EVOLUTION, Issue 2 2002
Nadia A. Chuzhanova
Abstract Complexity analysis is capable of highlighting those gross evolutionary changes in gene promoter regions (loosely termed "promoter shuffling") that are undetectable by conventional DNA sequence alignment. Complexity analysis was therefore used here to identify the modular components (blocks) of the orthologous ,-globin gene promoter sequences of 22 vertebrate species, from zebrafish to humans. Considerable variation between the ,-globin gene promoters was apparent in terms of block presence/absence, copy number, and relative location. Some sequence blocks appear to be ubiquitous, whereas others are restricted to a specific taxon. Block similarities were also evident between the promoters of the paralogous human ,-like globin genes. It may be inferred that a wide variety of different mutational mechanisms have operated upon the ,-globin gene promoter over evolutionary time. Because these include gross changes such as deletion, duplication, amplification, elongation, contraction, and fusion, as well as the steady accumulation of single base-pair substitutions, it is clear that some redefinition of the term "promoter shuffling" is required. This notwithstanding, and as previously described for the vertebrate growth hormone gene promoter, the modular structure of the ,-globin promoter region and those of its paralogous counterparts have continually been rearranged into new combinations through the alteration, or shuffling, of preexisting blocks. Some of these changes may have had no influence on promoter function, but others could have altered either the level of gene expression or the responsiveness of the promoter to external stimuli. The comparative study of vertebrate ,-globin gene promoter regions described here confirms the generality of the phenomenon of sequence block shuffling and thus supports the view that it could have played an important role in the evolution of differential gene expression. [source]

Nanosized Glass Frit as an Adhesion Promoter for Ink-Jet Printed Conductive Patterns on Glass Substrates Annealed at High Temperatures,

ADVANCED FUNCTIONAL MATERIALS, Issue 19 2008
Daehwan Jang
Abstract Ink-jet printed metal nanoparticle films have been shown to anneal at high temperatures (above 500,°C) to highly conductive metal films on glass or ceramic substrates, but they suffer from cracking and inadequate substrate adhesion. Here, we report printable conductive materials, with added nanosized glass frit that can be annealed at 500,°C to form a crack-free dense microstructure that adheres well to glass substrates. This overcomes the previous challenges while still retaining the desired high film conductivity. Controlling the particle characteristics and dispersion behavior plays an important role in successfully incorporating the glass frit into the conductive inks. [source]

Genotype,phenotype correlations in hereditary familial retinoblastoma,

HUMAN MUTATION, Issue 3 2007
Melissa Taylor
Abstract We studied 50 unrelated pedigrees with a family history of retinoblastoma (Rb) (165 carriers of a RB1 mutation) to delineate the spectrum of RB1 germline mutations in familial Rb and to identify genotype,phenotype correlations as well as putative modifiers. Patients were followed at Institut Curie and they were examined by an ophthalmologist, a pediatrician, and a geneticist. All cases of familial Rb were determined via genetic counseling. Clinical features included disease status, laterality, age at diagnosis, mutation type, follow-up, and disease,eye ratio (DER). To eliminate mosaic cases, first-generation carriers displaying low-penetrance (LP) Rb were excluded from the analysis. Complete penetrance was the rule for nonsense and frameshift mutations (25 families) and high penetrance was observed for large rearrangements (eight families). Promoter (two families) and missense (two families) mutations displayed heterogeneous phenotypes and LP. Variable penetrance was observed for splice abnormalities (13 families) and was explained by in/out of frame mutations or respect of functional domains. Surprisingly, two families with the LP g.45867G>T/IVS6+1G>T mutation presented data that conflicted with the data reported in previous publications, as unaffected carriers had paternally inherited mutant alleles. Moreover, RNA analyses suggested that the lack of penetrance in unaffected carriers could be explained by an increase in expression levels of the wild-type allele. This observation prompted us to define a new class "3" of LP alleles. We believe this is the first large-scale study of familial Rb with a high level of homogeneity in the clinical and genetic analysis of patients and their relatives, thereby allowing for reliable intrafamilial genotype,phenotype correlations. Our analysis suggests in some cases the influence of modifier factors probably involved in mRNA level regulation and/or pRB pathway regulation. Hum Mutat 28(3), 284,293, 2007. © 2006 Wiley-Liss, Inc. [source]

Michael Reaction of Nitroalkanes with ,-Nitroacrylates under a Solid Promoter: Advanced Regio- and Diastereoselective Synthesis of Nitro-Functionalized ,,,-Unsaturated Esters and 1,3-Butadiene-2-carboxylates

ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 9 2010
Alessandro Palmieri
Abstract A new class of nitro-functionalized ,,,-unsaturated esters has been prepared by a regio- and diastereoselective Michael addition of nitroalkanes to ,-nitroacrylates, performed at room temperature, under carbonate on polymer as promoter, and in the presence of ethyl acetate as eco-friendly solvent. Moreover, by the modular choice of the reaction conditions the method allows the synthesis of 1,3-butadiene-2-carboxylates. [source]

