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A 5-yr retrospective study (November 2006-December 2011) was conducted to determine the isolation frequency of Pasteurella multocida and Gallibacterium anatis and their antibiograms from chickens submitted to the Mississippi Poultry Research and Diagnostic Laboratory. The number of isolations of G. anatis increased over the last 5 yr in broiler and broiler breeder type chickens. For P. multocida, the number of isolations increased from 2006 to 2010, but decreased through 2011 with all isolations being from boiler breeder type chickens. Gallibacterium anatis demonstrated almost complete resistance to novobiocin, tylosin, lincosamide, and tetracycline antimicrobials with moderate to high sensitivity to sulfonamides, fluoroquinolones, and florfenicol. There was intermediate sensitivity for spectinomycin and erythromycin and variable resistance to ß-lactam and aminoglycoside antimicrobials. In sharp contrast, P. multocida showed moderate to high sensitivity to ß-lactam, novobiocin, and tetracycline antimicrobials, but had antibiograms similar to G. anatis for the other antimicrobials. Sensitivities were determined using minimum inhibitory concentration. This study examines the trends over a 5-yr period of the number of isolates of P. multocida and G. anatis and their sensitivities. These 2 pathogens produce very similar clinical signs and lesions (fowl cholera-like) in breeders despite having extremely antagonistic sensitivity patterns. This study shows the necessity for producers to attempt culture and sensitivity in suspect fowl cholera-like flocks before initiating antimicrobial treatment commonly used with P. multocida for fear that the culprit may actually be the more antimicrobial-resistant G. anatis.
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Antibacterianos/farmacologia , Galinhas , Farmacorresistência Bacteriana , Infecções por Pasteurellaceae/veterinária , Pasteurellaceae/efeitos dos fármacos , Doenças das Aves Domésticas/epidemiologia , Animais , Testes de Sensibilidade Microbiana/veterinária , Mississippi/epidemiologia , Infecções por Pasteurella/epidemiologia , Infecções por Pasteurella/microbiologia , Infecções por Pasteurella/veterinária , Pasteurella multocida/efeitos dos fármacos , Pasteurella multocida/isolamento & purificação , Pasteurellaceae/isolamento & purificação , Infecções por Pasteurellaceae/epidemiologia , Infecções por Pasteurellaceae/microbiologia , Doenças das Aves Domésticas/microbiologia , Estudos Retrospectivos , Estações do AnoRESUMO
INTRODUCTION: Although there has been considerable progress in the use of administrative data for applied health research, the family justice field lags behind. Better use of administrative data are essential to enhance understanding of how the family justice system is working, as well as the characteristics of, and outcomes for, children and families. The Family Justice Data Partnership (FJDP) supports this aim through analyses of core family justice and linked datasets in the SAIL Databank (Secure Anonymised Information Linkage). Cafcass Cymru provide expert advice for children involved in family court proceedings in Wales, ensuring decisions are made in the best interests of the child. We provide an overview of Cafcass Cymru data. We also describe and illustrate linkage to administrative datasets within SAIL. METHODS: Cafcass Cymru data was transferred to SAIL using a standardised approach to provide de-identified data with Anonymised Linking Fields (ALF) for successfully matched records. Three cohorts were created: all individuals involved in family court applications; all individuals with an ALF allowing subsequent health data linkage; and all individuals with a Residential Anonymised Linking Field (RALF) enabling area-level deprivation analysis. RESULTS: Cafcass Cymru application data are available for child protection matters (public law, range 2011-2019, n=12,745), and child arrangement disputes (private law, range 2005-2019, n=52,023). An 80% data linkage match rate was achieved. 40% had hospital admissions within two years pre or post application; 54% had emergency department attendances and 61% had outpatient appointments. Individuals were more likely to reside in deprived areas regardless of law type. CONCLUSION: Cafcass Cymru data can be accessed through the SAIL Databank. The FJDP will continue to enhance research opportunities for all to better understand the family justice system, and outcomes for those involved, such as health and wellbeing for children and family members.
