RESUMO
While it is well known that CD4(+) T cells and B cells collaborate for antibody production, our group previously reported that CD8(+) T cells down-regulate alloantibody responses following transplantation. However, the exact mechanism involved in CD8(+) T cell-mediated down-regulation of alloantibody remains unclear. We also reported that alloantibody production is enhanced when either perforin or FasL is deficient in transplant recipients. Here, we report that CD8(+) T cell-deficient transplant recipient mice (high alloantibody producers) exhibit an increased number of primed B cells compared to WT transplant recipients. Furthermore, CD8(+) T cells require FasL, perforin and allospecificity to down-regulate posttransplant alloantibody production. In vivo CD8-mediated clearance of alloprimed B cells was also FasL- and perforin-dependent. In vitro data demonstrated that recipient CD8(+) T cells directly induce apoptosis of alloprimed IgG1(+) B cells in co-culture in an allospecific and MHC class I-dependent fashion. Altogether these data are consistent with the interpretation that CD8(+) T cells down-regulate posttransplant alloantibody production by FasL- and perforin-dependent direct elimination of alloprimed IgG1(+) B cells.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteína Ligante Fas/metabolismo , Hepatócitos/imunologia , Isoanticorpos/imunologia , Perforina/metabolismo , Animais , Formação de Anticorpos , Western Blotting , Células Cultivadas , Citometria de Fluxo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Isoanticorpos/metabolismo , Transplante de Fígado , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transplante Homólogo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: We present the first reported case of a paraganglioma of the larynx occurring concurrently with squamous cell carcinoma in an irradiated neck. METHOD: We present a case report and a literature review of paraganglioma of the larynx. CASE REPORT: A 78-year-old woman was found to have a mass on the laryngeal surface of the epiglottis and non-functioning larynx, four months after radical radiotherapy for biopsy-proven squamous cell carcinoma. Repeated positron emission tomography computed tomography was highly suggestive of residual malignancy. Given these findings, and the fact that the patient was aspirating, she was treated with total laryngectomy, and was found to have a paraganglioma of the larynx. CONCLUSION: This is the first report of such a case. Its importance is three-fold in that it demonstrates: the coexistence of paraganglioma and squamous cell carcinoma; the difficulties that can be encountered in the diagnosis; and the use of radiotherapy in the treatment of paraganglioma.
Assuntos
Carcinoma de Células Escamosas/terapia , Epiglote/patologia , Neoplasias Laríngeas/diagnóstico por imagem , Neoplasias Primárias Múltiplas/diagnóstico por imagem , Paraganglioma/diagnóstico por imagem , Idoso , Biópsia , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/patologia , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Laríngeas/diagnóstico , Neoplasias Laríngeas/patologia , Neoplasias Laríngeas/terapia , Laringectomia , Laringoscopia , Imagem Multimodal , Esvaziamento Cervical , Neoplasias Primárias Múltiplas/patologia , Neoplasias Primárias Múltiplas/terapia , Paraganglioma/diagnóstico , Paraganglioma/patologia , Paraganglioma/cirurgia , Tomografia por Emissão de Pósitrons , Poluição por Fumaça de Tabaco , Tomografia Computadorizada por Raios X , Traqueostomia , Resultado do TratamentoRESUMO
BACKGROUND AND OBJECTIVE: Only a few clinical research studies have assessed different therapeutic approaches to oral malodor in subjects affected by periodontal diseases. The aim of this study was to evaluate the effects of periodontal treatment and tongue cleaning on oral malodor parameters in periodontitis and gingivitis patients. MATERIAL AND METHODS: The subjects were 102 periodontitis and 116 gingivitis patients with oral malodor. Oral malodor was measured by organoleptic test and Oral Chroma™. Oral health status, including tooth conditions, periodontal health, tongue coating and proteolytic activity of the BANA test in tongue coating were assessed. Subjects in each periodontal disease group were randomly assigned into two subgroups depending on the sequence of treatment: periodontal treatment and tongue cleaning. Oral malodor and oral health parameters were compared by groups and sequence of treatment. RESULTS: For subjects in the periodontitis group, there were statistically significant reductions in oral malodor after periodontitis treatment or tongue cleaning; however, major reductions were found after periodontitis treatment. For those in the gingivitis group, there were also statistically significant reductions in oral malodor after gingivitis treatment or tongue cleaning, but the most marked reductions were observed after tongue cleaning. At the completion of treatment, all oral malodor parameters fell below the threshold levels in all subgroups. CONCLUSION: The present study indicated that periodontal treatment played an important role and tongue cleaning contributed to a lesser extent to reduction in oral malodor in periodontitis patients. In contrast, tongue cleaning alone can be the primary approach to reduce oral malodor in gingivitis patients.