An In Vivo Model to Study Osteogenic Gene Regulation: Targeting an Avian Retroviral Receptor (TVA) to Bone With the Bone Sialoprotein (BSP) Promoter,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2005
Ling Li
Abstract To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene. Introduction: Tissue-specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5-kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue-specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant-negative form of the Cbfa1/Runx2 transcription factor on bone formation. Materials and Methods: We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9-kb mouse BSP promoter. The tissue-specific expression of the TVA gene was analyzed by RT-PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396-bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA-binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice. Results and Conclusion: Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu-RCASBP (A) vector, RT-PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased ,15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo. [source]

Variation in the TNF Gene Promoter and Risk of Osteolysis After Total Hip Arthroplasty

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2003
FRCS, J Mark Wilkinson PhD
Abstract Genetic factors may influence implant failure caused by osteolysis after THA. In an association study of 481 subjects after THA, we found that carriage of the TNF - 238A allele was associated with an increased incidence of osteolysis versus noncarriage (odds ratio, 1.7) and was independent of other risk factors. Genetic and environmental factors influence implant survival after THA. Introduction: Tumor necrosis factor (TNF) is thought to play a role in osteolysis, the major cause of implant failure after total hip arthroplasty (THA). Natural sequence variations at ,238 and ,308 in the TNF gene promoter are associated with differences in susceptibility to several TNF-mediated diseases. We tested whether these polymorphisms are associated with osteolysis after THA. Materials and Methods: A total of 481 whites (214 with failed versus 267 with intact implants) were recruited 11.7 ± 4 years after cemented THA. Genomic DNA was extracted from peripheral blood and genotyped for the ,238 and ,308 polymorphisms using the Taqman 5, nuclease method. Healthy controls (n = 500) from the background population were also genotyped to establish the local prevalence of these alleles. Results: The carriage of ,238A was 8.8% in the background population and 10.9% in the THA controls (p > 0.05). Carriage of ,238A in the osteolysis group was 17.3% (odds ratio, 1.7; 95% CI, 1.0,2.9). Carriage was highest (20.5%) in patients with more widespread osteolysis (OR, 2.1; 1.2,3.8). The association of ,238A with osteolysis was independent of other risk factors for osteolysis (logistic regression analysis: OR, 1.8; 1.0,3.2). Carriage of ,308A was not associated with osteolysis. Conclusion: Genetic, as well as environmental factors, influence implant failure after THA. Whether the TNF - 238 polymorphism causes a biological change that predisposes to loosening or is in linkage disequilibrium with such a locus is not yet known. [source]

Regulation of the Murine TRACP Gene Promoter

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2003
AI Cassady
Abstract The activity of the TRACP promoter has been investigated as a model of gene regulation in osteoclasts. The murine TRACP gene promoter contains potential binding sites for a number of transcription factors in particular, candidate sites for the Ets factor PU.1 and for the microphthalmia transcription factor (MiTF). These are of relevance to osteoclast biology because the PU.1 knockout mouse has an osteopetrotic phenotype, and MiTF, when mutated in the mi/mi mouse, also results in osteopetrosis. The binding sites for both of these factors have been identified, and they have been determined to be functional in regulating TRACP expression. A novel assay system using the highly osteoclastogenic RAW/C4 subclone of the murine macrophage cell line RAW264.7 was used to perform gene expression experiments on macrophage and osteoclast cell backgrounds. We have shown that TRACP expression is a target for regulation by the macrophage/osteoclast transcription factor PU.1 and the osteoclast commitment factor MiTF and that these factors act synergistically in regulating this promoter. This directly links two controlling factors of osteoclast differentiation to the expression of an effector of cell function. [source]

Down-Regulation of Procollagen ,1[I] Messenger RNA by Titanium Particles Correlates with Nuclear Factor ,B (NF-,B) Activation and Increased Rel A and NF-,B1 Binding to the Collagen Promoter

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2001
Kenneth A. Roebuck
Abstract Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein tyrosine phosphorylation (PTP), activates the transcription factor nuclear factor ,B (NF-,B), and causes an approximately 50% decrease in the steady-state messenger RNA (mRNA) level of procollagen ,1[I]. In this study, we identify three NF-,B binding sites within the human procollagen ,1[I] gene promoter, show that titanium particles stimulate their binding of the NF-,B subunits Rel A (p65) and NF-,B1 (p50), and find NF-,B activation correlates with collagen gene suppression by titanium particles in osteoblasts. Protein tyrosine kinase (PTK) inhibitors, which significantly reduce the suppressive effect of titanium particles on collagen gene expression, inhibited NF-,B binding activity showing that titanium particle stimulation of PTK signals in osteoblasts are critical for both NF-,B activation and collagen gene expression. The antioxidant pyrrolidine dithiocarbamate (PDTC), which also inhibits the titanium particle suppression of collagen, abrogated the titanium particle activation of NF-,B, suggesting the involvement of redox signals in NF-,B-mediated collagen gene expression. The RNA polymerase II inhibitor actinomycin D (Act D) decreased procollagen ,1[I] mRNA expression and effectively blocked the titanium-induced suppressive effect, suggesting that titanium particles activate a cascade of signals in osteoblasts, which result in a suppression of procollagen ,1[I] mRNA. Collectively, these results show that titanium particles can activate NF-,B signaling in osteoblasts and suggest that NF-,B binding to the collagen gene promoter has a functional role in the down-regulation of procollagen ,1[I] gene transcription. [source]

