230 resultados para Silenciamento de EGFP


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At surgical depths of anesthesia, inhalational anesthetics cause a loss of motor response to painful stimuli (i.e., immobilization) that is characterized by profound inhibition of spinal motor circuits. Yet, although clearly depressed, the respiratory motor system continues to provide adequate ventilation under these same conditions. Here, we show that isoflurane causes robust activation of CO(2)/pH-sensitive, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) of the rodent brainstem, in vitro and in vivo. In brainstem slices from Phox2b-eGFP mice, the firing of pH-sensitive RTN neurons was strongly increased by isoflurane, independent of prevailing pH conditions. At least two ionic mechanisms contributed to anesthetic activation of RTN neurons: activation of an Na(+)-dependent cationic current and inhibition of a background K(+) current. Single-cell reverse transcription-PCR analysis of dissociated green fluorescent protein-labeled RTN neurons revealed expression of THIK-1 (TWIK-related halothane-inhibited K(+) channel, K(2P)13.1), a channel that shares key properties with the native RTN current (i.e., suppression by inhalational anesthetics, weak rectification, inhibition by extracellular Na(+), and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO(2) levels. In these animals, isoflurane transiently enhanced activity of the respiratory system, an effect that was most prominent at low levels of respiratory drive and mediated primarily by an increase in respiratory frequency. These data indicate that inhalational anesthetics cause activation of RTN neurons, which serve an important integrative role in respiratory control; the increased drive provided by enhanced RTN neuronal activity may contribute, in part, to maintaining respiratory motor activity under immobilizing anesthetic conditions.

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A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation. Biotechnol. Bioeng. 2010; 107: 469-477. (C) 2010 Wiley Periodicals, Inc.

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Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. (C) 2011 Elsevier Inc. All rights reserved.

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Esta pesquisa aborda a experiência de avaliação realizada na e pela UNEMAT que tem legitimado, em sua prática, duas modalidades de avaliação: o PAIUNEMAT e o PROVÃO. A primeira, está sustentada pelos princípios do PAIUB e foi implantada a partir da decisão da própria universidade; a segunda, foi imposta pelo MEC. Este estudo teve como objetivo estudar estas modalidades de avaliação em desenvolvimento e entender as atitudes de silenciamento e pouca participação dos docentes no processo. Propôs-se a pensar sobre os motivos destas atitudes, partindo do pressuposto de que, quando estamos em silêncio, talvez, pretendamos dizer alguma coisa. Neste estudo o silêncio é entendido na perspectiva de Orlandi (1997) como algo que significa, que tem sentido. A avaliação é um processo de produção de conhecimento emancipatório, estudada na perspectiva teórica de Santos (1999, 2000), House (2000) e Saul (2000). Tomei como caminho à pesquisa etnográfica, em busca das vozes que foram entrelaçadas na tessitura dos fios do silêncio. Utilizei entrevistas (12) com professores do Campus de Cáceres e com gestores da UNEMAT, observações das relações estabelecidas entre PAIUNEMAT, gestores e comunidade acadêmica e análise de documentos do período de 1996 a 2000 referentes à avaliação institucional. Este estudo mostrou que o PAIUNEMAT é uma proposta que visa a produção de conhecimento emancipatório, nos modelos teóricos de Santos e Saul, mas que na prática ainda não se consolidou; e que o PROVÃO, uma proposta de avaliação do tipo análise de sistemas na perspectiva liberal utilitarista discutida por House, que visa a regulação, está se fortalecendo cada vez mais na instituição O silêncio dos docentes frente à avaliação apresenta-se como atitudes de não envolvimento, pouca participação, resistências, resignação, indiferença e algumas falas pelos corredores, atitudes que levam ao mais extremo comportamento de fuga e de autodefesa que é o silêncio. O silêncio dos docentes significa um descontentamento ao que está dado, ao PAIUNEMAT, porque não efetivou na prática a sua proposta; e ao PROVÃO, por ser uma imposição e não respeitar a identidade institucional. Este silêncio é político, é uma forma de resistência e pode ter um sentido de não querer se expor, medo de coerção frente às relações de poder que estão estabelecidas e que levam ao silenciamento e à resistência, comportamentos construídos histórico e culturalmente.