Assuntos
Gengivite/complicações , Gengivite/terapia , Halitose/terapia , Periodontite/complicações , Periodontite/terapia , Língua , Adulto , Benzoilarginina-2-Naftilamida , Testes Respiratórios , Distribuição de Qui-Quadrado , Índice de Placa Dentária , Raspagem Dentária , Feminino , Halitose/etiologia , Humanos , Sulfeto de Hidrogênio/análise , Masculino , Pessoa de Meia-Idade , Higiene Bucal , Índice Periodontal , Compostos de Sulfidrila/análise , Língua/química , Língua/microbiologia , Língua/patologiaRESUMO
Soybean rust, caused by the fungus Phakopsora pachyrhizi, was detected in the continental United States in 2004. Several new sources of resistance to P. pachyrhizi have been identified in soybean (Glycine max); however, there is limited information about their resistance when challenged with additional U.S. and international isolates. Resistance of 20 soybean (G. max) entries was compared after inoculation with 10 P. pachyrhizi isolates, representing different geographic and temporal origins. Soybean entries included 2 universal susceptible cultivars, 4 sources of soybean rust resistance genes (Rpp1-4), and 4 and 10 resistant entries selected from field trials in Paraguay and Vietnam, respectively. Of the known Rpp1-4 sources of resistance, plant introduction (PI) 459025B (Rpp4) produced reddish-brown (RB) lesions in response to all of the P. pachyrhizi isolates, while PI 230970 (Rpp2) produced RB lesions to all isolates except one from Taiwan, in response to which it produced a susceptible tan (TAN) lesion. PI 200492 (Rpp1) and PI 462312 (Rpp3) produced TAN lesions in response to most P. pachyrhizi isolates. The resistant entries selected from Paraguay and Vietnam varied considerably in their responses to the 10 P. pachyrhizi isolates, with M 103 the most susceptible and GC 84058-18-4 the most resistant. The reaction patterns on these resistant entries to the P. pachyrhizi isolates were different compared with the four soybean accessions with the Rpp genes, indicating that they contain novel sources of rust resistance. Among the P. pachyrhizi isolates, TW 72-1 from Taiwan and IN 73-1 from India produced the most susceptible and resistant reactions, respectively, on the soybean entries.
RESUMO
BACKGROUND: Increasing demand for dental services and a projected shortage in the oral health workforce in Victoria has focused attention on dental hygienists as one mechanism for increasing the supply of dental services. Understanding the dental hygienist workforce is essential in order to plan effectively for the future delivery of dental services in Victoria. METHODS: A postal survey of a random sample of Victorian dental hygienists was undertaken in 2006. Data on hygienists' demographic characteristics, current dental practice, history of career breaks, aspects of clinical practice and job satisfaction were collected. RESULTS: A response rate of 77 per cent was achieved. A total of 94.0 per cent of hygienists were currently working as a dental hygienist, working an average of 28.7 hours per week. Younger hygienists worked longer hours than their older colleagues. Career breaks were common, with 44.8 per cent reporting a career break of greater than one month, predominantly for child rearing, with a mean career break of 20.1 months. Hygienists reported a high level of satisfaction with most aspects of their employment. CONCLUSIONS: Victorian hygienists worked predominantly in private practices in metropolitan Melbourne, providing a range of preventive and periodontal services. Understanding the working patterns of dental hygienists is critical as hygienist numbers expand in the future, in order to undertake thorough evidence-based workforce planning.
