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‣ Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles

Borges, Olga; Cordeiro-da-Silva, Anabela; Tavares, Joana; Santarém, Nuno; Sousa, Adriano de; Borchard, Gerrit; Junginger, Hans E.
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Artigo de Revista Científica Formato: aplication/PDF
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Alginate coated chitosan nanoparticles were previously developed with the aim of protecting the antigen, adsorbed on the surface of those chitosan nanoparticles, from enzymatic degradation at mucosal surfaces. In this work, this new delivery system was loaded with the recombinant hepatitis B surface antigen (HBsAg) and applied to mice by the intranasal route. Adjuvant effect of the delivery system was studied by measuring anti-HBsAg IgG in serum, anti-HBsAg sIgA in faeces extracts or nasal and vaginal secretions and interferon-[gamma] production in supernatants of the spleen cells. The mice were primed with 10 [mu]g of the vaccine associated or not with nanoparticles and associated or not with 10 [mu]g CpG oligodeoxynucleotide (ODN) followed by two sequential boosts at three week intervals. The association of HBsAg with the alginate coated chitosan nanoparticles, administered intranasally to the mice, gave rise to the humoral mucosal immune response. Humoral systemic immune response was not induced by the HBsAg loaded nanoparticles alone. The generation of Th1-biased antigen-specific systemic antibodies, however, was observed when HBsAg loaded nanoparticles were applied together with a second adjuvant, the immunopotentiator, CpG ODN. Moreover...

‣ CpG-Induced Th1-Type Response in the Downmodulation of Early Development of Allergy and Inhibition of B7 Expression on T Cells of Newborn Mice

BRITO, Cyro A. de; FUSARO, Ana E.; VICTOR, Jefferson R.; RIGATO, Paula O.; GOLDONI, Adriana L.; MUNIZ, Bruno P.; DUARTE, Alberto J. S.; SATO, Maria N.
Fonte: SPRINGER/PLENUM PUBLISHERS Publicador: SPRINGER/PLENUM PUBLISHERS
Tipo: Artigo de Revista Científica
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Several differences have been described between neonatal and adult immune responses. The predisposition in early life to Th2-type response or tolerance makes it a susceptible period for infections and allergic sensitization. The aim of this work was to evaluate the effects of CpG-containing oligodeoxynucleotides on neonatal and adult immunization with ovalbumin and Blomia tropicalis extract and compare the CpG effects on B and T cells of neonatal and adult mice. Mice that received CpG showed reduced immunoglobulin E (IgE) antibody production in both neonatal and adult periods, in parallel to increased IgG2a antibody levels. We observed that spleen cells of mice that received CpG in early life produced increased amounts of interferon-gamma upon anti-CD3 stimulation. Negative regulation of IgE response was more pronounced in adult than neonate mice; further, CpG decreased anaphylactic antiovalbumin IgG1 only in adults. Also, an upregulation of toll-like receptor 9 expression was detected in adult B cells, but not in neonatal, upon CpG stimuli. Neonatal B cells showed enhanced interleukin (IL)-10 expression and decreased IL-6 levels than adult B cells in response to CpG. When we analyzed in vitro activation of CD4+ T cells, an increased expression of B7 molecules on T cells in neonates was suppressed by CpG. Altogether...

‣ CpG-ODN combined with Neospora caninum lysate, but not with excreted-secreted antigen, enhances protection against infection in mice

RIBEIRO, Damaso P.; FREITAS, Marina M. P.; CARDOSO, Mariana R. D.; PAJUABA, Ana C. A. M.; SILVA, Neide M.; MINEO, Tiago W. P.; SILVA, Joao S.; MINEO, Jose R.; SILVA, Deise A. O.
Fonte: ELSEVIER SCI LTD Publicador: ELSEVIER SCI LTD
Tipo: Artigo de Revista Científica
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CpG oligodeoxynucleotides (ODN) have shown to be potent immunoadjuvants for several pathogens, but there is limited information concerning their use in immunization protocols against neosporosis. This study aimed to evaluate the potential of CpG-ODN combined with Neosporar lysate antigen (NLA) or excreted-secreted antigen (NcESA) to induce protective immune response against Neospora caninum infection in mice. C57BL/6 mice were vaccinated subcutaneously three times at 2-week intervals with NLA, NLA+CpG, NcESA, NcESA+CpG, CpG (adjuvant control) or PBS (infection control). Serological assays showed an increased specific IgG2a response in animals immunized with either antigen plus adjuvant and elevated levels of the IgG1 isotype in those vaccinated with antigens alone. Splenocyte proliferative responses upon antigen stimulation were higher in groups immunized with NLA OF NcESA combined with CpG, showing increased IL-12 levels. Also, mice vaccinated with NcESA or NcESA+CpG demonstrated higher IFN-gamma levels and IFN-gamma/IL-10 ratio. After lethal challenge, mice immunized with NLA+CpG or NLA had lower Morbidity score and body weight changes in comparison to other groups, and animals did not succumb during acute infection. In contrast...

