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At least 631 records · Page 35Linked to original sources
PubMed2006

Targeted transgene expression in neuronal precursors: watching young neurons in the old brain.

Progress in the field of neurogenesis is limited by the lack of animal models allowing direct detection and analysis of living cells participating in neurogenesis. We engineered a transgenic mouse model that expresses the fluorescent reporter proteins enhanced green fluorescent protein or Discoma sp. reef coral red fluorescent protein under the control of the doublecortin (DCX) promoter, a gene specifically and transiently active in neuronal precursors and young neurons. The expression of the reporter proteins correlated with expression of the endogenous DCX protein, and with developmental and adult neurogenesis. Neurogenesis was unaffected by the presence of the fluorescent proteins. The transgenic mice allowed direct identification of the very few newly generated neurons present in the aged brain. We performed electrophysiological analysis and established that newly generated hippocampal granule cells in aged and young mice shared identical physiological properties. Hence, although the rate of neurogenesis tapers with ageing, a population of highly excitable young neurons indistinguishable to those found in younger animals is continuously generated. Therefore, maintenance of the fundamental properties of neuronal precursors even at advanced age suggests that stimulation of neurogenesis may constitute a valid strategy to counteract age-related neuronal loss and cognitive declines.

Age Factors↗
PubMed2003

Quinolone resistance due to reduced target enzyme expression.

We report for the first time low-level quinolone resistance mediated by decreased expression of topoisomerase IV in Staphylococcus aureus. A single-step mutant of wild-type S. aureus strain ISP794, P18 selected by using twice the MIC of premafloxacin, had four- and four- to eightfold greater MICs of premafloxacin and ciprofloxacin, respectively, than the wild type. Sequencing of parEC and gyrBA with their promoter regions revealed a point mutation (G-->A) 13 bp upstream of the start codon of parE. Genetic linkage studies showed that there was a high level of correlation between the mutation and the resistance phenotype, and allelic exchange confirmed the contribution of the mutation to resistance. Decreased expression of ParE and decreased steady-state levels of parEC transcripts in P18 and in resistant allelic exchange mutants were observed. The steady-state levels of gyrBA and topB transcripts were increased in P18 but not in two resistant allelic exchange mutants, and sequencing upstream of either gene did not reveal a difference between ISP794 and P18. The steady-state levels of topA transcripts were similar in the various strains. Growth competition experiments performed at 30, 37, and 41 degrees C with a susceptible allelic exchange strain and a resistant allelic exchange strain suggested that loss of fitness was associated with reduced levels of ParE at 41 degrees C. However, P18 had a growth advantage over ISP794 at all temperatures, suggesting that a compensatory mechanism was associated with the increased levels of gyrBA and topB transcripts. Thus, reduced levels of ParE appear to be compatible with cell survival, although there may be a fitness cost during rapid cell multiplication, which might be overcome by compensatory mechanisms without reversion of the resistance phenotype.

Blotting, Northern↗
PubMed2004

Identification of a mouse cytomegalovirus gene selectively targeting CD86 expression on antigen-presenting cells.

We and others have shown that infection of dendritic cells with murine cytomegalovirus (MCMV) leads to severe functional impairment of these antigen-presenting cells (D. M. Andrews, C. E. Andoniou, F. Granucci, P. Ricciardi-Castagnoli, and M. A. Degli-Esposti, Nat. Immunol. 2:1077-1084, 2001; S. Mathys, T. Schroeder, J. Ellwart, U. H. Koszinowski, M. Messerle, and U. Just, J. Infect. Dis. 187:988-999, 2003). Phenotypically, reduced surface expression of costimulatory molecules and major histocompatibility complex molecules was detected. In order to identify the molecular basis for the observed effects, we generated MCMV mutants with large deletions of nonessential genes. The study was facilitated by the finding that a monocyte-macrophage cell line displayed similar phenotypic alterations after MCMV infection. By analyzing the expression of cell surface molecules on infected cells, we identified a mutant virus which is no longer able to downmodulate the expression of the costimulatory molecule CD86. Additional mutants with smaller deletions allowed us to pin down the responsible gene to a certain genomic region. RNA analysis led to the identification of the spliced gene m147.5, encoding a protein with 145 amino acids. Experiments with an m147.5 mutant revealed that the protein affects CD86 expression only, suggesting that additional MCMV genes are responsible for downmodulation of the other surface molecules. Identification of viral gene products interfering with functionally important proteins of antigen-presenting cells will provide the basis to dissect the complex interaction of CMV with these important cells and to evaluate the biological importance of these viral genes in vivo.