Assuntos
Higienistas Dentários/psicologia , Satisfação no Emprego , Prática Profissional , Adulto , Higienistas Dentários/estatística & dados numéricos , Emprego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Salários e Benefícios , Fatores de Tempo , Vitória , Carga de TrabalhoRESUMO
BACKGROUND: Increasing the number of dental hygienists and expanding their scope of practice are two policy directions that are currently being explored to increase the supply of dental services in the context of projected oral health workforce shortages in Australia. Understanding factors relating to the employment of hygienists and the attitudes of the oral health workforce to dental hygiene practice are important in this policy debate. METHODS: A postal survey of a random sample of Victorian dentists, periodontists, orthodontists and hygienists was undertaken in 2006. Dentists and specialists were grouped into those whose practice employed or did not employ a hygienist. Data on the attitudes of dentists, specialists and hygienists towards various aspects of dental hygiene practice were explored. RESULTS: A response rate of 65.3 per cent was achieved. Hygienists believed that their employment made dental care more affordable (53.7 per cent) and improved access to dental care (88.1 per cent), while few dentists believed hygienists made care more affordable. Most hygienists believed they were capable of diagnosing periodontal disease and dental caries and formulating a treatment plan, but there was less support from employers and non-employers. Dentists were strongly opposed to independent practice for dental hygienists, although there was qualified support from employers for increasing the scope of practice for hygienists. CONCLUSIONS: Dentists who worked with hygienists acknowledged their contribution to increasing practice profitability, efficiency and accessibility of dental services to patients. Hygienists and employers supported increasing the scope of dental hygiene practice, however the majority of non-employers opposed any expansion.
Assuntos
Atitude do Pessoal de Saúde , Higienistas Dentários , Odontólogos/psicologia , Emprego , Prática Profissional , Adulto , Assistência Odontológica/economia , Cárie Dentária/diagnóstico , Higienistas Dentários/psicologia , Eficiência Organizacional , Feminino , Acessibilidade aos Serviços de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Ortodontia , Planejamento de Assistência ao Paciente , Doenças Periodontais/diagnóstico , Periodontia , Administração da Prática Odontológica/economia , Administração da Prática Odontológica/organização & administração , VitóriaRESUMO
Direct sequencing of mitochondrial DNA (mtDNA) D-loop (745 bp) and MTATPase6/MTATPase8 (857 bp) regions was used to investigate genetic variation within common carp and develop a global genealogy of common carp strains. The D-loop region was more variable than the MTATPase6/MTATPase8 region, but given the wide distribution of carp the overall levels of sequence divergence were low. Levels of haplotype diversity varied widely among countries with Chinese, Indonesian and Vietnamese carp showing the greatest diversity whereas Japanese Koi and European carp had undetectable nucleotide variation. A genealogical analysis supports a close relationship between Vietnamese, Koi and Chinese Color carp strains and to a lesser extent, European carp. Chinese and Indonesian carp strains were the most divergent, and their relationships do not support the evolution of independent Asian and European lineages and current taxonomic treatments.
Assuntos
Sequência de Bases/genética , Carpas/genética , DNA Mitocondrial , Variação Genética , Animais , Carpas/classificação , DNA Mitocondrial/isolamento & purificação , Haplótipos/genética , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
The development of precise connections in the mammalian brain proceeds through refinement of initially diffuse patterns, a process that occurs largely within critical developmental windows. To elucidate the molecular pathways that orchestrate these early periods of circuit remodeling, we have examined the role of a calcium- and cAMP-regulated transcriptional pathway. We show that there is a window of CRE/CREB-mediated gene expression in the developing thalamus, which precedes neocortical expression. In the LGN, this wave of gene expression occurs prior to visual experience, but requires retinal function. Mutant mice with reduced CREB expression show loss of refinement of retinogeniculate projections. These results suggest an important role of the CRE/CREB transcriptional pathway in the coordination of experience-independent circuit remodeling during forebrain development.
