RESUMO
Although the class I MHC receptors expressed by human and mouse natural killer (NK) cells have distinct molecular origins, they are functional analogues that are expressed in a variegated pattern. The murine Ly49 class I receptors contain bidirectional promoters that have been proposed to control the probabilistic expression of these genes. Whether similar elements are present in the human killer Ig-like receptor (KIR) genes is a fundamental question. A detailed analysis of the 2 kb intergenic region separating the KIR2DL4 gene and the adjacent KIR3DL1 gene revealed that additional promoter elements exist in the human KIR genes. Remarkably, the previously characterized KIR3DL1 proximal promoter possesses bidirectional promoter activity that maps to an 88 bp DNA fragment containing CREB, AML, Sp1 and Ets transcription factor binding sites. Individual KIR genes and alleles possess bidirectional promoters with distinct properties. Analysis of KIR(+)and KIR(-) NK cells and NK precursors indicates that reverse transcripts from the bidirectional promoter are found in cells that lack KIR protein expression, but are not present in mature KIR-expressing NK cells, suggesting that reverse transcription from the proximal promoter blocks gene activation in immature NK and precursor cells.
Assuntos
Regiões Promotoras Genéticas , Receptores Imunológicos/genética , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , DNA Antissenso/genética , DNA Complementar/genética , Humanos , Células Matadoras Naturais/imunologia , Camundongos , Dados de Sequência Molecular , Receptores KIR , Receptores KIR2DL4 , Receptores KIR3DL1RESUMO
OBJECTIVES: Intravitreal injection of steroids is a rapidly evolving treatment option for a variety of retinal pathologies. There is wide variation among ophthalmologists regarding dose of steroid and the technique of injection. Through this survey we aim to describe the current practice of injection of intravitreal steroid among ophthalmic consultants in the United Kingdom and review the literature. METHODS: A postal questionnaire was sent to all the 901 ophthalmic consultants in the United Kingdom. The questions covered frequency of intravitreal steroid injections, subspecialty interest, dosage and volume of steroid, patient preparation, surgeon preparation, and postoperative management. RESULTS: The response rate to the questionnaire was 63%. A total of 24.7% perform intravitreal steroid injections regularly with 90% of users performing five or fewer injections per month. In all, 94% use a dose of 4 mg. Among surgeons, 38% do not prescribe postoperative antibiotics or steroids and 59% check intraocular pressure either on the same or the next day. CONCLUSION: The relatively short experience with this technique has not yet allowed a clear picture to emerge of indications, complications or best practice administration. Published descriptions of intravitreal steroid injection techniques vary widely and variations are not evidence based. Observed UK practice is diverse.
Assuntos
Anti-Inflamatórios/administração & dosagem , Triancinolona/administração & dosagem , Câmara Anterior/cirurgia , Esquema de Medicação , Pesquisas sobre Atenção à Saúde/métodos , Humanos , Injeções/métodos , Pressão Intraocular/fisiologia , Medicina , Agulhas , Soluções Oftálmicas/uso terapêutico , Paracentese , Cuidados Pós-Operatórios/métodos , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/fisiopatologia , Especialização , Reino Unido , Corpo VítreoRESUMO
A more complete understanding of the transcriptional control of the human and murine class I MHC receptors will help to shed light on the mechanism of selective, stochastic, gene activation that operates in these gene families. Studies of the murine Ly49 class I MHC receptor genes have revealed an important role for distal transcripts originating upstream of the proximal promoter. To date, there have been no reports of distal promoters within the functionally analogous human KIR family of class I MHC receptors. In the current study, reverse transcriptase-polymerase chain reaction (RT-PCR) and RNase protection assays were used to reveal the presence of distal KIR transcripts initiating upstream of the previously characterized proximal KIR promoter. The intergenic promoter elements detected were associated with repetitive elements of the Alu and L1 families. Unlike the proximal KIR promoter, the distal promoter regions were not NK cell-specific. KIR genes expressed in a variegated manner produced a low level of distal transcripts containing a large 5' untranslated region. In contrast, the highly expressed KIR2DL4 gene possessed a higher level of spliced distal transcripts that were capable of producing KIR2DL4 protein. The identification of distal KIR promoter elements suggests that intergenic transcripts may influence the expression of KIR genes.