Prevalence of Actinobacillus actinomycetemcomitans in an ethnic adult Chinese population

JOURNAL OF CLINICAL PERIODONTOLOGY, Issue 9 2001
Kai Soo Tan
Abstract Aim: The aim of this study was to determine the prevalence and the structure of the leukotoxin promoter region of Actinobacillus actinomycetemcomitans in an ethnic Chinese population. Method: Subgingival plaque samples were collected from 42 patients with moderate to advanced periodontitis and 50 periodontally healthy patients. A. actinomycetemcomitans was detected directly from the crude subgingival plaque by PCR using leukotoxin gene specific primers. The presence of A. actinomycetemcomitans was determined by a single 285 bp PCR amplicon. Results:A. actinomycetemcomitans was found to be present in the subgingival plaque of 68 out of a total of 92 patients examined (74%). 29 out of the 42 periodontitis patients tested were carriers of A. actinomycetemcomitans (69%). Among the periodontally healthy patients studied, 39 out of 50 subjects possessed the bacteria (78%). PCR analysis of the promoter region of the ltx operon revealed that none of the 42 moderate to advanced periodontitis patients examined harboured A.actinomycetemcomitans strains with the JP2-like promoter of the ltx operon, known to enhance leukotoxin expression. 2 out of the 27 advanced periodontitis patients clinically diagnosed as suffering from rapidly progressive periodontitis were found to be carriers of the mildly toxic strain of A. actinomycetemcomitans with the characteristic 652-like promoter. Conclusions: The high prevalence of A. actinomycetemcomitans, regardless of whether the subgingival samples were analysed from patients with healthy or diseased periodontium suggests that this bacterial species is part of the normal oral flora of ethnic Chinese. Our preliminary results also suggested that subjects who harboured the mildly toxic strain of A. actinomycetemcomitans were potentially susceptible to aggressive forms of periodontitis. Zusammenfassung Das Ziel dieser Studie war es, in einer ethnischen Population von Chinesen die Prävalenz von Actinobacillus actinomycetemcomitans und die Struktur der Leukotoxin-Promoterregion zu bestimmen. Von 42 Patienten mit moderater bis fortgeschrittener Parodontitis und 50 parodontal gesunden Patienten wurden subgingivale Plaqueproben entnommen. A. actinomycetemcomitans wurde direkt in der unbehandelten subgingivalen Plaque durch PCR unter Verwendung eines Leukotoxingen-spezifischen Primers nachgewiesen. Das Vorhandensein von A. actinomycetemcomitans wurde mittels eines einzigen 285 bp-PCR-Amplikons bestimmt. Es wurde A. actinomycetemcomitans bei 68 von 92 untersuchten Patienten (74%) vorgefunden. 29 von 42 getesteten Parodontitispatienten waren Träger von A. actinomycetemcomitans (69%). Unter den Studierten parodontal gesunden Patienten besaßen 39 von 50 Personen das Bakterium (78%). Die PCR-Analyse der Promoterregion des ltx -Operons zeigte, dass keiner der 42 untersuchten Patienten mit moderater bis fortgeschrittener Parodontitis den A. actinomycetemcomitans mit dem JP2-ähnlichen Promoter des ltx -Operons, welches die Leukotoxinexpression verstärkt, besaß. Bei 2 der 27 Patienten mit fortgeschrittener Parodontitis wurde klinisch eine rasch fortschreitende Parodontitis diagnostiziert und es wurde der mit geringer Toxizität versehene Stamm des A. actinomycetemcomitans mit dem charakteristischen 652-ähnlichen Promoter vorgefunden. Bedingt durch die hohe Prävalenz von A. actinomycetemcomitans unabhängig davon, ob die Proben von Patienten mit gesundem oder erkranktem Parodontium stammen, lässt sich annehmen, dass diese Bakterienspezies bei ethnischen Chinesen ein Teil normalen Mundflora ist. Unsere vorläufigen Resultate lassen auch annehmen, dass Personen, die den mit geringer Toxizität versehenen Stamm des A. actinomycetemcomitans tragen eine potentielle Anfälligkeit für aggressive Formen der Parodontitis besitzen. Résumé Le but de l'étude présente a été de déterminer la fréquence globale et la structure de la région promoteur de leukotoxine de l'Actinobacillus actinomycetemcomitans (A.a.) dans une population chinoise. Des échantillons de plaque dentaire sous-gingivale ont été prélevés chez 42 patients avec parodontite modérée à avancée et chez 50 patients sains. L'A.a. a été détecté directement dans la plaque sous-gingivale par PCR en utilisant les sites spécifiques de gènes leukotoxines. La présence de l'A.a. a été déterminée par un amplicon PCR de 285 bp. L'A.a. a été décelé dans la plaque sous-gingivale de 68 des 92 patients examinés (74%). 29 des 42 patients avec parodontite ont été reconnus comme porteurs d'A.a. (69%). Parmi les patients sains étudiés, 39 des 50 sujets étaient porteurs de la bactérie (78%). L'analyse PCR de la région promoteur de operon ltx a révélé que des 42 patients avec parodontite modéréà avancée aucun n'avaient de souche A.a. avec le promoteur ressemblant au JP2 de l'operon ltx, reconnu pour acroître la leukotoxine. 2 des 27 patients avec parodontit avancée souffraient d'une parodontite progressant rapidement et étaient porteurs d'une souche moyennement toxique d'A.a. avec la caractéristique du promoteur ressemblant au 652. La fréquence globale importante d'A.a., sans tenir compte si les échantillons sous-gingivaux ont été analysés de patients avec un parodonte sain ou malade, suggère que ces espèces bactériennes font partie de la flore buccale normale de l'ethnie chinoise. Ces résultats indiquent également que les porteurs de la souche peu toxique d'A.a. seraient potentiellement susceptibles à des formes de parodontite agressive. [source]