‣ Stable assemblies of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity in vivo

Rozenfeld, Julio H. K.; Silva, Sandriana dos Ramos; Raneia, Priscila A.; Faquim-Mauro, Eliana; Carmona-Ribeiro, Ana M.
Fonte: ELSEVIER SCIENCE BV; AMSTERDAM Publicador: ELSEVIER SCIENCE BV; AMSTERDAM
Tipo: Artigo de Revista Científica
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The cationic lipid dioctadecyldimethylammonium bromide (DODAB) and the CpG oligonucleotide (CpG) have been separately used as potent immunoadjuvants driving Th1 responses. Here DODAB bilayer fragments (BF) and CpG (5 -TTGACGTTCG-3) assemblies have their physical properties and immunoadjuvant activity determined using ovalbumin (OVA) as a model antigen. At 0.1 mg/mL OVA, the dependence of DODAB BF/OVA size and zeta-potential on time and [DODAB] establishes 0.1 mMDODAB as suitable for obtaining stable and cationic DODAB BF/OVA assemblies. At 0.1 mMDODAB, 0.1 mg/mL OVA and 0.006 mMCpG, the zeta-potential is zero. At [CpG]>0.006 mM, good colloidal stability for the anionic assemblies is due to charge overcompensation. At 0.020 mM CpG, these DODAB BF/OVA/CpG assemblies are highly effective in vivo generating responses similar to those elicited by the stable and cationic DODAB BF/OVA. The anti-OVA DTH reaction and the secretion of IFN-gamma and IL-12 are 6, 42 and 9 times larger for the DODAB BF/OVA/CpG-immunized mice than the same responses by OVA-immunized mice, respectively. This work shows for the first time that charge of small assemblies is not important to determine the immune response. (C) 2011 Elsevier B. V. All rights reserved.; FAPESP; CNPq

‣ The effect of CpG-ODN on antigen presenting cells of the foal

Julia, M. Julia B.F.; Borges, Alexandre S.; Nydam, Daryl V.; Horohov, David W.; Hecker, Rolf; Matychak, Mary Beth
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica
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Background: Cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals. Methods: Peripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14-16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNα, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-κB p65 using a chemiluminescence assay. Results: The median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs...

‣ A new CPG model for the generation of modular trajectories for hexapod robots

Pinto, Carla M. A.; Rocha, Diana; Santos, Cristina; Matos, Vítor
Fonte: American Institute of Physics Publicador: American Institute of Physics
Tipo: Conferência ou Objeto de Conferência
Publicado em //2011 Português
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Legged robots are often used in a large variety of tasks, in different environments. Nevertheless, due to the large number of degrees-of-freedom to be controlled, online generation of trajectories in these robots is very complex. In this paper, we consider a modular approach to online generation of trajectories, based on biological concepts, namely Central Pattern Generators (CPGs). We introduce a new CPG model for hexapod robots’ rhythms, based in the work of Golubitsky et al (1998). Each neuron/oscillator in the CPG consists of two modules/primitives: rhythmic and discrete. We study the effect on the robots’ gaits of superimposing the two motor primitives, considering two distinct types of coupling.We conclude, from the simulation results, that the amplitude and frequency of periodic solutions, identified with hexapods’ tripod and metachronal gaits, remain constant for the two couplings, after insertion of the discrete part.