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Esta dissertação propõe um estudo sobre o funcionamento do discurso que se constrói em torno do feto, a partir dos diálogos que ocorrem nos exames de ultra-sonografia obstétrica. Um total de 30 exames foram gravados e transcritos, realizados em uma clínica particular e em um serviço público de saúde; gestantes de idade, nível social e escolaridade diferentes participaram da pesquisa, pacientes de quatro médicos que colaboraram com o trabalho. A análise foi construída a partir do referencial teórico e metodológico proposto pela análise de discurso da escola francesa; a teoria psicanalítica foi utilizada para aprofundar as questões pertinentes ao conceito de sujeito, autorizada pelos pontos de tensão existentes entre a psicanálise e análise de discurso. O funcionamento do discurso sobre o feto, no corpus analisado, apontou para a existência de duas formações discursivas: o saber médico, formação dominante, e o senso-comum, visível especialmente quando o desejo e a fantasia dos pais se manifestam com maior intensidade. Foram identificados enunciados que remetem ao sujeito do desejo inconsciente, instigando uma discussão entre psicanálise e análise de discurso. O discurso médico apareceu caracterizado pelos conceitos de normalidade e anormalidade, atrelado a uma promessa por parte da ciência de ver, prever e controlar que seduz os pais e destinado a participar da construção e reconfiguração do imaginário dos pais à medida em que fala sobre o feto. O silêncio da gestante foi analisado enquanto legitimador da posição do médico e, ao mesmo tempo, produto de uma política do silenciamento que administra o sentido. A análise de discurso foi contemplada pelas questões teóricas e metodológicas, na tentativa de compreender o funcionamento do discurso. À psicanálise reservaram-se as discussões teóricas e à psicologia do desenvolvimento foi proposta uma discussão de aspectos pertinentes à gestação na relação que se estabelece entre gestante, médico e tecnologia, dentro de uma abordagem discursiva.

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O jornalismo, entendido como construtor de sentidos sobre a realidade, é um discurso que deve representar a diversidade de pensamento da sociedade contemporânea. Nesta pesquisa, buscamos responder se os jornais O Globo e Folha de S. Paulo, principais jornais de referência no Brasil, tratam de forma equilibrada a pluralidade de vozes para falar sobre o tema da Aids. Usando conceitos da Análise de Conteúdo e Análise do Discurso, realizamos um mapeamento dos temas abordados nos textos e, na seqüência, das fontes de informação, verificando os movimentos de dominância e silenciamento dos diferentes grupos. Foram analisados 310 textos jornalísticos sobre HIV/Aids, o total publicado nesses dois jornais no ano de 2004. Verificamos que a Aids foi tratada, na maioria das vezes, como uma questão grave de saúde pública que deve ser combatida pelos países desenvolvidos. Mesmo com o domínio de fontes oficiais, através do conceito de enunciador identificamos que as vozes dominantes foram as que cobraram ações dos governos na luta contra a Aids. Registramos o cruzamento de enunciadores nos textos, o que caracterizou a pluralidade de vozes; contudo, a fala das pessoas vivendo com HIV/Aids praticamente não esteve presente nos jornais pesquisados. Desta forma, não houve um equilíbrio nas vozes em textos sobre HIV/Aids.

