RESUMO
Dementia, which affects a large and growing number of older people, presents particular challenges to primary care. There is an acknowledged need to develop interventions that address practitioners' needs for information and guidance regarding the diagnosis and management of dementia. This paper examines the potential usefulness and constraints of a Computer Decision Support System (CDSS) to assist practitioners in diagnosing and managing dementia. Questionnaire information was obtained from 97 primary care practitioners regarding their current practice and views on dementia care, priority given to training and familiarity with computer use. Implications of these findings for the relevance and value of CDSS are discussed. The paper is part of a larger ongoing study, the aim of which is the evaluation of three educational interventions for primary care practitioners.
Assuntos
Computadores , Tomada de Decisões , Demência/diagnóstico , Demência/terapia , Atenção Primária à Saúde , Software , Idoso , Feminino , Humanos , Masculino , Inquéritos e Questionários , Carga de Trabalho/estatística & dados numéricosRESUMO
BACKGROUND: Diagnosis and management of dementia is a complex process and primary care physicians are under-equipped to deal with uncertainties in the provision of optimal care for the patient. OBJECTIVE: To develop a computer decision support system (CDSS) which could assist physicians with diagnosis and management and improve patient care. METHODS: A design group including general practitioners derived logic pathways for diagnosis and management of dementia and validated them with a multiprofessional expert group. Logic pathways were used to construct a comprehensive CDSS rendered as a series of expert consultations. The CDSS was inserted into commercially available GP systems and bench and field-tested. RESULTS: The complexity of dementia diagnosis and management can be captured in logic pathways which can be expressed as decision trees within existing electronic patient records. The resulting CDSS appears useable in routine practice. CONCLUSION: The impact of this CDSS will be evaluated in a randomised controlled trial of educational interventions in primary care.
Assuntos
Sistemas de Apoio a Decisões Clínicas , Demência/diagnóstico , Demência/terapia , Sistemas Inteligentes , Medicina de Família e Comunidade , Idoso , Árvores de Decisões , Humanos , Reino Unido , Interface Usuário-ComputadorRESUMO
Tumor necrosis factor-alpha (TNF) induces apoptosis in confluent LLC-PK1 epithelial cells, but also activates NF-kappaB, a negative regulator of apoptosis. The presence of increased TNF-induced apoptosis causes a transient increase in epithelial permeability, but the epithelial barrier function recovers, as assessed by measuring the transepithelial electrical resistance, the paracellular flux of mannitol and by the electron microscopic evaluation of the penetration of the electron-dense dye ruthenium red across the tight junctions. The integrity of the epithelial cell layer is maintained by rearrangement of non-apoptotic cells in the monolayer and by the phagocytosis of apoptotic fragments. To study the role of NF-kappaB in an epithelium exposed to TNF, NF-kappaB was inhibited in LLC-PK1 epithelial cells with either the dietary compound, curcumin, or by transfection with a dominant negative mutant inhibitor I kappaB alpha. Replacement of serine 32 and 36 by alanine has been shown to prevent its phosphorylation and degradation, blocking NF-kappaB activation. Inhibition of NF-kappaB altered the morphology of TNF-induced apoptotic cells, which showed lack of fragmentation and membrane blebbings, and absence of phagocytosis by neighboring cells. TNF treatment of NF-kappaB-inhibited cells also caused altered distribution of the tight junction-associated protein ZO-1, increased epithelial leakiness, and impaired the recovery of the epithelial barrier function, which normally occurs 6 hours after TNF treatment of LLC-PK1 cells. These data demonstrate that NF-kappaB activation is required for the maintenance of the barrier function of an epithelium undergoing TNF-induced apoptosis.