In vitro Transient Expression System of Latex C-serum was used for Analysis of Hevein Promoter in Response to Abscisic Acid in Hevea brasiliensis

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 3 2008
Xiao-Wen Fei
Abstract Hevein has been found to be an essential element in coagulation of rubber particles in latex of rubber trees. In a previous study, we cloned a 1 241-bp fragment of a 5, upstream region of the hevein gene by genome walking. This fragment was analyzed by a 5, end nested deletion method in the present study, fused with a uidA (gus) gene to produce a series of tested constructs, which were transferred into C-serum of latex and the Gus activities were detected. Results showed that the fragment from ,749 to ,292 was sufficient for expression of gus gene in latex, and the fragment from ,292 to ,168 was crucial in response to abscisic acid inducement. In a transient transgenic test of rubber leaf with particle bombardment, construct Hev749 conferred gus -specific expression in veins, in which the latex tubes mainly distributed. This implies that the fragment from ,749 to ,292 was laticiferous-specific. [source]

Structure of the Mouse Glutamate Decarboxylase 65 Gene and Its Promoter

JOURNAL OF NEUROCHEMISTRY, Issue 4 2000
Preferential Expression of Its Promoter in the GABAergic Neurons of Transgenic Mice
Abstract: GABA is synthesized by glutamate decarboxylase (GAD), which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65 gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75 kb. The sequence of the first exon and the 5,-flanking region indicated the presence of potential neuron-specific cis -regulatory elements. We used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DNA fragment of the mGAD65 gene fused to the bacterial lacZ reporter gene. Transgenic mice showed high ,-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DNA fragment of the mGAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression. [source]

Ligand-independent Regulation of the hairless Promoter by Vitamin D Receptor,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2008
Andrew Engelhard
The characteristic alopecia associated with mutations in the hairless (hr) and vitamin D receptor (VDR) genes defines the resulting genetic disorders, known as atrichia and VDRRIIa rickets, as phenocopies. In both cases, the separation of the dermal papilla from the regressing hair follicle at the onset of the first catagen phase of the hair cycle and the development of dermal cysts and utricules subsequent to mutation of either gene suggests that their activities affect the same regulatory pathways. VDR functions as a hormonally activated transcription factor, and a role in transcription has been postulated for Hr due in part to its nuclear localization and homology with the GATA-1 zinc-finger domain. Therefore, we examined the hypothesis that VDR and Hr have a direct regulatory effect on each other via a transcriptional mechanism. Ectopic expression of the VDR repressed hr promoter activity in HaCaT cells and primary human keratinocytes (PHKs). While this repression occurs in the absence of 1,25 dihydroxyvitamin D3 (D3), the addition of ligand greatly augments the effect. However, we also demonstrate the rare phenomenon of ligand-independent promoter transactivation by VDR. We show that the full-length promoter is transactivated by VDR in a ligand-independent and cell type-specific manner, suggesting that direct transcriptional regulation of hr by the VDR accounts in part for the phenotypic overlap between atrichia and VDRRIIa rickets. [source]

Photochemical Internalization of Transgenes Controlled by the Heat-shock Protein 70 Promoter