Assuntos
DNA Intergênico/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Receptores Imunológicos/genética , Linhagem Celular , Clonagem Molecular , Humanos , Luciferases , Receptores Imunológicos/metabolismo , Receptores KIR , Receptores KIR2DL4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Análise de Sequência de DNARESUMO
Replication-dependent histone mRNAs end in a highly conserved stem-loop sequence rather than a polyA sequence. A 45-kDa stem-loop binding protein (SLBP), which specifically binds the stem-loop of histone mRNA, is present in both polyribosomes and nuclei. An identical 45-kDa protein, as determined by partial protease digestion, is cross-linked to a 30 nt RNA containing the 3' stem-loop from both nuclei and polyribosomes. The SLBP can also be detected by a Northwestern blot procedure using the 30 nt RNA as a probe. As judged from the Northwestern assay, more than 90% of the SLBP in the cell is found in the polyribosomes with the remaining SLBP localized to the nucleus. Only 5-10% of the SLBP could be extracted from the polyribosomes with salt. Treatment of the polyribosomes with micrococcal nuclease prior to salt extraction solubilized 5-10 times more SLBP as an RNA-protein complex. The SLBP could be subsequently partially purified from this complex.
Assuntos
Proteínas Nucleares/isolamento & purificação , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Fatores de Poliadenilação e Clivagem de mRNA , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Camundongos , Nuclease do Micrococo/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Células Tumorais CultivadasRESUMO
Cell cycle-regulated histone mRNAs end in a conserved 26-nt sequence that can form a stem-loop with a six-base stem and a four-base loop. The 3' end of histone mRNA has distinct functions in the nucleus and in the cytoplasm. In the nucleus it functions in pre-mRNA processing and transport, whereas in the cytoplasm it functions in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), present in both nuclei and polyribosomes, is likely the trans-acting factor that binds to the 3' end of mature histone mRNA and mediates its function. A nuclear extract that efficiently processes histone pre-mRNA was prepared from mouse myeloma cells. The factor(s) that bind to the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide containing the conserved stem-loop sequence. Using this depleted extract which is deficient in histone pre-mRNA processing, we show that SLBP found in polyribosomes can restore processing, suggesting that SLBP associates with histone pre-mRNA in the nucleus, participates in processing, and then accompanies the mature mRNA to the cytoplasm.
Assuntos
Histonas/genética , Proteínas Nucleares , Polirribossomos/genética , Precursores de RNA , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Fatores de Poliadenilação e Clivagem de mRNA , Animais , Sequência de Bases , Biotina/química , Extratos Celulares/química , Núcleo Celular/genética , Células Cultivadas , Sequência Conservada , Camundongos , Dados de Sequência Molecular , Mieloma Múltiplo/patologia , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , RNA Mensageiro , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
Histone mRNAs end in a conserved 26 nt sequence which can form a stem-loop with a six-base stem and a four base loop. The 3' end of histone mRNA functions in the nucleus in pre-mRNA processing and mRNA transport and in the cytoplasm in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), found in both the polyribosomes and the nucleus, binds to the 3' end of histone mRNA. A nuclear extract which efficiently processes histone pre-mRNA has been prepared from mouse myeloma cells. The factors which bind the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide. Using the depleted extract, we show that the SLBP found in the polyribosomes can function in histone pre-mRNA processing, suggesting that the SLBP associates with histone pre-mRNA in the nucleus and accompanies the mature mRNA to the cytoplasm.
Assuntos
Histonas/genética , Proteínas Nucleares , Polirribossomos/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA , Animais , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Precursores de RNA/química , Processamento Pós-Transcricional do RNARESUMO
The University of Michigan Hospitals began a quality management and improvement process in 1987 as the framework for all of its quality-related efforts and activities. The Infection Control Services department used total quality techniques to develop its mission statement, identify customers, identify customer requirements, and develop quality improvement objectives to meet the requirements. A service evaluation of customers resulted in specific improvement activities. An unexpected result of this evaluation was the difference noted between the Infection Control Services staff members' perception of customer requirements and these customers' actual needs. ICPs should use the continuous quality improvement tools and techniques to enhance their activities within their institutions, to better meet their customer needs, and to make sure that they are complementing their institution's mission.