‣ Matrix protein CCN1 induced by bacterial DNA and CpG ODN limits lung inflammation and contributes to innate immune homeostasis

Moon, Hyung-Geun; Qin, Zhaoping; Quan, Taihao; Xie, Lixin; Dela Cruz, Charles S.; Jin, Yang
Fonte: Harvard University Publicador: Harvard University
Tipo: Artigo de Revista Científica
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Summary To defend against pulmonary infections, lung epithelial cells are equipped with complex innate immunity closely linked to inflammation. Dysregulated innate immunity / inflammation leads to self-perpetuating lung injury. The CpG motif in bacterial DNA is one of the factors involved in bacterial infection-associated inflammation. Bacterial DNA and synthetic CpG oligonucleotide (ODN) induced CCN1 secretion from lung epithelial cells, functioning as a potential “braking” signal to prevent uncontrolled inflammatory responses. CpG ODN-induced ER stress resulted in Src-Y527 phosphorylation (pY527) and Src/CCN1 vWF domain dissociation. Src-Y527 activated caveolin-1 (cav-1) phosphorylation at Y14 and then modulated CCN1 secretion via pCav-1 interaction with CCN1 IGFbp domain. Functionally, secreted CCN1 promoted anti-inflammatory cytokine IL-10 release from epithelial cells via integrin αVβ6 PKC, and this subsequently suppressed TNF-α, MIP-2 secretion and neutrophil infiltration in the lungs. Collectively, bacterial DNA/CpG ODN-stimulated CCN1 secretion via BiP/GRP78-Src(Y527)-JNK-Cav-1(Y14) pathway and CpG-induced CCN1 conferred anti-inflammatory roles. Our studies suggested a novel paradigm by which the lung epithelium maintains innate immune homeostasis after bacterial infection.

‣ Correlação da expressão do gene CXCL12 com o perfil de metilação de ilhas de CpG na região promotora em linhagens tumorais de mama

Klassen, liliane Maria Bacaro
Fonte: Universidade Federal do Paraná Publicador: Universidade Federal do Paraná
Tipo: Dissertação Formato: application/pdf
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Resumo: O câncer de mama é o tipo mais comum e a principal causa de morte por câncer entre as mulheres no mundo todo. As metástases contribuem com 90% das causas de morte por câncer. O estudo de novos marcadores moleculares de câncer estão em amplo e atual desenvolvimento, tanto para diagnóstico como prognóstico da doença. Diversos estudos tem mostrado o papel de eventos epigenéticos entre eles a metilação do DNA, em regiões promotoras de genes como um evento importante no processo de tumorigênese em diversos tipos de câncer. No câncer de mama já foram descritos um grande número de genes envolvidos com controle da proliferação, adesão e migração celular que podem ser silenciados por mecanismos epigenéticos. O gene CXCL12 codifica para uma quimiocina ou citocina quimiotática, que é comprovadamente silenciado por metilação de sua região promotora em câncer gástrico, de cólon intestinal e de mama. O produto deste gene esta envolvido no processo migratório como molécula ligante de células tumorais que superexpressam o receptor CXCR4 e que migram e colonizam secundariamente pulmões ossos, fígado ou linfonodos que tem altos níveis de CXCL12. Nesse trabalho estudamos detalhadamente a região promotora do gene CXCL12 que contém 5 regiões ricas em dinucleotídeos CpGs (ilhas de CpG). As ilhas de CpG 1 ...

‣ Imunização de camundongos com antígenos de lisado total (NLA) e de excreção-secreção (NcESA) de Neospora caninum associados com oligodeoxinucleotídeo CpG como adjuvante

Ribeiro, Dâmaso Pacheco
Fonte: Universidade Federal de Uberlândia Publicador: Universidade Federal de Uberlândia
Tipo: Dissertação
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Neospora caninum é um protozoário do filo Apicomplexa que causa doenças neuromusculares em cães e abortos em bovinos, acarretando significativas perdas econômicas. Estudos em modelos murinos têm contribuído para caracterizar novos antígenos e estratégias para procedimentos de vacinação. Oligodeoxinucleotídeos (ODN) com seqüências CpG são considerados potentes imunoadjuvantes para vários patógenos, mas há limitada informação sobre sua utilização em vacinação contra neosporose. O objetivo deste estudo foi avaliar o potencial de antígenos de lisado total (NLA) e de excreção-secreção (NcESA) de N. caninum combinados com ODN-CpG na indução de resposta imune e proteção contra infecção por N. caninum em camundongos. Seis grupos de camundongos C57BL6 foram imunizados subcutaneamente com NLA, NLA+CpG, NcESA, NcESA+CpG, CpG (controle do adjuvante) ou PBS (controle da infecção), com três reforços em intervalos de 2 semanas. Análises sorológicas mostraram aumento na resposta de IgG2a anti-N. caninum em grupos de animais imunizados com qualquer antígeno combinado com adjuvante CpG e níveis elevados do isotipo IgG1 naqueles grupos contendo somente antígenos. Respostas proliferativas após estimulação antigênica in vitro foram mais elevadas nos grupos imunizados com antígenos associados com CpG...