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2006
Lina Prasmickaite
ABSTRACT Photochemical internalization (PCI) is a targeting technique that facilitates endosomal escape of macromolecules, such as transgenes, in response to photochemical treatment with endosome/lysosome-localized photosensitizers, such as disul-fonated meso-tetraphenylporphine (TPPS2a). In gene therapy this leads to enhanced transgene expression. Moreover, photochemical treatment generally activates transcription of stress-response genes, such as heat-shock proteins (HSPs), via stimulation of corresponding promoters. Therefore, we used HSP70 (HSPp; a promoter from the HSP family gene) and investigated whether the PCI stimulus could also activate HSPp and thereby stimulate transcription (expression) of the HSPp-controlled transgene internalized via PCI. Using human colorectal carcinoma and hepatoma cell lines in vitro, we showed that TPPS2a -based photochemical treatment enhances expression of cellular HSP70, which correlated with a photo-chemically enhanced expression (approximately 2-fold, at PCI-optimal doses) of the HSPp-controlled transgene integrated in the genome. Furthermore, PCI enhanced expression of the HSPp-controlled episomal transgene delivered as a plas-mid. However, in plasmid-based transfection, PCI-mediated enhancement with HSPp did not exceed the enhancement achieved with the constitutive active CMV promoter. In conclusion, we demonstrated that the PCI-relevant treatment initiates HSP70 response and that the HSP70 promoter can be used in combination with PCI, leading to PCI-enhanced expression of the HSPp-controlled transgene. [source]

Bound Transcription Factor Suppresses Photoproduct Formation in the NF-,B Promoter,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2001
Rita Ghosh
ABSTRACT The relationship between purified transcription factor p50 binding and ultraviolet light,induced DNA damage formation in the NF-,B promoter element was investigated. The effect of bound transcription factor on cyclobutane dimer formation was quantified using Maxam,Gilbert analysis of irradiated substrate digested with T4 phage endonuclease V. Two methods were employed for cleaving (6-4) photoproducts. Sites of (6-4) photoproducts cleaved by piperidine showed a general suppression in the presence of bound p50 protein similar to that observed for cyclobutane dimers. In contrast to piperidine, digestion with ultraviolet damage endonuclease (UVDE) from Saccharomyces pombe subsequent to cyclobutane dimer reversal by photolyase displayed a broader spectrum of damaged sites. Whereas some of these sites were suppressed by bound p50 protein, some remained unaffected and one site showed increased (6-4) photoproduct induction. These data illustrate the advantage of UVDE over piperidine for studying (6-4) photoproducts at the sequence level and suggest that this approach may be useful for footprinting transcription factor binding in other promoters. [source]

ORIGINAL ARTICLE: Haplotype-dependent Differential Activation of the Human IL-10 Gene Promoter in Macrophages and Trophoblasts: Implications for Placental IL-10 Deficiency and Pregnancy Complications

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2010
Surendra Sharma
Citation Sharma S, Stabila J, Pietras L, Singh AR, McGonnigal B, Ernerudh J, Matthiesen L, Padbury JF. Haplotype-dependent differential activation of the human IL-10 gene promoter in macrophages and trophoblasts: Implications for placental IL-10 deficiency and pregnancy complications. Am J Reprod Immunol 2010; 64: 179,187 Problem, Polymorphic changes in the IL-10 gene promoter have been identified that lead to altered IL-10 production. We hypothesized that because of these genotypic changes, the IL-10 promoter might be expressed in a cell type,specific manner and may respond differentially to inflammatory triggers. Method of study, We created reporter gene promoter constructs containing GCC, ACC, and ATA haplotypes using DNA from patients harboring polymorphic changes at ,1082 (G,A), ,819 (C,T), and ,592 (C,A) sites in the IL-10 promoter. These individual luciferase reporter constructs were transiently transfected into either primary term trophoblasts or THP1 monocytic cells. DNA-binding studies were performed to implicate the role of the Sp1 transcription factor in response to differential promoter activity. Results, Our results suggest that the GCC promoter construct was activated in trophoblast cells in response to lipopolysaccharide (LPS), as demonstrated by reporter gene expression, but not in monocytic cells. The ACC construct showed weaker activation in both cell types. Importantly, while the ATA promoter was constitutively activated in both cell types, its expression was selectively repressed in response to LPS, but only in trophoblasts. DNA-nuclear protein binding assays with nuclear extracts from LPS treated or untreated cells suggested a functional relevance for Sp1 binding differences at the ,592 position. Conclusions, These results demonstrate cell type,specific effects of the genotypic changes in the IL-10 gene promoter. These responses may be further modulated by bacterial infections or other inflammatory conditions to suppress IL-10 production in human trophoblasts. [source]

Efficient Gene Expression System Using the RTP801 Promoter in the Corpus Cavernosum of High-Cholesterol Diet-Induced Erectile Dysfunction Rats for Gene Therapy

