteínas Virales/biosíntesis , Latencia del Virus , Replicación ViralRESUMEN
Individually purified subunits have been used to reconstitute the action of the Escherichia coli DNA polymerase III holoenzyme (Pol III HE) at a replication fork formed in the presence of the primosome, the single-stranded DNA binding protein, and a tailed form II DNA template. Complete activity, indistinguishable from that of the intact DNA Pol III HE, could be reproduced with a combination of the DNA polymerase III core (Pol III core), the gamma.delta complex, and the beta subunit. Experiments where the Pol III core in reaction mixtures containing active replication forks was diluted suggested that the lagging-strand Pol III core remained associated continuously with the replication fork through multiple cycles of Okazaki fragment synthesis. Since the lagging-strand Pol III core must dissociate from the 3' end of the completed Okazaki fragment, this suggests that its association with the fork is via protein-protein interactions, lending credence to the idea that it forms a dimeric complex with the leading-strand Pol III core. An asymmetry in the action of the subunits was revealed under conditions (high ionic strength) that were presumably destabilizing to the integrity of the replication fork. Under these conditions, tau acted to stimulate DNA synthesis only when the primase was present (i.e. when lagging-strand DNA synthesis was ongoing). This stimulation was reflected by an inhibition of the formation of small Okazaki fragments, suggesting that, within the context of the model developed to account for the temporal order of steps during a cycle of Okazaki fragment synthesis, the presence of tau accelerated the transit of the lagging-strand Pol III core from the 3' end of the completed Okazaki fragment to the 3' end of the new primer.
Asunto(s)
ADN Polimerasa III/metabolismo , Replicación del ADN , ADN Bacteriano/biosíntesis , Escherichia coli/genética , ADN/metabolismo , Replicación del ADN/efectos de los fármacos , ADN Bacteriano/metabolismo , Electroforesis en Gel de Agar , Glutamatos/farmacología , Ácido Glutámico , Ribonucleósidos/metabolismoRESUMEN
Studies with a rolling-circle DNA replication system reconstituted in vitro with a tailed form II DNA template, the DNA polymerase III holoenzyme (Pol III HE), the Escherichia coli single-stranded DNA binding protein, and the primosome, showed that within the context of a replication fork, the oligoribonucleotide primers that were formed were limited to a length in the range of 9 to 14 nucleotides, regardless of whether they were subsequently elongated by the lagging-strand DNA polymerase. This is in contrast to the 8-60-nucleotide-long primers synthesized by the primosome in the absence of DNA replication on a bacteriophage phi X174 DNA template, although when primer synthesis and DNA replication were catalyzed concurrently in this system, the extent of RNA polymerization decreased. As described in this report, we therefore examined the effect of the DNA Pol III HE on the length of primers synthesized by primase in vitro in the absence of DNA replication. When primer synthesis was catalyzed either: i) by the primosome on a phi X174 DNA template, ii) by primase on naked DNA with the aid of the DnaB protein (general priming), or iii) by primase alone at the bacteriophage G4 origin, the presence of the DNA Pol III HE in the reaction mixtures resulted in a universal reduction in the length of the heterogeneous RNA products to a uniform size of approximately 10 nucleotides. dNTPs were not required, and the addition of dGMP, an inhibitor of the 3'----5' exonuclease of the DNA Pol III HE, did not alter the effect; therefore, neither the 5'----3' DNA polymerase activity nor the 3'----5' exonuclease activity of the DNA Pol III HE was involved. E. coli DNA polymerase I, and the DNA polymerases of bacteriophages T4 and T7 could not substitute for the DNA Pol III HE. The Pol III core plays a crucial role in mediating this effect, although other subunits of the DNA Pol III HE are also required. These observations suggest that the association of primase with the DNA Pol III HE during primer synthesis regulates its catalytic activity and that this regulatory interaction occurs independently of, and prior to, formation of a preinitiation complex of the DNA Pol III HE on the primer terminus.
Asunto(s)
Replicación del ADN , ADN Bacteriano/biosíntesis , Escherichia coli/genética , ARN Nucleotidiltransferasas/metabolismo , Bacteriófago phi X 174/metabolismo , ADN/metabolismo , ADN Polimerasa III/metabolismo , ADN Primasa , ADN Bacteriano/metabolismo , ADN de Cadena Simple/metabolismo , ADN Viral/biosíntesis , Electroforesis en Gel de AgarRESUMEN
The coordinated action of many enzymatic activities is required at the DNA replication fork to ensure the error-free, efficient, and simultaneous synthesis of the leading and lagging strands of DNA. In order to define the essential protein-protein interactions and model the regulatory pathways that control Okazaki fragment synthesis, we have reconstituted the replication fork of Escherichia coli in vitro in a rolling circle-type DNA replication system. In this system, in the presence of the single-stranded DNA binding protein, the helicase/primase function on the lagging-strand template is provided by the primosome, and the synthesis of DNA strands is catalyzed by the DNA polymerase III holoenzyme. These reconstituted replication forks synthesize equivalent amounts of leading- and lagging-strand DNA, move at rates comparable to those measured in vivo (600-800 nucleotides/s at 30 degrees C), and can synthesize leading strands in the range of 150-500 kilobases in length. Using this system, we have studied the cycle of Okazaki fragment synthesis at the replication fork. This cycle is likely to have several well defined decision points, steps in the cycle where incorrect execution by the enzymatic machinery will result in an alteration in the product of the reaction, i.e. in the size of the Okazaki fragments. Since identification of these decision points should aid in the determination of which of the enzymes acting at the replication fork control the cycle, we have endeavored to identify those reaction parameters that, when varied, alter the size of the Okazaki fragments synthesized. Here we demonstrate that some enzymes, such as the DnaB helicase, remain associated continuously with the fork while others, such as the primase, must be recruited from solution each time synthesis of an Okazaki fragment is initiated. We also show that variation of the concentration of the ribonucleoside triphosphates and the deoxyribonucleoside triphosphates affects Okazaki fragment size, that the control mechanisms acting at the fork to control Okazaki fragment size are not fixed at the time the fork is assembled but can be varied during the lifetime of the fork, and that alteration in the rate of the leading-strand DNA polymerase cannot account for the effect of the deoxyribonucleoside triphosphates.