Amino Acid Sequence↗
PubMed1992

Investigation of cell physiology in the animal using transgenic technology.

Over the past 10 years significant progress has been made in techniques for manipulating the genome of the animal. Production of transgenic mice has led to important insights into the regulation of gene expression, the molecular basis of cancer, immunology, and developmental biology. The tools necessary to generate transgenic mice are becoming widely available, making it possible to study a variety of problems. In this review a description of the strategies being used to address problems of interest in cell physiology using transgenic mice is given. Elucidation of the rules governing the regulation of gene expression now permits the targeted expression of a protein to a particular organ or cell type within an organ. Overexpression of proteins, expression of foreign or mutant proteins, mislocalization of proteins, and directed elimination of proteins are all procedures that can now be used to generate interesting animal models for physiological studies. The applications of these techniques to a variety of problems in normal and abnormal physiology are discussed in this review.

Animals↗
PubMed2001

Targeted transgenic expression of beta(2)-adrenergic receptors to type II cells increases alveolar fluid clearance.

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta-agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta(2)-adrenergic receptors (beta(2)-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta(2)-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta(2)-ARs than nontransgenic (NTG) mice (939 +/- 113 vs. 194 +/- 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was approximately 40% greater than that in untreated NTG mice (15 +/- 1.4 vs. 10.9 +/- 0.6%; P < 0.005) and approached that of NTG mice treated with the beta-agonist formoterol (19.8 +/- 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 +/- 0.5% but had no effect on NTG mice (11.5 +/- 1.0%). Na(+)-K(+)-ATPase alpha(1)-isoform expression was unchanged, whereas alpha(2)-isoform expression was approximately 80% greater in the TG mice. These findings show that beta(2)-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta(2)-ARs specifically expressed on ATII cells.

Adenylyl Cyclases↗
PubMed2005

Proteins and protein pattern differences between glioma cell lines and glioblastoma multiforme.

INTRODUCTION: Research into the pathogenesis, molecular signaling, and treatment of glioblastoma multiforme (GBM) has traditionally been conducted using cell lines derived from malignant gliomas. We compared protein expression patterns between solid primary GBMs and GBM cell lines to identify proteins whose expression may be altered in cell culture. METHODS: We cultured cell lines U87, U118, U251, and A172 and used tissue-selective microdissection of eight primary GBMs to obtain pure populations of tumor cells, which we studied using two-dimensional gel electrophoresis (2DGE) and examined using differential expression software. Select protein targets expressed differentially between GBM tumors and GBM cell lines were sequenced using tandem mass spectrometry. RESULTS: Analysis of the primary GBM tumor samples (n = 8) and the GBM cell lines revealed reproducibly similar proteomic patterns for each group, which distinguished tumors from the cell lines. Gels contained up to 500 proteins that were consistently identified in the pH 4 to 7 range. Comparison of proteins identified in the GBM tumors and in the cell lines showed approximately 160 proteins that were gained and 60 proteins that were lost on culture. Using normalized intensity patterns from the 2DGE images, ANOVA tests were done and statistically significant spots were identified. Seven proteins found in the cell lines were significantly increased when compared with the GBM tumors (P < 0.05), whereas 10 proteins were significantly decreased from cell lines compared with the GBM tumors. Proteins identified included transcription factors, tumor suppressor genes, cytoskeletal proteins, and cellular metabolic proteins. CONCLUSION: Global protein and proteomic differences were identified between primary GBM tumor samples and GBM cell lines. The proteins identified by 2DGE analysis elucidate some of the selection pressures of in vitro culture, help accentuate the advantages and limitations of cell culture, and may aid comprehension of gliomagenesis and enhance development of new therapeutics.

Brain Neoplasms↗
PubMed1998

Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation.

