RESUMEN
The incidence of Staphylococcus aureus bacteremia (SAB) is significantly higher in African American (AA) than in European-descended populations. We used admixture mapping (AM) to test the hypothesis that genomic variations with different frequencies in European and African ancestral genomes influence susceptibility to SAB in AAs. A total of 565 adult AAs (390 cases with SAB; 175 age-matched controls) were genotyped for AM analysis. A case-only admixture score and a mixed χ2(1df) score (MIX) to jointly evaluate both single-nucleotide polymorphism (SNP) and admixture association (P<5.00e-08) were computed using MIXSCORE. In addition, a permutation scheme was implemented to derive multiplicity adjusted P-values (genome-wide 0.05 significance threshold: P<9.46e-05). After empirical multiplicity adjustment, one region on chromosome 6 (52 SNPs, P=4.56e-05) in the HLA class II region was found to exhibit a genome-wide statistically significant increase in European ancestry. This region encodes genes involved in HLA-mediated immune response and these results provide additional evidence for genetic variation influencing HLA-mediated immunity, modulating susceptibility to SAB.
Asunto(s)
Bacteriemia/epidemiología , Bacteriemia/genética , Negro o Afroamericano/genética , Predisposición Genética a la Enfermedad , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/genética , Humanos , Incidencia , Polimorfismo de Nucleótido Simple , Staphylococcus aureusRESUMEN
Hematopoietic stem/progenitor cells (HSC/P) reside in the bone marrow in distinct anatomic locations (niches) to receive growth, survival and differentiation signals. HSC/P localization and migration between niches depend on cell-cell and cell-matrix interactions, which result from the cooperation of cytokines, chemokines and adhesion molecules. The CXCL12-CXCR4 pathway, in particular, is essential for myelopoiesis and B lymphopoiesis but the molecular mechanisms of CXCL12 action remain unclear. We previously noted a strong correlation between prolonged CXCL12-mediated focal adhesion kinase (FAK) phosphorylation and sustained pro-adhesive responses in progenitor B cells, but not in mature B cells. Although FAK has been well studied in adherent fibroblasts, its function in hematopoietic cells is not defined. We used two independent approaches to reduce FAK expression in (human and mouse) progenitor cells. RNA interference (RNAi)-mediated FAK silencing abolished CXCL12-induced responses in human pro-B leukemia, REH cells. FAK-deficient REH cells also demonstrated reduced CXCL12-induced activation of the GTPase Rap1, suggesting the importance of FAK in CXCL12-mediated integrin activation. Moreover, in FAK(flox/flox) hematopoietic precursor cells, Cre-mediated FAK deletion resulted in impaired CXCL12-induced chemotaxis. These studies suggest that FAK may function as a key intermediary in signaling pathways controlling hematopoietic cell lodgment and lineage development.
Asunto(s)
Linfocitos B/patología , Adhesión Celular , Quimiocinas CXC/farmacología , Quimiotaxis , Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Animales , Antígenos Ly/metabolismo , Diferenciación Celular , Quimiocina CXCL12 , Ensayo de Unidades Formadoras de Colonias , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Sistema Hematopoyético , Humanos , Integrasas/metabolismo , Lentivirus , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Interferencia de ARN , Receptores CXCR4 , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Deregulation of microRNAs (miRs) contributes to progression and metastasis of prostate and other cancers. miR-23b and -27b, encoded in the same miR cluster (miR-23b/-27b), are downregulated in human metastatic prostate cancer compared with primary tumors and benign tissue. Expression of miR-23b/-27b decreases prostate cancer cell migration, invasion and results in anoikis resistance. Conversely, antagomiR-mediated miR-23b and -27b silencing produces the opposite result in a more indolent prostate cancer cell line. However, neither miR-23b/-27b expression or inhibition impacts prostate cancer cell proliferation suggesting that miR-23b/-27b selectively suppresses metastasis. To examine the effects of miR-23b/-27b on prostate cancer metastasis in vivo, orthotopic prostate xenografts were established using aggressive prostate cancer cells transduced with miR-23b/-27b or non-targeting control miRNA. Although primary tumor formation was similar between miR-23b/-27b-transduced cells and controls, miR-23b/-27b expression in prostate cancer cells decreased seminal vesicle invasion and distant metastases. Gene-expression profiling identified the endocytic adaptor, Huntingtin-interacting protein 1-related (HIP1R) as being downregulated by miR-23b/-27b. Increased HIP1R expression in prostate cancer cells inversely phenocopied the effects of miR-23b/-27b overexpression on migration, invasion and anchorage-independent growth. HIP1R rescued miR-23b/-27b-mediated repression of migration in prostate cancer cells. HIP1R mRNA levels were decreased in seminal vesicle tissue from mice bearing miR-23b/-27b-transduced prostate cancer cell xenografts compared with scrambled controls, suggesting HIP1R is a key functional target of miR-23b/-27b. In addition, depletion of HIP1R led to a more rounded, less mesenchymal-like cell morphology, consistent with decreased metastatic properties. Together, these data demonstrate that the miR-23b/-27b cluster functions as a metastasis-suppressor by decreasing HIP1R levels in pre-clinical models of prostate cancer.