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INTRODUCTION: It is widely acknowledged that population health and administrative data, especially when linked at the individual level, hold great value for research. Cross-centre working between data centres providing access to such data has the potential to further increase this value by effectively expanding the data available for research. However, there is limited published information on how to address the challenges and achieve success. The aim of this paper is to explore perceived barriers and solutions to inform developments in cross-centre working across data centres. METHODS: We carried out a narrative literature review on data sharing and cross centre working. We used a mixed methods approach to assess the opinions of members of the public on cross-centre data sharing, and the views and experiences of among data centre staff connected with the UK Farr Institute for Health Informatics Research. RESULTS: The literature review uncovered a myriad of practical and cultural issues. Our engagement with a public group suggested that cross-centre working involving anonymised data being moved between established centres is considered acceptable. The main themes emerging from discussions with data centre staff were dedicated resourcing, practical issues, information governance and culture. CONCLUSION: In seeking to advance cross-centre working between data centres, we conclude that there is a need for dedicated resourcing, indicators to recognise data reuse, collaboration to solve common issues, and balancing necessary barrier removal with incentivisation. This will require on-going commitment, engagement and an academic culture change.
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BACKGROUND: The Secure Anonymised Information Linkage (SAIL) Databank is a national data safe haven of de identified datasets principally about the population of Wales, made available in anonymised form to researchers across the world. It was established to enable the vast arrays of data collected about individuals in the course of health and other public service delivery to be made available to answer important questions that could not otherwise be addressed without prohibitive effort. The SAIL Databank is the bedrock of other funded centres relying on the data for research. APPROACH: SAIL is a data repository surrounded by a suite of physical, technical and procedural control measures embodying a proportionate privacy-by-design governance model, informed by public engagement, to safeguard the data and facilitate data utility. SAIL operates on the UK Secure Research Platform (SeRP), which is a customisable technology and analysis platform. Researchers access anonymised data via this secure research environment, from which results can be released following scrutiny for disclosure risk. SAIL data are being used in multiple research areas to evaluate the impact of health and social exposures and policy interventions. DISCUSSION: Lessons learned and their applications include: managing evolving legislative and regulatory requirements; employing multiple, tiered security mechanisms; working hard to increase analytical capacity efficiency; and developing a multi-faceted programme of public engagement. Further work includes: incorporating new data types; enabling alternative means of data access; and developing further efficiencies across our operations. CONCLUSION: SAIL represents an ongoing programme of work to develop and maintain an extensive, whole population data resource for research. Its privacy-by-design model and UK SeRP technology have received international acclaim, and we continually endeavour to demonstrate trustworthiness to support data provider assurance and public acceptability in data use. We strive for further improvement and continue a mutual learning process with our contemporaries in this rapidly developing field.
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The UK Multiple Sclerosis Register (UKMSR) is a large cohort study designed to capture 'real world' information about living with multiple sclerosis (MS) in the UK from diverse sources. The primary source of data is directly from people with Multiple Sclerosis (pwMS) captured by longitudinal questionnaires via an internet portal. This population's diagnosis of MS is self-reported and therefore unverified. The second data source is clinical data which is captured from MS Specialist Treatment centres across the UK. This includes a clinically confirmed diagnosis of MS (by Macdonald criteria) for consented patients. A proportion of the internet population have also been consented at their hospital making comparisons possible. This dataset is called the 'linked dataset'. The purpose of this paper is to examine the characteristics of the three datasets: the self-reported portal data, clinical data and linked data, in order to assess the validity of the self-reported portal data. The internet (nâ¯=â¯11,021) and clinical (nâ¯=â¯3,003) populations were studied for key shared characteristics. We found them to be closely matched for mean age at diagnosis (clinicalâ¯=â¯37.39, portalâ¯=â¯39.28) and gender ratio (female %, portalâ¯=â¯73.1, clinicalâ¯=â¯75.2). The Two Sample Kolmogorov-Smirnov test was for the continuous variables to examine is they were drawn from the same distribution. The null hypothesis was rejected only for age at diagnosis (Dâ¯=â¯0.078, pâ¯<â¯0.01). The populations therefore, were drawn from different distributions, as there are more patients with relapsing disease in the clinical cohort. In all other analyses performed, the populations were shown to be drawn from the same distribution. Our analysis has shown that the UKMSR portal population is highly analogous to the entirely clinical (validated) population. This supports the validity of the self-reported diagnosis and therefore that the portal population can be utilised as a viable and valid cohort of people with Multiple Sclerosis for study.