THE JOURNAL OF SEXUAL MEDICINE, Issue 6 2008
Minhyung Lee PhD
ABSTRACT Introduction., The application of gene therapy for a nonlife-threatening disease, such as erectile dysfunction (ED), requires a higher safety level and more efficacious systems for gene transfer. Aim., To establish a novel technique for gene expression in a rat model of hypercholesterolemic ED that uses the RTP801 promoter, a hypoxia-inducible promoter. Methods., Two-month-old male Sprague,Dawley rats were fed a diet containing 4% cholesterol and 1% cholic acid, and age-matched control animals were fed a normal diet, for 3 months. Main Outcome Measures., Cavernous expression of hypoxia-inducible factor (HIF)-1, was evaluated by Western blot. After intracavernous injection of pSV-Luc or pRTP801-Luc, gene expression was evaluated by luciferase assay, and the gene expression area was evaluated by immunohistochemistry. Results., HIF-1, was up-regulated in the corpus cavernosum of hypercholesterolemic rats. Although pSV-Luc did not induce gene expression in either the control or the cholesterol group, pRTP801-Luc significantly induced gene expression in the cholesterol group and resulted in higher luciferase activity than did pSV-Luc up to 14 days after injection. Immunohistochemistry showed that the gene expression area was also greater in the pRTP801-Luc group than in the pSV-Luc group, but the difference was not as great as that in luciferase activity. This suggests that pRTP801-Luc exerts its effect mainly by inducing promoter activity under hypoxia, not by increasing the number of transfected cells. Conclusion., The RTP801 promoter-driven gene expression system increased gene expression in the corpus cavernosum tissue of rats with cholesterol-induced ED. This may be a useful system for the development of gene therapy in vasculogenic ED. Lee M, Ryu J-K, Piao S, Choi MJ, Kim HA, Zhang L-W, Shin H-Y, Jung HI, Kim I-H, Kim SW, and Suh J-K. Efficient gene expression system using the RTP801 promoter in the corpus cavernosum of high-cholesterol diet-induced erectile dysfunction rats for gene therapy. J Sex Med 2008;5:1355,1364. [source]

ABA-Hypersensitive Germination1 encodes a protein phosphatase 2C, an essential component of abscisic acid signaling in Arabidopsis seed

THE PLANT JOURNAL, Issue 6 2007
Noriyuki Nishimura
Summary The phytohormone abscisic acid (ABA) regulates physiologically important stress and developmental responses in plants. To reveal the mechanism of response to ABA, we isolated several novel ABA-hypersensitive Arabidopsis thaliana mutants, named ahg (ABA- hypersensitive germination). ahg1-1 mutants showed hypersensitivity to ABA, NaCl, KCl, mannitol, glucose and sucrose during germination and post-germination growth, but did not display any significant phenotypes in adult plants. ahg1-1 seeds accumulated slightly more ABA before stratification and showed increased seed dormancy. Map-based cloning of AHG1 revealed that ahg1-1 has a nonsense mutation in a gene encoding a novel protein phosphatase 2C (PP2C). We previously showed that the ahg3-1 mutant has a point mutation in the AtPP2CA gene, which encodes another PP2C that has a major role in the ABA response in seeds (Yoshida et al., 2006b). The levels of AHG1 mRNA were higher in dry seeds and increased during late seed maturation , an expression pattern similar to that of ABI5. Transcriptome analysis revealed that, in ABA-treated germinating seeds, many seed-specific genes and ABA-inducible genes were highly expressed in ahg1-1 and ahg3-1 mutants compared with the wild-type. Detailed analysis suggested differences between the functions of AHG1 and AHG3. Dozens of genes were expressed more strongly in the ahg1-1 mutant than in ahg3-1. Promoter,GUS analyses demonstrated both overlapping and distinct expression patterns in seed. In addition, the ahg1-1 ahg3-1 double mutant was more hypersensitive than either monogenic mutant. These results suggest that AHG1 has specific functions in seed development and germination, shared partly with AHG3. [source]

Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis

THE PLANT JOURNAL, Issue 1 2003
Lukas Bürkle
Summary Nucleobases and derivatives like cytokinins and caffeine are translocated in the plant vascular system. Transport studies in cultured Arabidopsis cells indicate that adenine and cytokinin are transported by a common H+ -coupled high-affinity purine transport system. Transport properties are similar to that of Arabidopsis purine transporters AtPUP1 and 2. When expressed in yeast, AtPUP1 and 2 mediate energy-dependent high-affinity adenine uptake, whereas AtPUP3 activity was not detectable. Similar to the results from cell cultures, purine permeases (PUP) mediated uptake of adenine can be inhibited by cytokinins, indicating that cytokinins are transport substrates. Direct measurements demonstrate that AtPUP1 is capable of mediating uptake of radiolabeled trans -zeatin. Cytokinin uptake is strongly inhibited by adenine and isopentenyladenine but is poorly inhibited by 6-chloropurine. A number of physiological cytokinins including trans- and cis- zeatin are also efficient competitors for AtPUP2-mediated adenine uptake, suggesting that AtPUP2 is also able to mediate cytokinin transport. Furthermore, AtPUP1 mediates transport of caffeine and ribosylated purine derivatives in yeast. Promoter,reporter gene studies point towards AtPUP1 expression in the epithem of hydathodes and the stigma surface of siliques, suggesting a role in retrieval of cytokinins from xylem sap to prevent loss during guttation. The AtPUP2 promoter drives GUS reporter gene activity in the phloem of Arabidopsis leaves, indicating a role in long-distance transport of adenine and cytokinins. Promoter activity of AtPUP3 was only found in pollen. In summary, three closely related PUPs are differentially expressed in Arabidopsis and at least two PUPs have properties similar to the adenine and cytokinin transport system identified in Arabidopsis cell cultures. [source]