Asunto(s)
MicroARNs/genética , Neoplasias de la Próstata/genética , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Proteínas de Microfilamentos , Invasividad Neoplásica/genética , Metástasis de la Neoplasia , Próstata/patología , Neoplasias de la Próstata/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A series of expression vectors have been developed which all contain an identical expression cassette comprised of the lacIq gene, the tac promoter, a multiple cloning site (MCS) and a downstream transcriptional terminator. This cassette has been inserted into four distinct plasmid backbones, each of which is from a separate incompatibility group and carries a different drug resistance gene. Therefore, different combinations of these expression plasmids can be stably maintained together.
Asunto(s)
Vectores Genéticos , Regiones Promotoras Genéticas , Escherichia coli/genética , Dosificación de Gen , Expresión Génica , Datos de Secuencia Molecular , Origen de RéplicaRESUMEN
As soon as RNA interference (RNAi) was found to work in mammalian cells, research quickly focused on harnessing this powerful endogenous and specific mechanism of gene silencing for human therapy. RNAi uses small RNAs, less than 30 nucleotides in length, to suppress expression of genes with complementary sequences. Two strategies can introduce small RNAs into the cytoplasm of cells, where they are active - a drug approach where double-stranded RNAs are administered in complexes designed for intracellular delivery and a gene therapy approach to express precursor RNAs from viral vectors. Phase I clinical studies have already begun to test the therapeutic potential of small RNA drugs that silence disease-related genes by RNAi. This review will discuss progress in developing and testing small RNAi-based drugs and potential obstacles.
Asunto(s)
Terapia Genética/métodos , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Animales , Esquema de Medicación , Diseño de Fármacos , Ingeniería Genética , Humanos , Modelos Animales , ARN Interferente Pequeño/farmacocinética , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Numerous experiments have indicated that the synthesis of RNA polymerase (beta beta' alpha 2 sigma 70) in Escherichia coli is autogenously regulated. In the present study, we have examined expression of the rpoB and rpoC genes which encode the beta and beta' subunits of RNA polymerase. These genes are the distal cistrons of the rplKAJLrpoBC ribosomal protein-RNA polymerase transcription unit. Both transcriptional (operon) and translational (gene) fusions of either rpoB or rpoC to the lacZ reporter were used to monitor their in vivo expression by inserting single copies of these fusions into the bacterial chromosome on integration-proficient lambda vectors. In order to examine the expression of the rpoBC genes in response to varied intracellular concentrations of the RNA polymerase subunits, a series of expression plasmids carrying the rpoB, rpoC, rpoA (alpha) and rpoD (sigma 70) genes in different combinations were then introduced into these cells. Elevated concentrations of either beta or beta' were found to repress the expression of both rpoB and rpoC at the translational level. However, the simultaneous increase in the concentration of all the subunits that comprise holoenzyme repressed the transcription of rpoBC. To determine the site of this repression, additional operon fusions were utilized which placed lacZ downstream of each of the transcriptional regulatory sites of this gene cluster, including two promoters, rplKp and rplJp, and a transcriptional attenuator in the rplL-rpoB intercistronic region. Expression from these fusions showed that transcriptional repression is achieved primarily by reducing initiation at both upstream promoters, coupled with a small increase in termination at the attenuator.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/biosíntesis , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica , Biosíntesis de Proteínas , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Represión Enzimática , Escherichia coli/genética , Retroalimentación , Genes Bacterianos , Operón Lac , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
A protocol has been developed that is capable of saturating regions hundreds of basepairs in length with linker scanning mutations. The efficacy of this method stems from the design of the linker scanning mutagenesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site for a different restriction endonuclease. The cleavage site for one endonuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette. Digestion with these endonucleases and subsequent ligation results in the replacement of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide. We have used this protocol to mutagenize a span of approximately 400 bp surrounding the start site of the gene for the beta subunit (rpoB) of Escherichia coli RNA polymerase. The translation of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta'. Analysis of the linker scanning mutations indicates that sequences extending a considerable distance both upstream and downstream of the start site are required for normal translation. Also a site that appears to be involved in translational repression by excess beta' has been identified.