Asunto(s)
Replicación del ADN , ADN Bacteriano/biosíntesis , Escherichia coli/genética , Secuencia de Bases , ADN/metabolismo , ADN Polimerasa III/metabolismo , ADN Primasa , ADN Bacteriano/metabolismo , Desoxirribonucleósidos/metabolismo , Electroforesis en Gel de Agar , Datos de Secuencia Molecular , ARN Nucleotidiltransferasas/metabolismo , Moldes GenéticosRESUMEN
To investigate the role of the priming apparatus at the replication fork in determining Okazaki fragment size, the products of primer synthesis generated in vitro during rolling-circle DNA replication catalyzed by the DNA polymerase III holoenzyme, the single-stranded DNA binding protein, and the primosome on a tailed form II DNA template were isolated and characterized. The abundance of oligoribonucleotide primers and the incidence of covalent DNA chain extension of the primer population was measured under different reaction conditions known to affect the size of the products of lagging-strand DNA synthesis. These analyses demonstrated that the factors affecting Okazaki fragment length could be distinguished by either their effect on the frequency of primer synthesis or by their influence on the efficiency of initiation of DNA synthesis from primer termini. Primase and the ribonucleoside triphosphates were found to stimulate primer synthesis. The observed trend toward smaller fragment size as the concentration of these effectors was raised was apparently a direct consequence of the increased frequency of primer synthesis. The beta subunit of the DNA polymerase III holoenzyme and the deoxyribonucleoside triphosphates did not alter the priming frequency; instead, the concentration of these factors influenced the ability of the lagging-strand DNA polymerase to efficiently utilize primers to initiate DNA synthesis. Maximum utilization of the available primers correlated with the lowest mean value of Okazaki fragment length. These data were used to draw general conclusions concerning the temporal order of enzymatic steps that operate during a cycle of Okazaki fragment synthesis on the lagging-strand DNA template.
Asunto(s)
Replicación del ADN , ADN Bacteriano/biosíntesis , Escherichia coli/genética , Autorradiografía , Catálisis , ADN/metabolismo , ADN Polimerasa III/metabolismo , ADN Primasa , ADN Bacteriano/metabolismo , Desoxirribonucleósidos/metabolismo , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , ARN Nucleotidiltransferasas/metabolismo , Ribonucleósidos/metabolismoRESUMEN
Replication forks formed during rolling-circle DNA synthesis supported by a tailed form II DNA substrate in the presence of the primosome, the single-stranded DNA binding protein, and the DNA polymerase III holoenzyme (Pol III HE) that had been reconstituted from the purified subunits, beta, tau, and the gamma.delta complex, at limiting (with respect to nucleotide incorporation) concentrations of the Pol III core (alpha, epsilon, and theta) produced aberrantly small Okazaki fragments, while the synthesis of the leading strand was unperturbed. These small Okazaki fragments were not arrayed in tandem along the lagging-strand DNA template, but were separated by large gaps. Similarly structured synthetic products were not manufactured by replication forks reconstituted with higher, saturating concentrations of the Pol III core. Replication forks producing these small fragments could respond, by modulating the size of the Okazaki fragments produced, to variations in the concentration of NTPs or the primase, conditions that affect the frequency of priming on the lagging strand, but not to variation in the concentration of dNTPs, conditions that affect the frequency of utilization of the primers. Significantly longer Okazaki fragments (greater than 7 kilobases) could be produced in the presence of a limiting amount of Pol III core at low concentrations of the primase. These observations indicated that the production of small Okazaki fragments was not a result of a debilitated lagging-strand Pol III core, but rather a function of the time available for nascent strand synthesis during the cycle of events that are required for the manufacture of an Okazaki fragment and that it was the association of primase with the replication fork that keyed this cycle.
Asunto(s)
Replicación del ADN , ADN Bacteriano/biosíntesis , Escherichia coli/genética , ARN Nucleotidiltransferasas/metabolismo , ADN/biosíntesis , ADN Primasa , ADN Bacteriano/metabolismo , Desoxirribonucleósidos/metabolismo , Electroforesis en Gel de Agar , Ribonucleósidos/metabolismoRESUMEN
The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.