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Uma significativa quantidade de proteínas vegetais apresenta-se compartimentalizada nas diversas estruturas celulares. A sua localização pode conduzir à elucidação do funcionamento dos processos biossintéticos e catabólicos e auxiliar na identificação de genes importantes. A fim de localizar produtos gênicos relacionados à resistência, foi utilizada a fusão de cDNAs de arroz (Oryza sativa L.) ao gene da proteína verde fluorescente (GFP). Os cDNAs foram obtidos a partir de uma biblioteca supressiva subtrativa de genes de arroz durante uma interação incompatível com o fungo Magnaporthe grisea. Estes cDNAs foram fusionados a uma versão intensificada de gfp e usados para transformar 500 plantas de Arabidopsis thaliana. Outras 50 plantas foram transformadas com o mesmo vetor, porém sem a fusão (vetor vazio). Foram obtidas aproximadamente 25.500 sementes oriundas das plantas transformadas com as fusões EGFP::cDNAs e 35.000 sementes das transformadas com o vetor vazio, produzindo, respectivamente, 750 e 800 plantas tolerantes ao herbicida glufosinato de amônio. Após a seleção, segmentos foliares das plantas foram analisados por microscopia de fluorescência, visando o estabelecimento do padrão de localização de EGFP. Foram observadas 18 plantas transformadas com a fusão EGFP::cDNAs e 16 plantas transformadas com o vetor vazio apresentando expressão detectável de GFP. Uma planta transformada com uma fusão EGFP::cDNA apresentou localização diferenciada da fluorescência, notadamente nas células guarda dos estômatos e nos tricomas. Após seqüenciamento do cDNA fusionado, foi verificado que esta planta apresentava uma inserção similar a uma seqüência codificante de uma quinase, uma classe de enzimas envolvidas na transdução de sinais em resposta à infecção por patógenos.

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In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

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This thesis is a result of a research on Natal/RN as a tourist destination. We understand that cities are chosen as tourist destinations beyond its cartographic localization, from other dimensions of meanings that, in its set, constitute images. These images are, probably, very different of the images constructed by native and resident populations, who possess relations of identity with the place. The knowledge of the meanings that others attribute to this city as tourist destination, bring us near to the symbolic bonds established by individuals or social groups on the act of their choices, as well as bring us near to the interaction process city-tourists where the expectations are confirmed or not. The images divulgated by the media also participate of the complex formation of the tourist image that is being constructed and available for the different public, in different social contexts. The tourism constitutes a symbolic asset of the modern society, being considered by the studious, as one of the most expressive phenomena of the modernity, for involving each year displacement and the interaction of thousand of people of different cultures in the entire world. All this people s mobilization points to practical social related to personal motivations, to the entailed desired to the idea to travel and to exceed borders. It is already consensus that tourism is a phenomenon of economic growth, generating jobs, income, professional, qualification, bringing improvements for the host cities. Since 1995, in Brazil, the tourism as a sector of the economy, passed to be considered one of the national priorities, and in this perspective, the national politics of the tourism invested in infrastructure of Brazilian cities with high tourist potential, objecting to increase the flows of Brazilian and foreign tourists. Owing to this fact, the country still invests in programs of tourist marketing, mainly divulging the images of the natural beauties of Brazil abroad. And for Brazilians, the campaigns appeal to rescue the feeling to be Brazilian, associating the idea to travel and know its country. Natal city possesses an excellent positioning in the tourist marketing, being predominantly divulgated in national and international level, for its naturalistic singularity, where the images of its natural enchantments as warm water beaches, white dunes, warm weather, constant breeze and an always blue sky are shown as the favorite scene on this city. From what was viewed above that the choice of a tourist destination articulates from a determined imaginary of a place, already constructed or in process of construction, we consider the knowledge of this imaginary a basic learning for the population of the city and especially, for educators, in the formation of professionals in this area and for tourism managers, elaborators of public politics. Based on this estimative, we developed this research that had as a general objective to identify the images that illustrate Natal city as a tourist destination - our objective of study, particularly the meanings and senses attributed by the tourist marketing (hotel s folders) and by the tourists that visited the city during this study. The discussions and reflections that had guided this research had been given from the theoretical link between imaginary and social representation, also considering some interfaces between the fields of communication and symbol. From the studied authors, Baczko (1985) clarifies that the study of social imaginary is directed for the mechanisms and structures of the social life, especially for the intervention accomplishes and efficient of the representations and symbols in the practical collectives, as well as in its direction and orientation . Following this same thought, Moscovici (1978) says that the social representation are produced in communicational and symbolic contexts, and these representations once that already constituted circulate socially as almost tangible entities. Based on this fundament and on the analyze of Barthes (1990), particularly in the approach given to the reading of photographic image, we could observe on hotel s folders that each page evidences senses and meanings of functionality of internal and external spaces, pointing to the way of leisure offered by the keepers of city which is the hotels. About, the leisure that they offer, it is directed to young public, giving meaning to the young myth of personalized leisure tourism on children, young and adults images. The image about security that hotels offer and the singular image of Natal city as a paradise place, provide an idealization of pleasure through the sun, dunes, and beaches and also due to the hospitability of the natives who are assigned as educated . For the tourist that participated on this research, Natal city is tied only by the imaginary of leisure and nature which constitute the emotional link of the relation media-city-tourist. And with such force and fullness of directions the city discloses without tensions and contradictions as a place protected by a mythical and sacred aura. The study also demonstrates us that the potiguar culture remains (almost) forgotten, due to the silenced in this imaginary. In this perspective, we highlight that this culture silence is very close related to the disvalue of education in its general meaning. We defend that the imaginary apprehended constitutes a new reading and a new looking and understanding the tourist reality that comes historically consolidating in this city. In this direction, we glimpse that this study and its future dismemberments can collaborate with the process of rescue the cultural values of the potiguar people, in the way that the meaning of tourist may be redefined, and the tourist image of the city can be also disclosed for its identities particularities of its culture