‣ Methylation of CpG sites in exon 2 of the Bcl-2 gene occurs in colorectal carcinoma

Babidge, W.; Butler, L.; Burton, M.; Cowled, P.
Fonte: Int Inst Anticancer Research Publicador: Int Inst Anticancer Research
Tipo: Artigo de Revista Científica
Publicado em //2001 Português
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BACKGROUND: Aberrant bcl-2 expression frequently occurs in colorectal carcinoma. The current study investigated if CpG sites in bcl-2 were methylated in colorectal carcinoma and if methylation correlated with loss of expression of bcl-2 mRNA. METHODS: Methylation was assessed in 23 matched normal mucosae and colonic carcinomas by Southern blotting with methylation-sensitive enzymes. Expression of bcl-2 mRNA was assessed by Northern blotting. RESULTS: A SacII site in exon 2 of the bcl-2 gene was methylated in 5 carcinomas, plus an adjacent HpaII sites in 1 tumour. SacII site in the bcl-2 promoter were not methylated. Elevated levels of bcl-2 mRNA were detected in 3 carcinomas, 5 showed decreased expression and 4 were unchanged. CONCLUSIONS: De novo methylation of CpG sites in exon 2 of the bcl-2 gene occurs during the development of colorectal carcinoma. However, there was no relationship between expression of bc1-2 mRNA and methylation of specific CpG sites.; Babidge WJ; Butler LM; Burton MA; Cowled PA; © 2001 International Institute of Anticancer Research

‣ Methylation levels of LINE-1 repeats and CpG island loci are inversely related in normal colonic mucosa

Iacopetta, B.; Grieu, F.; Phillips, M.; Ruszkiewicz, A.; Moore, J.; Minamoto, T.; Kawakami, K.
Fonte: Business Center Academic Societies Japan Publicador: Business Center Academic Societies Japan
Tipo: Artigo de Revista Científica
Publicado em //2007 Português
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Hypermethylation of CpG island loci within gene promoter regions is a frequent event in colorectal cancer that is often associated with transcriptional silencing and has been referred to as CIMP+. DNA hypomethylation can occur in concert with CIMP+, although these two phenomena appear not to be related in colorectal cancer. The authors investigated here whether the methylation level of LINE-1 repeats, a surrogate marker for genomic methylation, was associated with the level of CpG island methylation in colorectal cancers and in matching normal colonic mucosa from 178 patients. The MethyLight assay was used to quantitate the methylation of CpG islands within the MLH1, P16(INK4A), TIMP3, DAPK, APC, ER and MYOD genes. A real-time, methylation-specific polymerase chain reaction assay was also used to quantitate the methylation of LINE-1 repeats. In colorectal cancer, no associations were seen between methylation levels in LINE-1 repeats and CpG island loci, including a new CpG island panel that was recently proposed for CIMP+. In normal colonic mucosa, however, the methylation level of LINE-1 repeats was inversely correlated with CpG-island methylation of the MLH1, P16, TIMP3, APC, ER and MYOD genes. The methylation level of LINE-1 repeats in normal colonic mucosa also showed significant associations with common polymorphisms in the methylene tetrahydrofolate reductase and methylene tetrahydrofolate dehydrogenase genes involved in methyl group metabolism. Further investigation of genomic and CpG island methylation in normal colonic mucosa and the possible influences of environmental and genetic factors may provide new insights into the development of CIMP+ colorectal cancer.; Barry Iacopetta...