Acute lung injury is a frequent and treatment-limiting consequence of therapy with hyperoxic gas mixtures. To determine if IL-11 is protective in oxygen toxicity, we compared the effects of 100% O2 on transgenic mice that overexpress IL-11 in the lung and transgene (-) controls. IL-11 markedly enhanced survival in 100% O2 with 100% of transgene (-) animals dying within 72-96 h and > 90% of transgene (+) animals surviving for more than 10 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Significant differences in copper zinc superoxide dismutase and catalase activities were not noted and the levels of total, reduced and oxidized glutathione were similar in transgene (+) and (-) animals. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene (+) as versus (-) mice after 100% O2 exposure, and IL-11 diminished hyperoxia-induced expression of IL-1 and TNF. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals and IL-11 markedly diminished this cell death response. These studies demonstrate that IL-11 markedly diminishes hyperoxic lung injury. They also demonstrate this protection is associated with small changes in lung antioxidants, diminished hyperoxia-induced IL-1 and TNF production, and markedly suppressed hyperoxia-induced DNA fragmentation.

Animals↗
PubMed2003

The 5'-flanking region of the murine epididymal protein of 17 kilodaltons gene targets transgene expression in the epididymis.

A murine epididymal retinoic-acid-binding protein (mE-RABP) is specifically expressed in the mid/distal caput epididymidis and is androgen regulated. The murine epididymal protein of 17 kDa (mEP17) gene, a novel gene homologous to mE-RABP, is located within 5 kb of the 5'-flanking region of the mE-RABP gene. In contrast, expression of the mEP17 gene is restricted to the initial segment and regulated by factor(s) contained in testicular fluid. To identify cis-DNA regulatory element(s) involved in the tissue- and region-specific expression of the mEP17 gene in transgenic mice, we have studied the expression of a transgene containing 5.3 kb of the 5'-flanking region of the mEP17 gene (5.3mEP17) linked to chloramphenicol acetyltransferase (CAT) reporter gene. Significant caput epididymidis-specific CAT activity was detected in transgenic mouse lines; and CAT gene expression is restricted to the initial segment, as is the expression of the endogenous mEP17 gene. Ontogenic expression and testicular factor dependency also mimic that of endogenous mEP17 gene. These results suggest that the 5.3mEP17 fragment contains all the information required for spatial and temporal expression in the mouse epididymis. The 5.3mEP17 fragment will be useful to express a foreign gene of interest in the epididymis in an initial segment-specific manner.

Animals↗
PubMed2004

Histone deacetylase 1 is required to repress Notch target gene expression during zebrafish neurogenesis and to maintain the production of motoneurones in response to hedgehog signalling.

Histone deacetylases (Hdacs) are widely implicated as key components of transcriptional silencing mechanisms. Here, I show that hdac1 is specifically required in the zebrafish embryonic CNS to maintain neurogenesis. In hdac1 mutant embryos, the Notch-responsive E(spl)-related neurogenic gene her6 is ectopically expressed at distinct sites within the developing CNS and proneural gene expression is correspondingly reduced or eliminated. Using an hdac1-specific morpholino, I show that this requirement for hdac1 is epistatic to the requirement for Notch signalling. Consequently, hdac1-deficient embryos exhibit several defects of neuronal specification and patterning, including a dramatic deficit of hedgehog-dependent branchiomotor neurones that is refractory to elevated levels of hedgehog signalling. Thus, in the zebrafish embryo, hdac1 is an essential component of the transcriptional silencing machinery that supports the formation and subsequent differentiation of neuronal precursors.

Animals↗
PubMed2005

Host and parasite-derived IKK activities direct distinct temporal phases of NF-kappaB activation and target gene expression following Toxoplasma gondii infection.

Activation of NF-kappaB by the intracellular pathogen Toxoplasma gondii is associated with the localization of phosphorylated IkappaB alpha to the parasitophorous vacuole membrane (PVM). This is mediated by a parasite-derived IkappaB kinase (TgIKK) activity and is independent of host IKK function. In the present study, we examined the roles of host IKK and parasite-derived TgIKK on the temporal modulation of NF-kappaB activation. Despite the presence of TgIKK activity at the PVM, nuclear translocation of NF-kappaB and subsequent gene expression exhibited a requirement for the host IKK complex. A detailed kinetic analysis of NF-kappaB activation revealed a biphasic, hierarchical and temporally regulated response. We propose a novel paradigm for the modulation of NF-kappaB-dependent gene expression by T. gondii that involves both the host IKK complex and TgIKK activity at different phases of infection. Thus, T. gondii effectively alters gene expression in a temporal dimension by exploiting the NF-kappaB signaling machinery and subsequently rewiring the activation circuits of the infected host cell.