Assuntos
Apoptose , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Western Blotting , Permeabilidade da Membrana Celular , Curcumina/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Imunofluorescência , Rim/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Mutagênese , Fosfoproteínas/metabolismo , Rutênio Vermelho/metabolismo , Suínos , Junções Íntimas/metabolismo , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Proteína da Zônula de Oclusão-1RESUMO
XR-1 is a CHO mutant cell line defective in double strand break repair and V(D)J recombination. These defects are due to a deletion of the XRCC4 gene which encodes a 38-kDa nuclear phosphoprotein. Recent studies have shown that XRCC4 interacts with and enhances the activity of DNA ligase IV in vitro. In this study we investigate the effect of the absence of XRCC4 on the level of DNA ligase IV in XR-1 cells. Western blot analysis indicates that levels of DNA ligase IV protein are almost undetectable in these cells, however, introduction of the XRCC4 cDNA into XR-1 resulted in a return to wild type levels of the protein. Furthermore, analysis of DNA ligase IV mRNA showed equivalent levels in both XR-1 and XRCC4 transfected XR-1 indicating that the altered level of DNA ligase IV is not due to a change in the expression of the gene. These data strongly suggest that an important function of XRCC4 is to stabilize the DNA ligase IV protein.
Assuntos
DNA Ligases/metabolismo , Proteínas de Ligação a DNA/genética , Estabilidade Enzimática/genética , Animais , Células CHO , Cricetinae , DNA Ligase Dependente de ATP , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Proteínas Nucleares/genética , RNA Mensageiro/análise , Recombinação Genética/genética , Transfecção/genéticaRESUMO
XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.
Assuntos
Antígenos Nucleares , Sistema Nervoso Central/embriologia , DNA Helicases , Proteínas de Ligação a DNA/metabolismo , Subpopulações de Linfócitos/citologia , Neurônios/citologia , Animais , Apoptose , Padronização Corporal , Ciclo Celular , Diferenciação Celular , Linhagem Celular , Sistema Nervoso Central/citologia , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário e Fetal , Fibroblastos , Rearranjo Gênico , Genes Essenciais , Autoantígeno Ku , Subpopulações de Linfócitos/imunologia , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Radiação IonizanteRESUMO
MIP-1 alpha is a secreted chemokine which can inhibit hematopoietic stem cells and modulate inflammatory responses. It is also an inhibitor of HIV replication in CD8+ T-cells, MIP-1 alpha is expressed in transformed B cells and can also be induced during cellular activation of CD4+ T-cells and monocytes. We have previously identified a new transcription factor family (the MNP family) whose expression is crucial for the induction of MIP-1 alpha transcription during cellular activation. Monocytes and transformed B-cells normally express MNP-1 strongly and MNP-2 weakly, while T-cells strongly express only MNP-2. In this communication we show evidence identifying a new member of the MNP transcription family, MNP-3, in PMA differentiated HL60 cells.
Assuntos
Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células HL-60 , Humanos , Dados de Sequência Molecular , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Polyamines are small polycationic molecules that have been implicated in cell growth, differentiation and transformation. A possible mechanism for their involvement in these processes is via their influence on the expression of growth promoting genes. In this study we used an ornithine decarboxylase (ODC) overexpressing retroviral system to achieve high intracellular levels of polyamines in epidermal cells. We then looked at the effect this environment had on transcription from several promoter-reported gene constructs, including c-myc and ODC itself. In transient transfection studies elevated polyamines significantly enhanced the transcriptional activity of all promoters tested. In addition, reporter gene expression was induced approximately 3 fold in stable transformants containing an integrated c-myc or ODC promoter-reporter gene construct suggesting that elevated levels of polyamines, such as those found in transformed cells, have a stimulatory effect on gene expression.
Assuntos
Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Regiões Promotoras Genéticas , Animais , Linhagem Celular , Epiderme , Regulação da Expressão Gênica , Genes Reporter , Luciferases/biossíntese , Camundongos , Ornitina Descarboxilase/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes/biossíntese , Retroviridae , TransfecçãoRESUMO
Regulation of FGF-4 gene expression is controlled both by elements in the promoter and by an enhancer domain located in the untranslated region of the third exon. We have determined that transcription factor NF-Y binds to the FGF-4 promoter. We further show by mutational analysis that binding of NF-Y is essential for FGF-4 enhancer activity but has minimal effect on activity of the FGF-4 promoter alone.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Fatores de Crescimento de Fibroblastos/biossíntese , Fatores de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Carcinoma Embrionário , Linhagem Celular , Clonagem Molecular , Primers do DNA , Fator 4 de Crescimento de Fibroblastos , Luciferases/biossíntese , Metilação , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
Murine macrophage inflammatory protein 1 alpha (MIP-1 alpha) and its human equivalent (GOS19, LD78, or AT464) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived MIP-1 alpha/GOS19 inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of MIP-1 alpha/GOS19. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the GOS19 promoter. One member, ICK-1A, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the MIP-1 alpha nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells.