‣ LL-37 promotes rapid sensing of CpG oligodeoxynucleotides by B lymphocytes and plasmacytoid dendritic cells

Hurtado, P.; Peh, C.
Fonte: Amer Assoc Immunologists Publicador: Amer Assoc Immunologists
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
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LL-37 is a cationic antimicrobial peptide derived from neutrophils and keratinocytes. It plays an important role in protection against bacterial infection in the skin and mucosal surfaces. However, its role within the blood compartment remains unclear given that serum inhibits its bactericidal property. In this study, we show that LL-37 promotes very rapid and highly efficient sensing of CpG motifs in bacterial DNA by human B lymphocytes and plasmacytoid dendritic cells (pDCs) in serum-containing media and in whole blood. LL-37 allowed detection of CpG oligodeoxynucleotide (ODN) within minutes of exposure. Without LL-37, 20-30 times more CpG was required to produce the same effect. The promotion of CpG detection by LL-37 was independent of the backbone of the ODN, as the effect was observed not only in ODNs with modified phosphorothioate backbone, but also in ODNs with natural phosphodiester backbone, as found in genomic DNA. Unmethylated CpG motifs within the phosphodiester ODN and LL-37-mediated delivery are required for pDCs to respond. In keeping with the above, cells responded to CpG-rich bacterial DNA and LL-37, but not to human DNA and LL-37. The ability of LL-37 to enhance delivery of CpG to stimulate immune cells is independent of its amphipathic structure and its bactericidal property. LL-37 aids the delivery of CpG to B cells and pDCs...

‣ Collaboration between CpG sites is needed for stable somatic inheritance of DNA methylation states

Haerter, J.; Lovkvist, C.; Dodd, I.; Sneppen, K.
Fonte: Oxford Univ Press Publicador: Oxford Univ Press
Tipo: Artigo de Revista Científica
Publicado em //2014 Português
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Inheritance of 5-methyl cytosine modification of CpG (CG/CG) DNA sequences is needed to maintain early developmental decisions in vertebrates. The standard inheritance model treats CpGs as independent, with methylated CpGs maintained by efficient methylation of hemimethylated CpGs produced after DNA replication, and unmethylated CpGs maintained by an absence of de novo methylation. By stochastic simulations of CpG islands over multiple cell cycles and systematic sampling of reaction parameters, we show that the standard model is inconsistent with many experimental observations. In contrast, dynamic collaboration between CpGs can provide strong error-tolerant somatic inheritance of both hypermethylated and hypomethylated states of a cluster of CpGs, reproducing observed stable bimodal methylation patterns. Known recruitment of methylating enzymes by methylated CpGs could provide the necessary collaboration, but we predict that recruitment of demethylating enzymes by unmethylated CpGs strengthens inheritance and allows CpG islands to remain hypomethylated within a sea of hypermethylation.; Jan O. Haerter, Cecilia Lövkvist, Ian B. Dodd and Kim Sneppen; Published online 27 November 2013

‣ Prediction of CpG-island function: CpG clustering vs. sliding-window methods

Hackenberg, Michael; Barturen, Guillermo; Carpena, Pedro; Luque-Escamilla, Pedro Luis; Previti, Christopher; Oliver, Jos?? Luis
Fonte: Biomed Central Publicador: Biomed Central
Tipo: Artigo de Revista Científica
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[Background] Unmethylated stretches of CpG dinucleotides (CpG islands) are an outstanding property of mammal genomes. Conventionally, these regions are detected by sliding window approaches using %G + C, CpG observed/expected ratio and length thresholds as main parameters. Recently, clustering methods directly detect clusters of CpG dinucleotides as a statistical property of the genome sequence. [Results] We compare sliding-window to clustering (i.e. CpGcluster) predictions by applying new ways to detect putative functionality of CpG islands. Analyzing the co-localization with several genomic regions as a function of window size vs. statistical significance (p-value), CpGcluster shows a higher overlap with promoter regions and highly conserved elements, at the same time showing less overlap with Alu retrotransposons. The major difference in the prediction was found for short islands (CpG islets), often exclusively predicted by CpGcluster. Many of these islets seem to be functional, as they are unmethylated, highly conserved and/or located within the promoter region. Finally, we show that window-based islands can spuriously overlap several, differentially regulated promoters as well as different methylation domains, which might indicate a wrong merge of several CpG islands into a single...