Active Transport, Cell Nucleus↗
PubMed1996

Drugs that target gene expression: an overview.

Over the last few years, it has become apparent that regulation of many normal physiological pathways, as well as the response to a wide variety of disease states, involves specific changes in gene transcription. The concept of drugs that regulate gene transcription is now firmly established. There are multiple, highly effective drugs in widespread use whose primary mechanism of action is to affect gene transcription. A major goal at Oncogene Science is to develop drugs that can modulate the normal physiological responses associated with disease by specifically increasing or decreasing the transcription of key genes. In this article, we review several examples of drugs that have been shown to act by regulating gene transcription and describe the approach Oncogene Science has pioneered.

Drug Evaluation, Preclinical↗
PubMed2003

Sequentiality and processivity of nuclear receptor coregulators in regulation of target gene expression.

A series of data has accumulated over the past five years that raises questions about our current understanding of the transcriptional process and its regulation. Following the discovery of coactivators for nuclear receptors (NRs), a large number of these molecules have been reported in the literature. This perspective will summarize some opinions on the significance of this large number of factors.

Journal Article↗
PubMed2006

Targeting the expression of platelet-derived growth factor receptor by reactive stroma inhibits growth and metastasis of human colon carcinoma.

The stromal cells within colon carcinoma express high levels of the platelet-derived growth factor receptor (PDGF-R), whereas colon cancer cells do not. Here, we examined whether blocking PDGF-R could inhibit colon cancer growth in vivo. KM12SM human colon cancer cells were injected subcutaneously (ectopic implantation) into the cecal wall (orthotopic implantation) or into the spleen (experimental liver metastasis) of nude mice. In the colon and liver, the tumors induced active stromal reaction, whereas in the subcutis, the stromal reaction was minimal. Groups of mice (n=10) received saline (control), the tyrosine kinase inhibitor imatinib, irinotecan, or a combination of imatinib and irinotecan. Four weeks of treatment with imatinib and irinotecan significantly inhibited tumor growth (relative to control or single-agent therapy) in the cecum and liver but not in the subcutis. The combination therapy completely inhibited lymph node metastasis. Imatinib alone or in combination with irinotecan inhibited phosphorylation of PDGF-Rbeta of tumor-associated stromal cells and pericytes. Combination therapy also significantly decreased stromal reaction, tumor cell proliferation, and pericyte coverage of tumor microvessels and increased apoptosis of tumor cells and tumor-associated stromal cells. These data demonstrate that blockade of PDGF-R signaling pathways in tumor-associated stromal cells and pericytes inhibits the progressive growth and metastasis of colon cancer cells.

Animals↗
PubMed2001

Targeted antizyme expression in the skin of transgenic mice reduces tumor promoter induction of ornithine decarboxylase and decreases sensitivity to chemical carcinogenesis.

To directly evaluate the role of increased ornithine decarboxylase (ODC) and polyamines in mouse skin carcinogenesis, we used bovine keratin 5 (K5) and keratin 6 (K6) promoter elements to direct the expression of antizyme (AZ) to specific skin cell populations. AZ is a multifunctional regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. K5-AZ mice treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) at 0 and 24 h exhibit increases in epidermal and dermal ODC activity that are reduced in magnitude. K6-AZ mice treated similarly do not show any increased ODC activity or protein after a second application due to TPA-induced expression of AZ protein. Epidermal and dermal polyamine content, particularly spermidine, is reduced in untreated K5-AZ mice and TPA-treated K5-AZ and K6-AZ mice. Susceptibility to 7,12-dimethylbenz(a)anthracene/TPA carcinogenesis was also investigated for two K6-AZ transgenic lines [K6-AZ(52) and K6-AZ(18)] and a single K5-AZ line. K6-AZ(52) mice had a substantial delay in tumor onset and a >80% reduction in tumor multiplicity compared with normal littermates. K6-AZ(18) and K5-AZ mice also developed fewer papillomas than littermate controls (35% and 50%, respectively), and the combination of these lines to produce double transgenic animals yielded an additive decrease (70%) in tumor multiplicity. These mice demonstrate for the first time that AZ suppresses tumor growth in an animal cancer model and provide a valuable model system to evaluate the role of ODC and polyamines in skin tumorigenesis.

9,10-Dimethyl-1,2-benzanthracene↗