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Esclerose Múltipla/epidemiologia , Sistema de Registros , Adulto , Feminino , Humanos , Internet , Masculino , Pessoa de Meia-Idade , Autorrelato , Reino UnidoRESUMO
The sympathetic nervous system exerts a trophic-mitogenic influence on C-1300 mouse neuroblastoma. We now report that sympathetic axotomy suppresses growth of the S-20 clonal line of neuroblastoma but does not influence the growth in vivo of two other clonal lines, NIE-115 and C-46. Sympathetic ganglia-conditioned medium significantly increases proliferation of S-20 cells in vitro. Growth of NIE-115 and C-46 clonal neuroblastoma lines is not influenced by sympathetic ganglia-conditioned medium. We postulate that the sympathetic nervous system secretes a mitogenic-trophic factor that favors growth of C-1300 neuroblastoma in vivo. Sensitivity to this factor varies between neuroblastoma clonal lines.
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Neuroblastoma/patologia , Sistema Nervoso Simpático/fisiologia , Animais , Divisão Celular , Células Cultivadas , Meios de Cultura , Gânglios Simpáticos/fisiologia , Camundongos , RatosRESUMO
The sympathetic nervous system modulates the growth of C-1300 mouse neuroblastoma in vivo and in vitro. We now report that a mitogenic/trophic factor that augments growth of C-1300 neuroblastoma and of the S-20 neuroblastoma clonal line is present in freshly excised sympathetic cervical ganglia from newborn rats, but is not detectable in homogenates from sympathetic ganglia obtained from adult rats.
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Neuroblastoma/patologia , Sistema Nervoso Simpático/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Células Cultivadas , Substâncias de Crescimento/fisiologia , RatosRESUMO
The frizzled (fz) gene of Drosophila is essential for the development of normal tissue polarity in the adult cuticle of Drosophila. In fz mutants the parallel array of hairs and bristles that decorate the cuticle is disrupted. Previous studies have shown the fz encodes a membrane protein with seven putative transmembrane domains, and that it has a complex role in the development of tissue polarity, as there exist both cell-autonomous and cell nonautonomous alleles. We have now examined a larger number of alleles and found that 15 of 19 alleles display cell nonautonomy. We have examined these and other alleles by Western blot analysis and found that most fz mutations result in altered amounts of Fz protein, and many also result in a Fz protein that migrates aberrantly in SDS-PAGE. We have sequenced a subset of these alleles. Cell nonautonomous fz alleles were found to be associated with mutations that altered amino acids in all regions of the Fz protein. Notably, the four cell-autonomous mutations were all in a proline residue located in the presumptive first cytoplasmic loop of the protein. We have also cloned and sequenced the fz gene from D. virilis. Conceptual translation of the D. virilis open reading frame indicates that the Fz protein is unusually well conserved. Indeed, in the putative cytoplasmic domains the Fz proteins of the two species are identical.
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Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos , Mutação , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Metanossulfonato de Etila , Receptores Frizzled , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Estrutura Molecular , Mutagênicos , Ratos , Receptores Acoplados a Proteínas G , Homologia de Sequência de AminoácidosRESUMO
The function of the frizzled (fz) gene is essential for the development of the normal pattern of hairs on the Drosophila wing. In the absence of fz function hairs develop, but they display an abnormal polarity. Mutations in fz result in an altered subcellular location for the assembly of the F-actin filled prehair that becomes the adult cuticular hair. This observation led to the suggestion that fz and other tissue polarity genes form a regulatory pathway that controls the initiation of prehairs. We have isolated a cold-sensitive fz allele and found that the cold-sensitive period for fz in the pupal wing starts in the early pupae and ends prior to the first sign of prehair morphogenesis. This cold-sensitive mutation is due to a missense mutation in a putative transmembrane domain. Western blot analysis shows that the accumulation of the mutant protein is not cold sensitive, consistent with the supposition that it is the activity of the mutant protein that is cold sensitive. Our data argue that fz has a regulatory function in specifying where the prehair forms, but no role in the actual morphogenesis of the prehair.