Assuntos
Axônios/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Corpos Geniculados/fisiologia , Integrases/metabolismo , Retina/fisiologia , Tálamo/fisiologia , Transcrição Gênica , Proteínas Virais/metabolismo , Vias Visuais/fisiologia , Envelhecimento , Animais , Cruzamentos Genéticos , Enucleação Ocular , Feminino , Heterozigoto , Integrases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tálamo/crescimento & desenvolvimento , Proteínas Virais/genética , beta-Galactosidase/genéticaRESUMO
OBJECTIVE: To analyze retrospectively results of reverse transcription polymerase chain reaction (RT-PCR) testing and demographic information in patients with known or suspected Ewing sarcoma/primitive neuroectodermal tumor family of tumors referred to the National Cancer Institute and to describe factors influencing the determination of molecular marker status. PATIENTS AND METHODS: Tumor samples from 76 patients from February 1997 to December 1999 were analyzed. In all cases, the diagnosis of this family of tumors was confirmed by histopathologic review. RESULTS: In 58 patients, the presence of a translocation associated with this family of tumors was confirmed using RT-PCR. Specifically, there were 45 Ewing sarcoma (EWS)-FLI type 1 translocations, four EWS-FLI type 2 translocations, five EWS-ERG translocations, and four less common EWS-FLI variants. Of patients with a confirmed translocation, four were confirmed only after nested RT-PCR techniques were used. In five patients who initially underwent needle biopsy, the diagnosis was confirmed only after open biopsy or repeat needle biopsy was undertaken. Samples from 18 patients were translocation-negative. Of these, seven samples were deemed inadequate for RT-PCR testing as a result of inappropriate tissue handling or the presence of necrotic material. Five patients were found to have a different diagnosis after complete histopathologic and molecular characterization. Six samples remained, in which adequate tissue was obtained with no evidence of a characteristic translocation. CONCLUSIONS: In apparently translocation-negative samples, close attention should be given to the possibility of an alternative diagnosis, the potential need for nested RT-PCR, and the possibility of an inadequate sample. Strong consideration should be given to the use of open biopsy as opposed to needle biopsy to avoid the need for repeat biopsies and the potential for inaccurate assessment of molecular marker status.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Ósseas/diagnóstico , Proteínas de Fusão Oncogênica/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/diagnóstico , Fatores de Transcrição/análise , Antígeno 12E7 , Adolescente , Adulto , Antígenos CD/análise , Biomarcadores Tumorais/genética , Biópsia , Biópsia por Agulha , Neoplasias Ósseas/química , Neoplasias Ósseas/epidemiologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Moléculas de Adesão Celular/análise , Criança , Pré-Escolar , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/ultraestrutura , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 22/ultraestrutura , DNA de Neoplasias/genética , Diagnóstico Diferencial , Erros de Diagnóstico , Feminino , Humanos , Masculino , Neoplasias de Bainha Neural/diagnóstico , Neuroblastoma/diagnóstico , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1 , Proteína EWS de Ligação a RNA , Estudos Retrospectivos , Sarcoma/diagnóstico , Sarcoma de Ewing/química , Sarcoma de Ewing/epidemiologia , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Fatores de Transcrição/genética , Translocação Genética , Tumor de Wilms/diagnósticoRESUMO
Although the gene responsible for multiple endocrine neoplasia, type 1 (MEN1) has been identified recently, the function of its gene product, menin, is not known. To examine menin's biological role, we created an N-terminal tagged fusion protein to follow the distribution of menin in the cell. In all cell lines tested, menin was found both in the nucleus and the cytoplasm, but its localization was dependent on the phase of the cell cycle; during a nondividing phase, menin was found in the nucleus; during and immediately after cell division, it was found in the cytoplasm. To confirm the cellular localization seen with the N-terminal tagged protein, we developed and purified peptide-specific antibodies. One of these antibodies (NCI 624), which recognizes a domain (aa 383-395) of menin, was used in immunofluorescence studies to corroborate the N-terminal tagging results. Further confirmation of menin localization was obtained in a pituitary tumor cell line derived from a familial MEN1 patient, which contained a mixed cell population with either none, or one functional copy of the MEN1 gene. Our results indicate that menin functions principally as a nuclear protein but may be found in the cytoplasm during cell division.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos/genética , Imunofluorescência , Células HeLa , Humanos , Dados de Sequência Molecular , Neoplasias Hipofisárias/metabolismo , Sitios de Sequências Rotuladas , Frações Subcelulares/metabolismo , Distribuição Tecidual/fisiologia , Células Tumorais CultivadasRESUMO
Neuronal activity-dependent processes are believed to mediate the formation of synaptic connections during neocortical development, but the underlying intracellular mechanisms are not known. In the visual system, altering the pattern of visually driven neuronal activity by monocular deprivation induces cortical synaptic rearrangement during a postnatal developmental window, the critical period. Here, using transgenic mice carrying a CRE-lacZ reporter, we demonstrate that a calcium- and cAMP-regulated signaling pathway is activated following monocular deprivation. We find that monocular deprivation leads to an induction of CRE-mediated lacZ expression in the visual cortex preceding the onset of physiologic plasticity, and this induction is dramatically downregulated following the end of the critical period. These results suggest that CRE-dependent coordinate regulation of a network of genes may control physiologic plasticity during postnatal neocortical development.