Assuntos
Citocinas/biossíntese , Citocinas/genética , Células-Tronco Hematopoéticas/metabolismo , Monocinas/biossíntese , Monocinas/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , Regulação da Expressão Gênica , Humanos , Proteínas Inflamatórias de Macrófagos , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Plasmídeos , Linfócitos T/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
We report the independent isolation of a rearranged FGF-4 gene from a patient with chronic myeloid leukaemia. We show that the FGF-4 gene has been truncated 30 nucleotides 3' to the coding sequence and has been fused to the RNA processing signals from a putative unknown gene on chromosome 15. We demonstrate that the promoter region of the FGF-4 gene is active in NIH3T3 cells and is indeed necessary for transformation. Using the luciferase reporter assay we have shown that the FGF-4 5' flanking sequences possess easily detectable promoter activity in both F9 and HeLa cell lines. 5' deletion analysis of the FGF-4 promoter has delineated regions containing cis-acting elements of functional importance. These regulatory regions are common to both embryonal and somatic cell lines. Electrophoretic mobility shift assay, using nuclear extracts from F9 and HeLa cells, has allowed detection of DNA-protein interactions occurring in the functionally significant regions. Subsequent comparison of the human and murine FGF-4 promoters show that the regions of functional significance are highly conserved. We suggest that the FGF-4 gene may be suppressed through a distal suppressor locus and becomes active when separated from this suppressor.
Assuntos
Transformação Celular Neoplásica , Fatores de Crescimento de Fibroblastos/biossíntese , Fatores de Crescimento de Fibroblastos/genética , Rearranjo Gênico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Células 3T3 , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , Primers do DNA , Embrião de Mamíferos , Fator 4 de Crescimento de Fibroblastos , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Teratoma , TransfecçãoRESUMO
Down regulation of the c-myc gene is a prerequisite for the differentiation of a number of cell types. Studies have shown that two mechanisms of inactivation are involved in c-myc repression: a block of the elongation of RNA polymerase followed by transcriptional inactivation mediated through promoter sequences. In this study DMS in vivo footprinting was performed on the P2 promoter region of c-myc in differentiated and undifferentiated HL60 cells. A differentiation-specific footprint was observed at G residues immediately upstream of the TATA box. This observation occurred only in cells differentiated for 48 hours or more and hence is likely to be involved in the repression of c-myc by promoter inactivation.
Assuntos
Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Genes myc , Regiões Promotoras Genéticas , Sequência de Bases , Northern Blotting , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Primers do DNA , Dimetil Sulfóxido/farmacologia , Humanos , Leucemia Promielocítica Aguda , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Neoplásico/análise , RNA Neoplásico/biossíntese , TATA Box , Células Tumorais CultivadasRESUMO
Methylation of a plasmid containing the SV40 promoter linked to the chloramphenicol acetyl transferase (CAT) gene, with either murine DNA methylase or methylase SssI results in inhibition of the expression of the reporter gene after transfection into cultured cells. Methylation of the plasmid with the methylases HhaI and HpaII has no effect on the expression of this gene. Protein-DNA interactions in the SV40 promoter are not affected by the presence of methylcytosine suggesting that inactivation results from the formation of an inactive chromatin structure that is dependent on the high CG content of the plasmid.
Assuntos
Metilases de Modificação do DNA/metabolismo , Vetores Genéticos , Regiões Promotoras Genéticas , Vírus 40 dos Símios/genética , Transcrição Gênica , Animais , Sequência de Bases , Carcinoma Krebs 2/enzimologia , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Regulação Viral da Expressão Gênica , Cinética , Metilação , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
In Burkitt's lymphoma (BL) a consistent chromosomal translocation involving the c-myc proto-oncogene locus on chromosome 8 and one of the immunoglobulin loci located on chromosomes 14, 22 or 2 results in the aberrant expression of c-myc. In six out of ten published BL sequences the translocated c-myc gene contains mutations and/or deletions in an Spl site in the first exon. We show by competition gel mobility shift assays and association constant comparison that the binding of Spl to this site is altered in four of these clones. The affinity of Spl for this site is increased 5-10 fold in Burkitt's lymphoma BL22 and decreased approximately 5 fold in BL2. Mutations at this site in cell lines Raji and Daudi also show a decrease in Sp1 binding when compared with the wild type sequence.