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We believe that the dissatisfaction arising from the lack of belief in the possibilities of change in the workplace, which cause difficulties to achieve professional results in the professional psychological distress that currently fits into the context of mental health. This is a qualitative, descriptive and representational research aiming to discover how the professional nurses represent the very psychological distress from work in the hospital environment. Aided and supported by specific objectives of identifying factors that generates this suffering and strategies for defense and confronting these professionals in the hospital. 22 nurses participated in this research, officials of the University Hospital Onofre Lopes, located in the city of Natal / RN, with length of service in the institution more than one year and less than five, and they accepted, by signing the Term of Free and Informed Consent, participate in the study. We use plurimethodological approach: a questionnaire, a semi-structured interview and the design-story with a theme adapted from Trinca with the support of the Theory of Social Representations and that nurses do in their psychological distress of the Central Core. We reviewed the data from the results generated by the ALCESTE software, based on hierarchical categorization downward, leading seven classes used as categories: Work process: completeness vs. incompleteness; labor contradiction of the nurse; qualitative aspects of interpersonal relationships; hospital surveillance: Challenges, muteness and neglect; Expectations, conflicts and feelings in the work process; Leisure: the other side of the work process, and Suffering generating aspects of in the work process. We consider the analysis of quarters generated by the program, which SLQ houses in the central core of the representations; the SRQ and the DLQ the intermediaries elements and the DRQ the peripheral elements that nurses do in their psychological distress. We analytically adequate results in the three belonging dimensions of social representations: the Subjectivity, the Intersubjectivity and Trans-subjectivity. We infer that the interpersonal relationship, the extra work, the deviation in the role of nurse show themself as the factors responsible for psychological distress of it. In that sense, the central core of SR of this profession is based on the level of trans-subjectivity and understood as a Social Representation controversy