Assuntos
Envelhecimento/fisiologia , Cálcio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , AMP Cíclico/metabolismo , Plasticidade Neuronal/fisiologia , Transcrição Gênica , Córtex Visual/fisiologia , Animais , Corpos Geniculados/fisiologia , Camundongos , Camundongos Transgênicos , Privação Sensorial/fisiologia , Transcrição Gênica/fisiologia , Visão Monocular/fisiologiaRESUMO
Gastrinomas and insulinomas are frequent in multiple endocrine neoplasia type 1 (MEN1). The MEN1 tumor suppressor gene was recently identified. To elucidate the etiological role of the MEN1 gene in sporadic enteropancreatic endocrine tumorigenesis, we analyzed tumors (28 gastrinomas and 12 insulinomas) from 40 patients for MEN1 gene mutations and allelic deletions. One copy of the MEN1 gene was found to be deleted in 25 of 27 (93%) sporadic gastrinomas and in 6 of 12 (50%) sporadic insulinomas. MEN1 gene mutations were identified in 9 of 27 (33%) sporadic gastrinomas and 2 of 12 (17%) insulinomas and were not seen in corresponding germ-line DNA sequence. A specific MEN1 mutation was detected in one gastrinoma and in the corresponding germ-line DNA of a patient who had no family history of MEN1. Somatic MEN1 gene mutations and deletions play a critical role in the tumorigenesis of sporadic gastrinomas and may also contribute to the development of a subgroup of insulinomas.
Assuntos
Gastrinoma/genética , Genes Supressores de Tumor/genética , Insulinoma/genética , Neoplasias do Jejuno/genética , Neoplasia Endócrina Múltipla Tipo 1/genética , Mutação/genética , Neoplasias Pancreáticas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Deleção de Genes , Mutação em Linhagem Germinativa , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-IdadeRESUMO
The estrogen receptor (ER) is a transcription factor involved in steroid hormone signal transduction in higher eukaryotes. The receptor also functions as a ligand-dependent transcriptional activator when introduced into Saccharomyces cerevisiae (baker's yeast), which suggests that at least some of the components of the signal transduction pathway are conserved between yeast and mammalian cells, and, moreover, allows the possibility of using this simple eukaryotic organism to dissect receptor function. However, whether the ER actually activates transcription in a mechanistically similar fashion in yeast and mammalian cells is unclear, since it has been reported that the transactivation function within the hormone binding domain (TAF-2) does not function in yeast. In this report, we have characterized the activity of the transactivation functions of the ER in yeast. Our results indicate that both TAF-2 and the N-terminal transactivation region (TAF-1) are functional in yeast and contribute synergistically to the receptor's total activity. These results are consistent with those obtained in mammalian cells. Furthermore, we show that in yeast the antagonistic effects of the antiestrogen nafoxidine arise from a modulation of the synergistic interactions of TAF-1 and TAF-2, and not simply from an inactivation of TAF-2 by antihormone. Finally, we characterize the effect of ER deletion mutants on chromatin structure in yeast. Our data lend support to the view that the formation of competent transcriptional initiation complexes requires a precise disruption of chromatin structure.
Assuntos
Estradiol/farmacologia , Regulação Fúngica da Expressão Gênica , Receptores de Estrogênio/fisiologia , Proteínas Recombinantes/fisiologia , Saccharomyces cerevisiae/genética , Transativadores/fisiologia , Ativação Transcricional , Animais , Sítios de Ligação , Cromatina/ultraestrutura , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Mamíferos/genética , Nafoxidina/farmacologia , Regiões Promotoras Genéticas , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Transativadores/genéticaRESUMO
In cells, steroid hormone receptors interact with target enhancer elements on nucleosomes to regulate transcription of genes. To elucidate how nucleosomes can potentially regulate the interactions of steroid receptors with steroid response elements, we have examined the effects of nucleosome positioning and histone source on the binding of the progesterone receptor to DNA elements on nucleosomes reconstituted in vitro. We find that the affinity of the receptor for its response element is dependent on the position of the element within the nucleosome, but not on the histone source, active or inactive chromatin. Our results suggest that the strength of DNA-histone interactions within the nucleosome modulates the binding of progesterone receptor to response elements. Thus, nucleosome positioning is likely to influence the function of steroid receptors in vivo.