‣ The Impact of CpG Island on Defining Transcriptional Activation of the Mouse L1 Retrotransposable Elements

Lee, Sung-Hun; Cho, Soo-Young; Shannon, M. Frances; Fan, Jun; Rangasamy, Danny
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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BACKGROUND L1 retrotransposable elements are potent insertional mutagens responsible for the generation of genomic variation and diversification of mammalian genomes, but reliable estimates of the numbers of actively transposing L1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of L1 elements, several phylogenetic and L1Xplore analyses in the mouse genome suggest that 1,500-3,000 active L1 elements currently exist and that they are still expanding in the genome. Conversely, the human genome contains only 150 active L1 elements. In addition, there is a discrepancy among the nature and number of mouse L1 elements in L1Xplore and the mouse genome browser at the UCSC and in the literature. To date, the reason why a high copy number of active L1 elements exist in the mouse genome but not in the human genome is unknown, as are the potential mechanisms that are responsible for transcriptional activation of mouse L1 elements. METHODOLOGY/PRINCIPAL FINDINGS We analyzed the promoter sequences of the 1,501 potentially active mouse L1 elements retrieved from the GenBank and L1Xplore databases and evaluated their transcription factors binding sites and CpG content. To this end, we found that a substantial number of mouse L1 elements contain altered transcription factor YY1 binding sites on their promoter sequences that are required for transcriptional initiation...

‣ CpGcluster: a distance-based algorithm for CpG-island detection

Hackenberg, Michael; Previti, Christopher; Luque-Escamilla, Pedro Luis; Carpena, Pedro; Mart??nez-Aroza, Jos??; Oliver, Jos?? Luis
Fonte: Biomed Central Publicador: Biomed Central
Tipo: Artigo de Revista Científica
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[Background] Despite their involvement in the regulation of gene expression and their importance as genomic markers for promoter prediction, no objective standard exists for defining CpG islands (CGIs), since all current approaches rely on a large parameter space formed by the thresholds of length, CpG fraction and G+C content. [Results] Given the higher frequency of CpG dinucleotides at CGIs, as compared to bulk DNA, the distance distributions between neighboring CpGs should differ for bulk and island CpGs. A new algorithm (CpGcluster) is presented, based on the physical distance between neighboring CpGs on the chromosome and able to predict directly clusters of CpGs, while not depending on the subjective criteria mentioned above. By assigning a p-value to each of these clusters, the most statistically significant ones can be predicted as CGIs. CpGcluster was benchmarked against five other CGI finders by using a test sequence set assembled from an experimental CGI library. CpGcluster reached the highest overall accuracy values, while showing the lowest rate of false-positive predictions. Since a minimum-length threshold is not required, CpGcluster can find short but fully functional CGIs usually missed by other algorithms. The CGIs predicted by CpGcluster present the lowest degree of overlap with Alu retrotransposons and...

‣ Methylation of a CpG island within the uroplakin lb promoter: A possible mechanism for loss of uroplakin lb expression in bladder carcinoma

Varga, A.; Leondardos, L.; Jackson, P.; Marreiros, A.; Cowled, P.
Fonte: Nature America Inc Publicador: Nature America Inc
Tipo: Artigo de Revista Científica
Publicado em //2004 Português
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Uroplakin Ib is a structural protein on the surface of urothelial cells. Expression of uroplakin Ib mRNA is reduced or absent in many transitional cell carcinomas (TCCs) but molecular mechanisms underlying loss of expression remain to be determined. Analysis of the uroplakin Ib promoter identified a weak CpG island spanning the proximal promoter, exon 1, and the beginning of intron 1. This study examined the hypothesis that methylation of this CpG island regulates uroplakin Ib expression. Uroplakin Ib mRNA levels were determined by reverse transcription polymerase chain reaction and CpG methylation was assessed by bisulfite modification of DNA, PCR, and sequencing. A correlation was demonstrated in 15 TCC lines between uroplakin Ib mRNA expression and lack of CpG methylation. In support of a regulatory role for methylation, incubating uroplakin Ib-negative lines with 5-aza-2'-deoxycytidine reactivated uroplakin Ib mRNA expression. A trend between uroplakin Ib mRNA expression and CpG methylation was also observed in normal urothelium and bladder carcinomas. In particular, loss of uroplakin Ib expression correlated with methylation of a putative Sp1/NFkappaB binding motif. The data are consistent with the hypothesis that methylation of specific sites within the uroplakin Ib promoter may be an important factor in the loss of uroplakin Ib expression in TCCs.; Andrea E Varga...

