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Fold Induction (fold + induction)
Selected AbstractsMolecular cloning of the Matrix Gla Protein gene from Xenopus laevisFEBS JOURNAL, Issue 7 2002Functional analysis of the promoter identifies a calcium sensitive region required for basal activity To analyze the regulation of Matrix Gla Protein (MGP) gene expression in Xenopus laevis, we cloned the xMGP gene and its 5, region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. The Xenopus MGP (xMGP) gene is organized into five exons, one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active, resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5, end of the xMGP promoter revealed a minimal activating element in the sequence from ,70 to ,36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation, demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from ,180 to ,36, the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach the major site of factor binding was demonstrated to be included in the minimal activating promoter fragment from ,70 to ,36 bp. In addition, in transient transfection experiments we could show that this element mediates calcium dependent transcription and increasing concentrations of extracellular calcium lead to a significant dose dependent activation of reporter gene expression. [source] Naturally Occurring Regulatory T cells (CD4+, CD25high, FOXP3+) in the Antrum and Cardia are Associated with Higher H. pylori Colonization and Increased Gene Expression of TGF-,1HELICOBACTER, Issue 4 2008Arne Kandulski Abstract Background:Helicobacter pylori causes gastric inflammation. Despite the induction of H. pylori -specific B- and T cells, the immune response is not sufficient to clear the infection. Regulatory T cells (Treg cells) suppress the activation and proliferation of antigen-specific T cells and mediate immunologic tolerance. FOXP3 was shown to be expressed in a subset of Treg cells known as ,naturally occurring Treg cells'. These cells have not been sufficiently studied in context to H. pylori -induced inflammation in human gastric mucosa. Materials and methods: The study included 76 patients stratified according to the presence of H. pylori. Gene expression levels of FOXP3, transforming growth factor (TGF)-,1, and interleukin-10 were analyzed by quantitative real-time polymerase chain reaction in biopsies from gastric antrum, corpus, and cardia. FOXP3 expression was also analyzed by immunohistochemistry. Differences in expression levels were analyzed by comprehensive statistical analyses and correlated with clinical and histomorphologic parameters. Results:H. pylori -positive patients revealed a 19- to 25-fold induction of FOXP3 transcript levels in antrum and cardia (p < .02). FOXP3 transcript levels correlated positively with inflammation (p < .04) and TGF-,1 transcript levels (p < .001). Furthermore, a positive correlation between FOXP3+ Treg cells and H. pylori colonization was demonstrated. Conclusion: This study demonstrates that H. pylori -induced gastritis is associated with a recruitment of naturally occurring FOXP3+ Treg cells that correlates with the degree of bacterial colonization and mucosal TGF-,1 expression. Together, these data support the hypothesis that naturally FOXP3+ Treg cells play a role in the lifelong persistence of H. pylori infection in humans. [source] Differential platelet-activating factor synthesis by monocytes and polymorphonuclear leukocytes from subjects with localized aggressive periodontitisJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2007C. R. Shin Background and Objective:, Platelet-activating factor is elevated in localized aggressive periodontitis. We previously demonstrated that the elevated level of platelet-activating factor in localized aggressive periodontitis is at least partially attributable to low levels of platelet-activating factor acetylhydrolase, the enzyme that catabolizes platelet-activating factor. The objective of this study was to determine if platelet-activating factor synthesis was also elevated in localized aggressive periodontitis. To test this, platelet-activating factor synthesis was quantified in the monocytes and polymorphonuclear neutrophils of periodontally healthy patients and of subjects with localized aggressive periodontitis. Material and Methods:, Cells were labeled with [3H]acetate and treated with vehicle or stimulated with calcium ionophore A23187. Platelet-activating factor was extracted and quantified by scintillation counting. Results:, For both subject groups, resting monocytes and polymorphonuclear neutrophils produced platelet-activating factor, and calcium ionophore A23187 stimulated platelet-activating factor production in both cell types. However, calcium ionophore A23187-activated monocytes from subjects with localized aggressive periodontitis produced less platelet-activating factor than did activated periodontally healthy monocytes (p < 0.0001), suggesting an aberrant calcium ionophore A23187 response in monocytes from subjects with localized aggressive periodontitis. Indeed, when the data were expressed as fold induction of platelet-activating factor synthesis in response to calcium ionophore A23187, monocytes from subjects with localized aggressive periodontitis exhibited only a fourfold increase in platelet-activating factor synthesis, whereas calcium ionophore A23187-stimulated monocytes from periodontally healthy, chronic periodontitis and generalized aggressive periodontitis subjects produced ,,12 times more platelet-activating factor than did resting monocytes. In contrast, both resting and activated localized aggressive periodontitis polymorphonuclear neutrophils synthesized more platelet-activating factor than did periodontally healthy polymorphonuclear neutrophils. Conclusion:, These data suggest that high levels of platelet-activating factor in subjects with localized aggressive periodontitis result from both increased synthesis and reduced catabolism. While localized aggressive periodontitis polymorphonuclear neutrophils contribute to increased platelet-activating factor mass through synthesis, the contribution of monocytes is probably the result of reduced catabolism by platelet-activating