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Gene Targets (gene + target)
Selected AbstractsA host species-informative internal control for molecular assessment of African swine fever virus infection rates in the African sylvatic cycle Ornithodoros vectorMEDICAL AND VETERINARY ENTOMOLOGY, Issue 4 2009A. D. S. BASTOS Abstract African swine fever virus (ASFV) infection in adult Ornithodoros porcinus (Murry 1877, sensuWalton 1979) ticks collected from warthog burrows in southern and East Africa was assessed using a duplex genomic amplification approach that is informative with respect to the invertebrate host species and infecting sylvatic cycle virus. DNA extracted from individual ticks was used as template for the simultaneous amplification of a C-terminal 478-bp ASFV p72 gene region and a ,313-bp fragment of the tick mitochondrial 16S rRNA gene, under optimized reaction conditions. Within-warthog burrow infection rates ranged from 0% to 43% using this approach, and phylogenetic analysis of 16S gene sequences revealed the presence of three geographically discrete O. porcinus lineages, but no support for subspecies recognition. False negatives are precluded by the inclusion of host species-informative primers that ensure the DNA integrity of cytoplasmically located genome extracts. In addition, infection rate estimates are further improved as false positives arising from carry-over contamination when performing a two-step nested polymerase chain reaction are negated by the one-step approach. Phylogenetic comparison of full-length virus gene sequences with the partial C-terminal p72 gene target confirmed the epidemiological utility of the latter in a sylvatic setting. The method is therefore of particular value in studies assessing the prevalence and diversity of ASFV in relation to the African sylvatic tick vector and holds potential for investigating the role of alternative tick species in virus maintenance and transmission. [source] Cell-free production of transducible transcription factors for nuclear reprogramming,BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009William C. Yang Abstract Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli -based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target. Biotechnol. Bioeng. 2009; 104: 1047,1058. © 2009 Wiley Periodicals, Inc. [source] Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2005Linping Wang Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15INK4b), and Gas3/PMP22. The expression of p21 and p15INK4b contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death. [source] The noncoding RNA, miR-126, suppresses the growth of neoplastic cells by targeting phosphatidylinositol 3-kinase signaling and is frequently lost in colon cancersGENES, CHROMOSOMES AND CANCER, Issue 11 2008Chunguang Guo MicroRNAs (miRNA/miR) are a class of small noncoding RNAs implicated in the pathogenesis of various malignancies. In the current study, using micro(RNA) arrays, we found a ubiquitous loss of miR-126 expression in colon cancer lines when compared to normal human colon epithelia. Reconstitution of miR-126 in colon cancer cells resulted in a significant growth reduction as evidenced in clonogenic assays. A search for miR-126 gene targets revealed p85,, a regulatory subunit involved in stabilizing and propagating the phosphatidylinositol 3-kinase (PI3K) signal, as one of the potential substrates. Restoration of miR-126 in cancer cells induced a ,3-fold reduction in p85, protein levels, with no concomitant change in p85,, a gene that is functionally related to p85, but not a supposed target of miR-126. Additionally, using reporter constructs, we show that the p85,-3, untranslated region is directly targeted by miR-126. Furthermore, this miR-126 mediated reduction of p85, was accompanied by a substantial reduction in phosphorylated AKT levels in the cancer cells, suggesting an impairment in PI3K signaling. Finally, in a panel of matched normal colon and primary colon tumors, each of the tumors demonstrated miR-126 down-regulation together with an increase in the p85, protein level. Taken together, we propose that miR-126 regulates PI3K signaling partly by targeting p85,, and that the loss of miR-126 may provide a selective growth advantage during colon carcinogenesis. © 2008 Wiley-Liss, Inc. [source] Muscle precursor cells isolated from aged rats exhibit an increased tumor necrosis factor-, responseAGING CELL, Issue 1 2009Simon J. Lees Summary Improving muscle precursor cell (MPC, muscle-specific stem cells) function during aging has been implicated as a key therapeutic target for improving age-related skeletal muscle loss. MPC dysfunction during aging can be attributed to both the aging MPC population and the changing environment in skeletal muscle. Previous reports have identified elevated levels of tumor necrosis factor-, (TNF-,) in aging, both circulating and locally in skeletal muscle. The purpose of the present study was to determine if age-related differences exist between TNF-,-induced nuclear factor-kappa B (NF-,B) activation and expression of apoptotic gene targets. MPCs isolated from 32-month-old animals exhibited an increased NF-,B activation in response to 1, 5, and 20 ng mL,1 TNF-,, compared to MPCs isolated from 3-month-old animals. No age differences were observed in the rapid canonical signaling events leading to NF-,B activation or in the increase in mRNA levels for TNF receptor 1, TNF receptor 2, TNF receptor-associated factor 2 (TRAF2), or Fas (CD95) observed after 2 h of TNF-, stimulation. Interestingly, mRNA levels for TRAF2 and the cell death-inducing receptor, Fas (CD95), were persistently upregulated in response to 24 h