‣ Variable promoter region CpG island methylation of the putative tumor suppressor gene Connexin 26 in breast cancer

Tan, L.W.; Bianco-Miotto, T.; Dobrovic, A.
Fonte: Oxford Univ Press Publicador: Oxford Univ Press
Tipo: Artigo de Revista Científica
Publicado em //2002 Português
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Intercellular communication via gap junctions is a mechanism for tumor suppression. Connexin 26 (Cx26) is a structural component of gap junctions expressed by breast epithelial cells. Expression levels of Cx26 are reduced in many breast tumors. Methylation-sensitive single-stranded conformation analysis showed variable methylation in the promoter region CpG island in 11 out of 20 (55%) breast cancer patients. Heterogeneity in methylation patterns was observed both between and within tumors. The degree of methylation ranged from a few CpG dinucleotides to almost all the CpG dinucleotides in the analyzed region. The most frequently methylated CpG was in an Sp1 site known to be important for Cx26 gene expression. One of eight breast cancer cell lines (MD-MBA-453) was methylated in the promoter region and did not express Cx26. Treatment of MDA-MB-453 with 5-aza-2'-deoxycytidine resulted in the re-expression of Cx26 mRNA. Methylation of the promoter region is likely to be an important mechanism in modulating the expression of Cx26 in breast cancer.; Lor-wai Tan, Tina Bianco and Alexander Dobrovic; © 2002 Oxford University Press

‣ Incorporation of CpG Oligodeoxynucleotides into α2-Macroglobulin: Development of a Novel Vaccine Adjuvant Delivery Mechanism

Anderson, Ryan Berger
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação Formato: 1502502 bytes; application/pdf
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Bacterial DNA is immunostimulatory, and the motifs responsible for this activity are unmethylated CpG dinucleotides. Following cellular uptake, CpG-containing oligodeoxynucleotides (CpG ODN) are trafficked to the endosome where they bind Toll-like receptor 9 (TLR9) to initiate a signaling cascade that culminates in the release of numerous pro-inflammatory cytokines. Because of their immunostimulatory properties, CpG ODN are being clinically evaluated as treatments and vaccine adjuvants for infectious diseases, cancer, and allergic disorders. α2-Macroglobulin (α2M) is a human plasma protein that binds and modulates the activity of a variety of cytokines, growth factors, enzymes, and antigens. Upon proteolytic activation, α2M is converted to its receptor recognized form, α2M*, and rapidly binds to and is internalized by immune competent cells expressing the α2M* endocytic receptor, LRP, and is then trafficked to the endosome. Based on these interactions, α2M seems to play an important role at sites of infection and inflammation by controlling the level of proteinase activity, modulating cytokine signals, and enhancing antigen processing for the adaptive immune response. Here, we report the first evidence that α2M* binds and forms stable complexes with nucleic acids. We have characterized the mechanisms and stoichiometry of this interaction...

‣ Genes con islas CpG amplificados con la mezcla formamida, albúmina sérica bovina y dimetilsulfóxido

Martínez-López,Miriam Carolina; Ruiz,Freddy De la Cruz; Cortes-Peñaloza,José Luis; Cadena-Sandoval,Daniel; Zamarrón-Licona,Erasmo
Fonte: Acta bioquímica clínica latinoamericana Publicador: Acta bioquímica clínica latinoamericana
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2012 Português
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Diversos aditivos químicos han sido utilizados para garantizar la polimerización de genes con islas CpG. El objetivo de este trabajo fue diseñar una mezcla potenciadora de PCR para amplificar genes con islas CpG. Con ese fin se analizaron fragmentos de los genes IRS2 y HNF1a con el programa EMBOSS CpG Report. Los iniciadores se diseñaron con el programa Primer 3 y se analizaron con el programa e-PCR. Se usaron tres aditivos químicos: Albúmina sérica bovina (0,1µg/µL), dimetilsulfóxido (5%) y formamida (5%) para 5 ensayos de PCR: dos usando un solo aditivo, dos combinando dos aditivos y uno combinando tres aditivos. Las amplificaciones con las mezclas se realizaron con las enzimas Taq Nativa, taq Recombinante y Taq Platinum. La calidad de los amplicones se probó por secuenciación. Fragmentos sin islas CpG (HNF-1a) amplificaron con las tres enzimas, sin el uso de los aditivos pero presentaron problemas de pureza en la secuenciación. Los fragmentos del gen IRS2 con islas CpG amplificaron sólo con la combinación de tres aditivos dimetilsulfóxido, albúmina sérica bovina y formamida, independientemente de la enzima usada, las secuencias fueron limpias. Se concluye que la mezcla de tres aditivos es una solución que permite obtener amplicones de alta calidad en genes con islas CpG...