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Broadly speaking, the concept of gene therapy involves the transfer of a genetic material into a cell, tissue, or organ in order to cure a disease or at least improve the clinical status of a patient. Making it simple, gene therapy consists in the insertion of functional genes into cells containing defective genes by substituting, complementing or inhibiting them. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, it is a key issue to create systems able to transfer and protect the DNA until it reaches the target, the vectors. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easily produced, present controllable stability and enable transfection. The aim of this work was to develop an emulsion for gene therapy and evaluate its ability to compact nucleic acids by the development of a complex with the plasmid pIRES2-EGFP. The first step was to determine the Hydrophilic Lipophilic Balance (HLB) of the Captex® 355 (oily internal phase of the emulsion) through long and short term stability assays. Based on the results, emulsions composed of Captex® 355, Tween 20® and Span 60® with 10.7 HLB were produced by three different methods: phase inversion, spontaneous emulsification and sonication. The results showed that the lowest diameter and best stability of the emulsions were achieved by the sonication method. The cationic emulsions were made by adding DOTAP to the basic emulsion. Its association with pIRES2-EGFP was evaluated by electrophoresis. Several rates of emulsion and DNA were evaluated and the results showed that 100% of the complex was formed when the rate DOTAP/DNA(nmol/µg) was 130. In conclusion, the overall results show the ability of the proposed emulsion to compact pIRES2-EGFP, which is a requirement to a successful transfection. Therefore, such formulation may be considered a promising candidate for gene therapy

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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There is a molecular crosstalk between the trophoblast and maternal immune cells of bovine endometrium. The uterine cells are able to secrete cytokine/chemokines to either induce a suppressive environment for establishment of the pregnancy or to recruit immune cells to the endometrium to fight infections. Despite morphological differences between women and cows, mechanisms for immune tolerance during pregnancy seem to be conserved. Mechanisms for uterine immunesuppression in the cow include: reduced expression of major histocompatability proteins by the trophoblast; recruitment of macrophages to the pregnant endometrium; and modulation of immune-related genes in response to the presence of the conceptus. Recently, an eGFP transgenic cloned embryo model developed by our group showed that there is modulation of foetal proteins expressed at the site of syncytium formation, suggesting that foetal cell can regulate not only by the secretion of specific factors such as interferon-tau, but also by regulating their own protein expression to avoid excessive maternal recognition by the local immune system. Furthermore, foetal DNA can be detected in the maternal circulation; this may reflect the occurrence of an invasion of trophoblast cells and/or their fragment beyond the uterine basement membrane in the cow. In fact, the newly description of exosome release by the trophoblast cell suggests that could be a new fashion of maternal-foetal communication at the placental barrier. Additionally, recent global transcriptome studies on bovine endometrium suggested that the immune system is aware, from an immunological point of view, of the presence of the foetus in the cow during early pregnancy.

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Osteoblast-derived IL-6 functions in coupled bone turnover by supporting osteoclastogenesis favoring bone resorption instead of bone deposition. Gene regulation of IL-6 is complex occurring both at transcription and post-transcription levels. The focus of this paper is at the level of mRNA stability, which is important in IL-6 gene regulation. Using the MC3T3-E1 as an osteoblastic model, IL-6 secretion was dose dependently decreased by SB203580, a p38 MAPK inhibitor. Steady state IL-6 mRNA was decreased with SB203580 (2 μM) ca. 85% when stimulated by IL-1β (1-5 ng/ ml). These effects require de novo protein synthesis as they were inhibited by cycloheximide. p38 MAPK had minor effects on proximal IL-6 promoter activity in reporter gene assays. A more significant effect on IL-6 mRNA stability was observed in the presence of SB203580. Western blot analysis confirmed that SB203580 inhibited p38 MAP kinase, in response to IL-1β in a dose dependent manner in MC3T3-E1 cells. Stably transfected MC3T3-E1 reporter cell lines (MC6) containing green fluorescent protein (GFP) with the 3′untranslated region of IL-6 were constructed. Results indicated that IL-1β, TNFα, LPS but not parathyroid hormone (PTH) could increase GFP expression of these reporter cell lines. Endogenous IL-6 and reporter gene eGFP-IL-6 3′UTR mRNA was regulated by p38 in MC6 cells. In addition, transient transfection of IL-6 3′UTR reporter cells with immediate upstream MAP kinase kinase-3 and -6 increased GFP expression compared to mock transfected controls. These results indicate that p38 MAPK regulates IL-1β-stimulated IL-6 at a post transcriptional mechanism and one of the primary targets of IL-6 gene regulation is the 3′UTR of IL-6.