Assuntos
Elementos Facilitadores Genéticos , Nucleossomos/química , Progesterona/química , Receptores de Progesterona/química , Animais , Sequência de Bases , Sítios de Ligação , Galinhas , DNA/química , Eritrócitos/química , Histonas/química , Dados de Sequência Molecular , Progesterona/genética , Ligação ProteicaRESUMO
The activation of gene transcription by nuclear receptors is invariably associated with alterations in chromatin structure at hormone-responsive elements of target genes. To identify the molecular functions underlying receptor-mediated chromatin structure alterations we have evaluated the effects of DNA binding and transactivation of estrogen receptor derivatives on the promoter chromatin structure of estrogen-responsive reporter minichromosomes in Saccharomyces cerevisiae. We report here that the DNase I-hypersensitive chromatin structure at the promoter region is not simply a consequence of estrogen receptor binding to estrogen-responsive elements but is greatly enhanced by transactivation functions. These chromatin structure alterations are dependent on the presence of more than one estrogen-responsive element as well as downstream promoter sequences and appear to be correlated with transcriptional competence of the promoter. Our results imply that a disruption of chromatin structure at promoters is associated with the establishment of active transcription complexes. Since RNA polymerase cannot initiate transcription on nucleosomal DNA in vitro (Lorch, Y., Lapointe, J.W., and Kornberg, R.D. (1987) Cell 49, 203-210) this local disruption of chromatin structure may represent a nucleosome-free window, allowing initiation to occur in vivo.
Assuntos
Cromatina , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/genética , Ativação Transcricional , Sequência de Bases , Western Blotting , DNA Fúngico/metabolismo , Genes Fúngicos , Cinética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Receptores de Estrogênio/genética , Transcrição GênicaRESUMO
Biochemical over-expression of the human estrogen receptor was achieved using a Saccharomyces cerevisiae expression system. The receptor was produced as a novel ubiquitin fusion protein. This fusion protein is short lived in the cell and is processed to produce unfused receptor shortly after folding. Conventional high copy expression plasmids produced receptor to about 0.04% of the total soluble protein. By incorporating a defective leu2 allele into these vectors, an additional 5-fold increase in receptor production was obtained. The recombinant receptor was undergraded, soluble and biologically active. Conventional methods of disrupting cells using glass beads had a detrimental effect on the ability of the receptor to bind hormone. Enzymatic digestion of the cell wall followed by hypotonic shock liberates the receptor that quantitatively binds estrogen.
Assuntos
Receptores de Estrogênio/genética , Saccharomyces cerevisiae/genética , Alelos , Animais , Western Blotting , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Estrogênios/metabolismo , Expressão Gênica , Genes Fúngicos , Humanos , Plasmídeos , Testes de Precipitina , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , beta-Galactosidase/metabolismoRESUMO
We describe in this report experiments in vivo that demonstrate that antiestrogens promote DNA binding of the estrogen receptor without efficiently inducing transcription. When the receptor is modified to carry a foreign unregulated transactivation domain, transcription can be induced efficiently by both estrogen and antiestrogens. Under apparent saturation conditions, antihormone-receptor complexes binding to responsive enhancer elements elicit only a low level of transcription. In addition, we show that both estrogen and an antiestrogen, nafoxidine, effect very similar alterations in chromatin structure at a responsive promoter. These results indicate that in vivo steroid receptor action can be regulated subsequent to the DNA binding step, by regulating interactions with the target transcriptional machinery. In this regard, antihormones can function by establishing receptor-DNA complexes that are transcriptionally nonproductive.
Assuntos
Elementos Facilitadores Genéticos , Antagonistas de Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Cromatina/ultraestrutura , Clonagem Molecular , Proteínas de Ligação a DNA/fisiologia , Estradiol/farmacologia , Humanos , Técnicas In Vitro , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae , Transdução de Sinais , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
Five cases of oesophageal stricture secondary to caustic ingestion are presented. The colon, as a total bypass graft, was applied in all patients; two with right hemicolon and three with isoperistaltic transverse colon. The colon was placed subcutaneously, with the operation being performed as a one-stage procedure. A good functional result of the colon bypass, could be achieved in all persons.