TNF-, treatment in MPCs isolated from 32-month-old animals, compared to 3-month-old animals. Our data indicate that age-related differences may exist in the regulatory mechanisms responsible for NF-,B inactivation, which may have an effect on TNF-,-induced apoptotic signaling. These findings improve our understanding of the interaction between aged MPCs and the changing environment associated with age, which is critical for the development of potential clinical interventions aimed at improving MPC function with age. [source] The Freud-1/CC2D1A family: Transcriptional regulators implicated in mental retardationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2007Anastasia Rogaeva Abstract The CC2D1A gene family consists of two homologous genes, Freud-1/CC2D1A and Freud-2/CC2D1B, that share conserved domains, including several DM14 domains that are specific to this protein family, a C-terminal helix-loop-helix domain, and a C2 calcium-dependent phospholipid binding domain. Although the function of Freud-2 is unknown, Freud-1 has been shown to function as a transcriptional repressor of the serotonin-1A receptor gene that binds to a novel DNA element (FRE, 5,-repressor element). The DNA binding and repressor activities of Freud-1 are inhibited by calcium-calmodulin-dependent protein kinase. Recently, a deletion in the CC2D1A gene has been linked to nonsyndromic mental retardation. This deletion results in the truncation of the helix-loop-helix DNA binding and the C2 domains, crucial for Freud-1 repressor activity, and hence is predicted to generate an inactive or weakly dominant negative protein. The possible mechanisms by which inactivation of Freud-1 could lead to abnormal cortical development and cognitive impairment and the potential roles of Freud-1 gene targets are discussed. © 2007 Wiley-Liss, Inc. [source] Interleukin-1 modulates periprosthetic tissue formation in an intramedullary model of particle-induced inflammationJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2005Noah J. Epstein Abstract Interleukin-1 (IL-1) is a proinflammatory cytokine that has been implicated in wear-debris associated total joint replacement failure. We hypothesized that the absence of the IL-1 type-1 receptor would mitigate the inflammatory response to titanium particles and decrease periprosthetic inflammatory tissue in a murine intramedullary rod model. Methods: An intramedullary rod with and without commercially pure titanium particles was placed in the femora of 24 wild type mice (WT) and 24 mice lacking a functional type-1 receptor to IL-1. Femora were analyzed histologically and by ELISA of organ culture explant supernatants. Results: The presence of titanium particles in WT mice stimulated increased expression of interleukin-6 (IL-6) and macrophage chemoattractant protein-1 (MCP-1) relative to rod only controls. In contrast, IL-6 and MCP-1 expression were diminished in IL-lrl-KO mice exposed to titanium particles. Additionally, the formation of a periprosthetic fibro-inflammatory membrane in IL-lrl-KO mice was blunted at 2 weeks when compared to that in wild-type mice. Inflammatory changes and the quality of periprosthetic bone of IL-lrl-KO mice was similar to WT mice in response to titanium particles. Conclusions: These results implicate IL-1 as an important modulator in the local inflammatory response to intramedullary titanium particles. MCP-1 appears to be significantly modulated in IL-lrl-KO mice in response to titanium particles. This may be responsible, in part, for the diminished periprosthetic membrane observed in IL-lrl-KO mice at 2 weeks. Expansion of this murine model of intramedullary particle-induced inflammation to other gene targets may contribute to a more mechanistic understanding of wear-debris associated prosthesis failure. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] Is quantitative PCR for the pneumolysin (ply) gene useful for detection of pneumococcal lower respiratory tract infection?CLINICAL MICROBIOLOGY AND INFECTION, Issue 6 2009G. Abdeldaim Abstract The pneumolysin (ply) gene is widely used as a target in PCR assays for Streptococcus pneumoniae in respiratory secretions. However, false-positive results with conventional ply -based PCR have been reported. The aim here was to study the performance of a quantitative ply -based PCR for the identification of pneumococcal lower respiratory tract infection (LRTI). In a prospective study, fibreoptic bronchoscopy was performed in 156 hospitalized adult patients with LRTI and 31 controls who underwent bronchoscopy because of suspicion of malignancy. Among the LRTI patients and controls, the quantitative ply -based PCR applied to bronchoalveolar lavage (BAL) fluid was positive at ,103 genome copies/mL in 61% and 71% of the subjects, at ,105 genome copies/mL in 40% and 58% of the subjects, and at ,107 genome copies/mL in 15% and 3.2% of the subjects, respectively. Using BAL fluid culture, blood culture, and/or a urinary antigen test, S. pneumoniae was identified in 19 LRTI patients. As compared with these diagnostic methods used in combination, quantitative ply -based PCR showed sensitivities and specificities of 89% and 43% at a cut-off of 103 genome copies/mL, of 84% and 66% at a cut-off of 105 genome copies/mL, and of 53% and 90% at a cut-off of 107 genome copies/mL, respectively. In conclusion, a high cut-off with the quantitative ply -based PCR was required to reach acceptable specificity. However, as a high cut-off resulted in low sensitivity, quantitative ply -based PCR does not appear to be clinically useful. Quantitative PCR methods for S. pneumoniae using alternative gene targets should be evaluated. [source] | |||||||||||||