Carbon Monoxide as a Promoter for its own Oxidation on a Gold Electrode,

ANGEWANDTE CHEMIE, Issue 7 2010
Paramaconi Rodríguez Dr.
Treffpunkt Gold: Das vorgestellte Modell für die CO-Oxidation an Goldelektroden in alkalischem Medium geht davon aus, dass das CO die Adsorption des Oxidationsmittels verstärkt. Dies erklärt die Reaktionsordnung von größer 1 und die Beobachtung, dass gelöstes CO leichter oxidiert wird als adsorbiertes CO. DFT-Rechnungen bestätigen, dass CO und OH jeweils die Bindung des anderen Adsorbats auf Au(111) verstärken. C,schwarz, O,rot, H,grau. [source]

Heterologous Protein Production from the Inducible MET25 Promoter in Saccharomyces cerevisiae

BIOTECHNOLOGY PROGRESS, Issue 2 2005
Steven P. Solow
Heterologous protein production late in Saccharomyces cerevisiae fermentations is often desirable because it may help avoid the unintentional selection of more rapidly growing, non-protein-expressing cells or allow for the expression of toxic proteins. Here, we describe the use of the MET25 promoter for the production of human serum albumin (HSA) and HSA-fusion proteins in S. cerevisiae. In media lacking methionine, the MET25 promoter yielded high expression levels of HSA and HSA fused to human glucagon, human growth hormone, human interferon ,, and human interleukin-2. More importantly, we have shown that this system can be used to delay heterologous protein production until late log phase of the growth of the culture and does not require the addition of an exogenous inducer. [source]

Hypermethylation of the TSLC1 Gene Promoter in Primary Gastric Cancers and Gastric Cancer Cell Lines

CANCER SCIENCE, Issue 8 2002
Teiichiro Honda
The TSLC1 (tumor suppressor in lung cancer,1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non-small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non-cancerous gastric tissues, by bisulfite-SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO-III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non-cancerous gastric tissues, suggesting that this hypermethylation is a cancer-specific alteration. KATO-III and ECC10 cells retained two alleles of TSLC1, both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi-allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers. [source]

Promoter Methylation of TSLC1 and Tumor Suppression by Its Gene Product in Human Prostate Cancer

CANCER SCIENCE, Issue 6 2002
Hiroshi Fukuhara
We recently identified TSLC1, a tumor suppressor gene in human lung cancer. Gene silencing by promoter methylation has been observed frequently in adenocarcinoma of the lung, liver, and pancreas. Here, we demonstrate that TSLC1 expression is also absent or markedly reduced in 3 of 4 prostate cancer cell lines. Promoter sequences of TSLC1 were heavily methylated in PPC-1 cells that lacked TSLC1 expression, supporting the idea that promoter methylation is strongly correlated with complete loss of gene expression. Promoter sequences of TSLC1 were also methylated significantly in 7 of 22 (32%) primary prostate cancers. Hypermethylation of the promoter occurred not only in advanced tumors, but also in relatively early-stage tumors. Restoration of TSLC1 expression substantially suppressed tumor formation of PPC-1 cells in nude mice. These findings indicate that alteration of TSLC1 is involved in prostate cancer. [source]

ChemInform Abstract: An Efficient Protocol for Multicomponent Stereoselective Synthesis of 3-Amino-2(1H)-pyridinones Using CeCl3×7H2O/NaI as a Reaction Promoter.

CHEMINFORM, Issue 4 2009
Lal Dhar S. Yadav
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source]

ChemInform Abstract: epi-Cinchona-Based Thiourea Organocatalyst Family as an Efficient Asymmetric Michael Addition Promoter: Enantioselective Conjugate Addition of Nitroalkanes to Chalcones and ,,,-Unsaturated N-Acylpyrroles.

CHEMINFORM, Issue 33 2008
Benedek Vakulya
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source]

Tf2O as a Rapid and Efficient Promoter for the Dehydrative Friedel,Crafts Acylation of Aromatic Compounds with Carboxylic Acids.

CHEMINFORM, Issue 39 2007
Mohammd Mehdi Khodaei
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source]

Palladium-Catalyzed Alkylation of Aryl C,H Bonds with sp3 Organotin Reagents Using Benzoquinone as a Crucial Promoter.

CHEMINFORM, Issue 19 2006
Xiao Chen
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source]

A New Biginelli Reaction Procedure Using Potassium Hydrogen Sulfate as the Promoter for an Efficient Synthesis of 3,4-Dihydropyrimidin-2(1H)-one.