RESUMO
In vitro transcription competition with oligonucleotides has shown that a down regulating factor can be displaced by a methylated oligonucleotide covering a specific region of the avian vitellogenin II gene promoter (Proc. Natl. Acad. Sci USA, (1990) 87, 3047-3051). Gel mobility shift and competition assays show that a protein binding preferentially to methylated DNA (MDBP-2) is present in fractionated hen and rooster nuclear extracts. The protein(s) bind to the methylated sequence 5' TTCACCTTmCGCTATG-AGGGGGATCATACTGG' 3' (nucleotide positions +2 to +32) of the vitellogenin II promoter and not to other methylated DNA sequences. Contact points of the MDBP-2 with DNA were studied by DNA binding interference experiments with partially depurinated and depyrimidinated oligonucleotides. The protein has an approximate molecular weight of 40 KDa and is mainly found in the liver and oviduct. Proteolytic clipping bandshift assays of the MDBP-2 from rooster and hen liver nuclear extracts indicate that the protein from the two sources are different. In vitro transcription experiments show that the addition of a purified nuclear fraction containing the addition of a purified nuclear dependent manner the transcription of vitellogenin II gene.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Vitelogeninas/genética , Animais , Sequência de Bases , Ligação Competitiva , Galinhas , DNA , Proteínas de Ligação a DNA/efeitos da radiação , Feminino , Regulação da Expressão Gênica , Fígado/metabolismo , Masculino , Metilação , Dados de Sequência Molecular , Peso Molecular , Proteínas Nucleares/efeitos da radiação , Especificidade de Órgãos , Regiões Promotoras Genéticas/efeitos da radiação , Caracteres Sexuais , Transcrição Gênica , Raios UltravioletaRESUMO
DNA methylases from mouse and pea have been purified and characterized. Both are high molecular mass enzymes that show greater activity with hemimethylated than unmethylated substrate DNA. Both methylate cytosines in CpG preferentially, but not exclusively and show similar kinetics of methylation, which makes it difficult to saturate all possible sites on the DNA, but procedures are described that circumvent this problem.
Assuntos
Metilases de Modificação do DNA/metabolismo , Fabaceae/enzimologia , Plantas Medicinais , Animais , Sequência de Bases , Cromatografia de Afinidade , Cromatografia em Gel , Metilases de Modificação do DNA/isolamento & purificação , Cinética , Fígado/enzimologia , Metilação , Camundongos , Dados de Sequência Molecular , Peso Molecular , Especificidade por SubstratoAssuntos
DNA (Citosina-5-)-Metiltransferases/isolamento & purificação , Camundongos/metabolismo , Animais , Reações Cruzadas , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/imunologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Sistema Digestório/enzimologia , Rim/enzimologia , Metilação , Peso Molecular , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/enzimologia , Especificidade de Órgãos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
We have purified the DNA methylase from mouse ascites tumour cells to a specific activity of 11,500 units per mg protein using denatured Micrococcus luteus DNA as methyl acceptor. Methyl groups are transferred to cytosines almost exclusively in CpG dinucleotides. The purified enzyme contains two polypeptides of molecular mass 185 and 160 kDa, and an antiserum raised in a rabbit to the purified enzyme specifically reacts with these two proteins in crude extracts. The two proteins can be partially separated by affinity chromatography when activity is associated with the 185 kDa protein which can be proteolytically degraded to give polypeptides of 170 and later 100 and 50 kDa. Only the 185 kDa methylase is lost when cells are treated with azadeoxycytidine and this is the predominant form firmly bound in the nucleus of dividing cells. Antibody bound to the 185 kDa band in protein blots will itself bind native DNA methylase, which can be detected by its binding 14C-labelled, azacytosine-containing DNA.