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Proteínas de Drosophila , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Hormônios de Inseto/genética , Proteínas de Membrana/genética , Asas de Animais/embriologia , Actinas/química , Alelos , Sequência de Aminoácidos , Animais , Arginina/genética , Western Blotting , Drosophila/genética , Receptores Frizzled , Genes Reguladores , Glicina/genética , Cabelo/embriologia , Hormônios de Inseto/fisiologia , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Morfogênese , Mutação Puntual , Estrutura Secundária de Proteína , Pupa/embriologia , Pupa/genética , Receptores Acoplados a Proteínas G , TemperaturaRESUMO
OBJECTIVES: To investigate why and how patients decide to attend accident and emergency (A&E) departments, and to assess their satisfaction with the experience, in a predominantly rural west Wales population. METHODS: This was a semi-structured follow up telephone interview of patients who walked in to A&E in one of four general hospitals in west Wales and were triaged as Manchester Triage score 4 or 5. Patients were recruited by nurses during the period July-November 2002. The study sample consisted of 176 male and 145 female patients, mean (SD) age 36.6 (20.0) years. The main outcome measure was a quantitative and qualitative description of the recalled experiences of A&E attenders, the circumstances of their attendance, and their satisfaction with the experience. RESULTS: Of the study sample, 78% attended with injury or illnesses of recent origin, and 50% with actual or presumed musculoskeletal injury, 73% of which were sustained within 10 miles of home. Travel to hospital was by private transport for 86%, average distance 7.4 miles. The majority (90%) were registered with a local GP, but 32% felt A&E was the obvious choice, and a further 44% considered their GP inaccessible to their needs. Patients' reasons for seeking health care at A&E were similar to those described in an English urban study. Waiting times were rarely excessive; 80% left within 2 hours, and patient satisfaction was generally high. Among the 87 patients (27%) who reported a less satisfactory experience, 48 (55%) of these complained of dismissive attitudes of doctors. CONCLUSIONS: Anecdotal accounts of abuse of A&E services and unreasonable patient expectations gain the status of "urban legends" within the medical profession. Among the predominantly settled rural population in west Wales, there is little evidence of unreasonable patient expectations, and most patients report high satisfaction levels. Patients' bad experiences most frequently arise from a dismissive attitude on the part of medical staff. These attitudes are often consequent on an A&E culture that views some patients' attendances as less appropriate than others.
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Serviço Hospitalar de Emergência/normas , Aceitação pelo Paciente de Cuidados de Saúde/psicologia , Satisfação do Paciente , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Tomada de Decisões , Feminino , Mau Uso de Serviços de Saúde/estatística & dados numéricos , Pesquisa sobre Serviços de Saúde , Humanos , Lactente , Entrevistas como Assunto , Masculino , Pessoa de Meia-Idade , Relações Médico-Paciente , População Rural/estatística & dados numéricos , Transporte de Pacientes/estatística & dados numéricos , País de GalesRESUMO
PURPOSE: To provide an overview of essential elements of good governance of data linkage for health-related research, to consider lessons learned so far and to examine key factors currently impeding the delivery of good governance in this area. Given the considerable hurdles which must be overcome and the changing landscape of health research and data linkage, a principled, proportionate, risk-based approach to governance is advocated. DISCUSSION: In light of the considerable value of data linkage to health and well-being, the United Kingdom aspires to design and deliver good governance in health-related research. A string of projects have been asking: what does good governance look like in data linkage for health research? It is argued here that considerable progress can and must be made in order to develop the UK's contribution to future health and wealth economies, particularly in light of mis-start initiatives such as care.data in NHS England. Discussion centres around lessons learned from previous successful health research initiatives, identifying those governance mechanisms which are essential to achieving good governance. CONCLUSION: This article suggests that a crucial element in any step-increase of research capability will be the adoption of adaptive governance models. These must recognise a range of approaches to delivering safe and effective data linkage, while remaining responsive to public and research user expectations and needs as these shift and change with time and experience. The targets are multiple and constantly moving. There is not--nor should we seek--a single magic bullet in delivering good governance in health research.
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Confidencialidade , Registros Eletrônicos de Saúde , Ética em Pesquisa , Armazenamento e Recuperação da Informação , Animais , Confidencialidade/ética , Registros Eletrônicos de Saúde/legislação & jurisprudência , Registros Eletrônicos de Saúde/normas , Humanos , Armazenamento e Recuperação da Informação/ética , Armazenamento e Recuperação da Informação/métodos , Consentimento Livre e Esclarecido , Relações Interprofissionais , Pesquisa , Medicina Estatal , Reino UnidoRESUMO
The pharmacodynamics of MK-912, a benzofuroquinolizine alpha-adrenoceptor antagonist, were evaluated in healthy male volunteers. Eight subjects were treated with single oral doses of 0.1, 1.0, and 2.0 mg MK-912 and with a placebo in a four-period, double-blind, balanced, crossover study. Hemodynamic effects were observed with the 2.0 mg dose of MK-912 (peak increase from baseline in systolic and diastolic blood pressure +/- SEM, 14.8/9.2 +/- 2.9/2.1 mm Hg; peak increase in heart rate, 6.3 +/- 2.1 beats/min; p less than 0.05 versus placebo). Plasma concentrations of 3-methoxy-4-hydroxyphenylglycol (MHPG, a catecholamine metabolite) were increased 29% +/- 7% and 40% +/- 10% above baseline 2 hours after administration of 1.0 and 2.0 mg MK-912, respectively (p less than 0.01 compared with placebo). A modest dose-dependent reduction (5% to 10%) in fasting plasma glucose concentration was observed 1/2 to 1 hour after administration of 1.0 and 2.0 mg MK-912 (p less than 0.05 compared with placebo), without significant change in plasma insulin values. MK-912 was well tolerated, although it did have a mild anxiogenic effect. MK-912 is a potent, orally active agent with a pharmacologic profile consistent with alpha 2-adrenoceptor antagonism.