factor acetylhydrolase. [source] Ethanol-Mediated Fetal Dysmorphology and its Relationship to the Ontogeny of Maternal Liver MetallothioneinALCOHOLISM, Issue 6 2009Peter Coyle Background:, Fetal zinc (Zn) deficiency arising from ethanol-induction of the Zn-binding protein metallothionein (MT) in the mother's liver has been proposed as a mechanism of teratogenicity. Here, we determine the ontogeny of MT and Zn homeostasis in rats and mice and then examine the effect of acute ethanol exposure in early embryonic development on this relationship. The protective effect of Zn against ethanol-mediated fetal dysmorphology is also examined. Methods:, Study 1: Maternal liver MT and Zn homeostasis was determined in Sprague,Dawley rats and C57BL/6J mice throughout gestation. Study 2: Rats were administered ethanol (25% in saline, intraperitoneal 0.015 ml/g) or vehicle alone on gestational day (GD) 9. Maternal liver MT and Zn, and plasma Zn was determined over the ensuing 24 hours. Study 3: Pregnant rats were treated with ethanol and Zn (s.c. 2.5 ,g Zn/g) on GD9 and fetal dysmorphology was assessed on GD 19. Results:, Study 1: Maternal liver MT began to rise around GD 9 peaking on GD 15 before falling to nonpregnant levels around term. The pregnancy-related increase in MT was associated with a fall in plasma Zn which was significantly lower on GD 15 thereafter returning to nonpregnant levels by parturition. Study 2: Ethanol administered to pregnant rats on GD 9 resulted in a 10-fold induction of MT in the maternal liver and was associated with a 33% rise in liver Zn and a 30% fall in plasma Zn, 16 hours after treatment. Study 3: Ethanol treatment on GD 9 resulted in a significant increase in craniofacial malformations which were prevented by concurrent Zn treatment. Conclusions:, The findings indicate that maternal liver MT levels are lowest in early gestation (before GD 10) making this a sensitive period where ethanol-induction of MT can affect fetal Zn homeostasis and cause fetal dysmorphology. The study further provides evidence of a protective role for Zn against ethanol-mediated teratogenicity. [source] Metabolism of isometamidium in hepatocytes isolated from control and inducer-treated ratsJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2006I. BOIBESSOT Little is known about the metabolism and mechanism of action of the trypanocide, isometamidium (ISM), the major drug used for prophylaxis of trypanosomiasis. We have investigated its metabolism and distribution in isolated rat hepatocytes using liquid chromatography-mass spectrometry and confocal laser scanning microscopy (CLSM). Two putative metabolites were formed, which were proposed to be a mono-acetyl derivative and an oxidized metabolite (SII). This is the first demonstration of the hepatic metabolism of ISM, as previous in vivo studies were hampered by dose-limiting toxicity and insensitive analytical methods. The intrinsic fluorescence of the drug enabled its intracellular uptake to be followed by CLSM. It is taken up rapidly into the nucleolus, nuclear membrane and endoplasmic reticulum within 5 min, and retained in the nucleus for at least 24 h. Persistent binding of ISM to cellular macromolecules may contribute to its prophylactic effect in vivo. Pretreatment of rats with 3-methylcholanthrene, phenobarbitone (PB) or the widely used pyrethroid pesticide, deltamethrin, resulted in an increase in metabolism of ISM to the proposed SII after 1 h incubation with hepatocytes. 3-methylcholanthrene was the most potent inducer, causing a maximal 19.5-fold induction of SII formation after exposure of hepatocytes to ISM for 1 h compared with formation by control hepatocytes. In comparison, at the 1 h timepoint deltamethrin pre-treatment caused a 10.2-fold induction, and PB only 8.2 fold. [source] Tetracycline-regulated bone morphogenetic protein 2 gene expression in lentivirally transduced primary rabbit chondrocytes for treatment of cartilage defectsARTHRITIS & RHEUMATISM, Issue 7 2010Daniela Wübbenhorst Objective Treatment of cartilage defects is still challenging, primarily because of the poor self-healing capacity of articular cartilage. Gene therapy approaches have gained considerable attention, but, depending on the vector system used, they can lead to either limited or unrestrained gene expression, and therefore regulation of gene expression is necessary. This study was undertaken to construct an efficient tetracycline (Tet),regulated, lentivirally mediated system for the expression of growth factor bone morphogenetic protein 2 (BMP-2) in primary rabbit chondrocytes that will allow for the induction and termination of growth factor gene expression once cartilage regeneration is complete. Methods Chondrogenic ATDC5 cells and primary rabbit chondrocytes were lentivirally transduced with different tetracycline-on (Tet-On),regulated, self-inactivating vectors for the induction of expression of enhanced green fluorescent protein (eGFP) or BMP-2, using either a 1-vector system or a 2-vector system. Results Expression of eGFP was induced on ATDC5 cells and chondrocytes. The highest induction rate and highest level of gene expression were reached when the spleen focus-forming virus long terminal repeat promoter was used to drive the reverse transactivator expression, after the addition of doxycycline, in chondrocytes. An up to 20-fold induction of Tet-mediated BMP-2 expression was observed on ATDC5 cells. The extent of induction and expression level of BMP-2 in chondrocytes were similar between the 1-vector system, and 2-vector system,infected cells (mean ± SD 15.5 ± 1.1 ng/ml and 14.6 ± 0.4 ng/ml, respectively). In addition, prolonged induction and switching-off of BMP-2 expression, as well as repeated induction, were demonstrated. Production of proteoglycans, as shown by Alcian blue staining, demonstrated the functionality of the lentivirally expressed BMP-2 under induced conditions. Conclusion The lentivirally mediated Tet-On system is an effective strategy for efficient, repeatedly inducible expression of BMP-2 in primary rabbit chondrocytes. Therefore, use of this system in in vivo experiments may be a promising approach as a treatment strategy for cartilage defects. [source] | ||||||||||