CHEMINFORM, Issue 36 2004
Shujiang Tu
Abstract For Abstract see ChemInform Abstract in Full Text. [source]

Characterization of sleep,wake patterns in a novel transgenic mouse line overexpressing human prepro-orexin/hypocretin

ACTA PHYSIOLOGICA, Issue 3 2010
K. A. Mäkelä
Abstract Aim:, Orexin/hypocretin peptides are expressed in the lateral hypothalamus and involved in the regulation of autonomic functions, energy homeostasis and arousal states. The sleep disorder narcolepsy, which is characterized by excessive daytime sleepiness and occurrence of sudden rapid eye movement (REM) sleep, is associated with a loss of orexin neurones. Our study investigated the effects of orexins on sleep,wake patterns in a novel transgenic mouse line overexpressing the human prepro-orexin (hPPO) gene under the control of its endogenous promoter. Methods:, Orexin overexpression was investigated by PCR, Southern and Western blotting as well as immunohistochemistry. Polysomnographic recordings were performed for analyses of sleep,wake patterns and for electroencephalographic activity during 24 h baseline and during and after 6 h of sleep deprivation (SD). Results:, Transgenic hPPO mice had increased expression of human prepro-orexin (hPPO) and orexin-A in the hypothalamus. Transgene expression decreased endogenous orexin-2 receptors but not orexin-1 receptors in the hypothalamus without affecting orexin receptor levels in the basal forebrain, cortex or hippocampus. Transgenic mice compared with their wild type littermates showed small but significant differences in the amount of waking and slow wave sleep, particularly during the light,dark transition periods, in addition to a slight reduction in REM sleep during baseline and during recovery sleep after SD. Conclusion:, The hPPO-overexpressing mice show a small reduction in REM sleep, in addition to differences in vigilance state amounts in the light/dark transition periods, but overall the sleep,wake patterns of hPPO-overexpressing mice do not significantly differ from their wild type littermates. [source]

Thyroid hormone receptor , can control action potential duration in mouse ventricular myocytes through the KCNE1 ion channel subunit

ACTA PHYSIOLOGICA, Issue 2 2010
A. Mansén
Abstract Aims:, The reduced heart rate and prolonged QTend duration in mice deficient in thyroid hormone receptor (TR) ,1 may involve aberrant expression of the K+ channel ,-subunit KCNQ1 and its regulatory ,-subunit KCNE1. Here we focus on KCNE1 and study whether increased KCNE1 expression can explain changes in cardiac function observed in TR,1-deficient mice. Methods:, TR-deficient, KCNE1-overexpressing and their respective wildtype (wt) mice were used. mRNA and protein expression were assessed with Northern and Western blot respectively. Telemetry was used to record electrocardiogram and temperature in freely moving mice. Patch-clamp was used to measure action potentials (APs) in isolated cardiomyocytes and ion currents in Chinese hamster ovary (CHO) cells. Results:, KCNE1 was four to 10-fold overexpressed in mice deficient in TR,1. Overexpression of KCNE1 with a heart-specific promoter in transgenic mice resulted in a cardiac phenotype similar to that in TR,1-deficient mice, including a lower heart rate and prolonged QTend time. Cardiomyocytes from KCNE1-overexpressing mice displayed increased AP duration. CHO cells transfected with expression plasmids for KCNQ1 and KCNE1 showed an outward rectifying current that was maximal at equimolar plasmids for KCNQ1-KCNE1 and decreased at higher KCNE1 levels. Conclusion:, The bradycardia and prolonged QTend time in hypothyroid states can be explained by altered K+ channel function due to decreased TR,1-dependent repression of KCNE1 expression. [source]

A case study of shell at Sakhalin: having a whale of a time?

CORPORATE SOCIAL RESPONSIBILITY AND ENVIRONMENTAL MANAGEMENT, Issue 3 2008
Subhasis Ray
Abstract This is a case study on the world's largest oil and gas project, at the Sakhalin Islands, Russia. Shell is the key promoter of this project. The case highlights the sustainability challenges that Shell faced when working on the mega-project. By their very nature, all such projects involve disruptions in the environmental and social fabric of the project site. NGOs often take up these issues and create international headlines, bringing pressure on the management team. The Russian government also changed its stand over a period of time. While many of these issues are valid in their own way, they often create managerial dilemmas. Traditional management approaches to community development and environmental conservation fell short of stakeholder expectations at Sakhalin. The issue of saving around 100 endangered whales put a cloud of doubt over this $20 billion project. The case highlights strategic issues involved in crafting sustainability strategies at mega-projects, possible pitfalls and the challenge of balancing project execution and stakeholder commitments against an unstable political backdrop. As Shell plans to start many exploration projects in bio-diversity rich parts of the world, the Sakhalin project acts as a pilot to and reminder of social responsibility challenges to big multi-nationals. Copyright © 2007 John Wiley & Sons, Ltd and ERP Environment. [source]