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Antagonistas Adrenérgicos alfa/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Quinolizinas/farmacologia , Adulto , Afeto/efeitos dos fármacos , Análise de Variância , Glicemia/metabolismo , Relação Dose-Resposta a Droga , Método Duplo-Cego , Humanos , Insulina/sangue , Masculino , Metoxi-Hidroxifenilglicol/sangueRESUMO
Three new angiotensin converting-enzyme inhibitors were given orally to 20 men in single doses ranging from 1.25 to 40 mg. Two of them induced comparable marked inhibition of both the blood pressure response to exogenous angiotensin I and plasma converting-enzyme activity. Onset of action was relatively slow, but 21 to 24 hr after drug plasma converting-enzyme activity was still clearly reduced. The third was less active. There was a close correlation between blood pressure response on administration of angiotensin I and plasma converting-enzyme activity. There were no adverse effects. These new drugs are interesting because of their long duration of action. The measurement of plasma converting-enzyme activity seems useful for monitoring efficacy of converting-enzyme blockade and compliance to therapy.
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Angiotensina I/antagonistas & inibidores , Inibidores da Enzima Conversora de Angiotensina , Angiotensinas/antagonistas & inibidores , Adulto , Aldosterona/sangue , Angiotensina II/sangue , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Masculino , Peptidil Dipeptidase A/sangue , Renina/sangueRESUMO
We found previously that [d]-alpha-tocopherol (alpha-T) and [d]-gamma-tocopherol (gamma-T) are lipid antioxidants (thiobarbituric acid test) in model systems containing arachidonic acid (AA), cumene hydroperoxide, and Fe3+ and in smooth muscle cell (SMC) cultures challenged with AA. We now show that [d]-alpha-tocopherylquinone (alpha-TQ), [d]-delta-tocopherylquinone (delta-TQ), and [d]-gamma-tocopherylquinone (gamma-TQ) are antioxidants at low concentrations and prooxidants at high concentrations in the model system. Prooxidant activity is greater with gamma-TQ than either alpha-TQ or delta-TQ. Low concentrations of alpha-TQ, delta-TQ, and gamma-TQ are also antioxidants in SMC cultures challenged with AA. Unlike alpha-TQ, partially substituted gamma-TQ and glutathione (GSH) form a Michael adduct which has been purified and characterized. We found previously that alpha-T, gamma-T, and alpha-TQ are mitogenic in SMC. We now report that both delta-TQ and gamma-TQ but not alpha-TQ show concentration-dependent cytotoxicity (changes in morphology, propidium iodide stain) in SMC cultures. Cytotoxicity is greater with gamma-TQ than delta-TQ. An acute lymphoblastic leukemia (ALL) cell line shows greater chemosensitivity (MTT and Neutral Red assays) to gamma-TQ than to either doxorubicin (DOX) or vinblastine (VLB). An ALL cell line resistant to both DOX and VLB retains the same chemosensitivity to gamma-TQ as the drug-sensitive ALL cell line. ALL cell lines are unaffected by either alpha-TQ or the GSH Michael adduct of gamma-TQ. These data show that partially substituted tocopheryl quinones capable of forming Michael adducts are potential chemotherapeutic agents for multidrug-resistant cancer cells.
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Antioxidantes/farmacologia , Morte Celular/efeitos dos fármacos , Quinonas/farmacologia , Vitamina E/farmacologia , Animais , Ácido Araquidônico/farmacologia , Derivados de Benzeno/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Glutationa/química , Cobaias , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Tumorais Cultivadas , Vitamina E/administração & dosagem , Vitamina E/análogos & derivadosRESUMO
Mevinolin (MK-803) is a potent inhibitor of HMG-CoA reductase. After a placebo run-in period, mevinolin 5,15 or 50 mg, or placebo was given twice daily for 7-11 days under double -blind conditions ot 4 groups of 6 normocholesterolemic male volunteers. After 7 days, mean serum cholesterol fell 14%, 25% and 24% on 5, 15 and 50 mg, respectively, which was significantly greater than the fall on placebo (4%) in the case of the two higher doses (P less than 0.01). Serum triglycerides did not change significantly. Mevinolin was generally well-tolerated and there were no serious adverse effects.
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Anticolesterolemiantes/farmacologia , Colesterol/sangue , Inibidores de Hidroximetilglutaril-CoA Redutases , Naftalenos/farmacologia , Adulto , Humanos , Lovastatina , Masculino , Naftalenos/administração & dosagem , Naftalenos/efeitos adversosRESUMO
We describe a rapid procedure using propidium iodide (PI) as a nuclear counterstain in immunofluorescence studies where cell surface or intracellular antigens are localized with fluorescein-conjugated antisera. In fixed monolayer preparations, all cell nuclei fluoresce red and can be seen simultaneously with cellular antigens that fluoresce green. Counterstaining with PI therefore makes possible quantification of the proportion of cells present in culture that stain immunocytochemically for a specific antigen.
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Núcleo Celular/análise , Fenantridinas , Propídio , Células Cultivadas , Fluoresceína-5-Isotiocianato , Fluoresceínas , Imunofluorescência , Rodaminas , Coloração e Rotulagem , TiocianatosRESUMO
A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.
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Sobrevivência Celular , Fluoresceínas , Fenantridinas , Propídio , Coloração e Rotulagem/métodos , Animais , Contagem de Células , Histocitoquímica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Baço/citologia , Azul TripanoRESUMO
A supravital staining procedure for the identification of macrophages in cell suspension using a modification of a standard cytochemical assay for alpha-naphthyl acetate esterase (ANAE) activity is described. Macrophages are stained an intense red-brown after 5 min incubation in a buffer using ANAE as the substrate and hexazonium pararosaniline as the coupler for the azo dye. There is close agreement in the number of ANAE-positive cells found and the number of macrophages identified in smears by morphological criteria, by phagocytosis, and by the presence of Fc receptors. Therefore, this stain provides a quick, inexpensive method to estimate the number of macrophages present in suspensions of lymphocytic tissues from rats and mice.
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Hidrolases de Éster Carboxílico/metabolismo , Macrófagos/enzimologia , Naftol AS D Esterase/metabolismo , Animais , Feminino , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Endogâmicos , Coloração e Rotulagem/métodosRESUMO
PURPOSE: To examine families ascertained for late-onset primary open-angle glaucoma (POAG) to determine mutations in the gene coding for myocilin. METHODS: The diagnosis of late-onset POAG was defined as age at diagnosis more than 35 years, intraocular pressure (IOP) 22 mm Hg or more in both eyes or 19 mm Hg or more while the patient was taking two glaucoma medications, glaucomatous optic neuropathy in both eyes, and visual field loss consistent with optic nerve damage in at least one eye of the proband. Two of three criteria were required in other family members. DNA from all families was screened for polymorphisms in myocilin using single-strand conformation polymorphism analysis. All polymorphisms were sequenced for mutations. RESULTS: Eighty-three affected people in 29 families with late-onset POAG were screened for mutations. Three mutations, two novel missense (Thr377Met and Glu352Lys) and one nonsense (Gln368STOP), were identified. The missense mutations did not segregate with the disease phenotype in these families. The nonsense mutation was found in 3 of 29 unrelated families with POAG. All affected family members and 8 of 12 in whom glaucoma was suspected had the Gln368STOP mutation. All people with this mutation had elevated IOP, and 78% had POAG by age 70. CONCLUSIONS: Three mutations were identified in the gene coding for myocilin in families with late-onset POAG. Of these, the Gln368STOP mutation was highly associated with the development of glaucoma. All people with this mutation had glaucoma or elevated IOP by age 70. In the United States, the Gln368STOP mutation in myocilin is strongly associated with the development of late-onset POAG. However, factors in addition to the presence of this mutation seem to play a role in the development of ocular hypertension and glaucoma in these families.