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1.
Healthc Policy ; 14(2): 22-30, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30710438

RESUMO

Introduction: The concept, "most responsible provider" has a specific definition in the Canadian National Discharge Abstract Database (DAD). Variation exists in how care providers are defined in administrative data. Methods: We compared chart data with administrative data to understand how "most responsible provider" was identified in these two data sources. Results: We found a 3% discrepancy between data sources. Differences between data sources were attributable to transfers in care that occurred at birth. Discussion: "Most responsible provider" should consider the full trajectory of care when assigning outcomes in order to understand how to best support optimal health among low-risk births.


Assuntos
Cesárea/estatística & dados numéricos , Registros Médicos/estatística & dados numéricos , Alocação de Recursos/estatística & dados numéricos , Resultado do Tratamento , Adulto , Canadá , Feminino , Humanos , Gravidez
3.
Clin Pract Cases Emerg Med ; 1(3): 183-186, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29849283

RESUMO

Isolated pelvic deep vein thromboses (DVT) are rare and difficult to diagnose, but they are more common in pregnant women and carry an increased risk of embolization. Pulmonary embolism is the most common non-obstetric cause of death in pregnancy. Compression ultrasound is the first-line imaging test for suspected lower extremity DVT, but it cannot usually aid in directly visualizing or easily diagnosing isolated pelvic DVT. Nonetheless, point-of-care ultrasound (POCUS) may provide valuable clues to help rule in pelvic DVT and expedite initiation of anticoagulant therapy. Such findings include increased venous diameter, increased resistance to compression, visible venous reflux, and blunted phasicity. This case presents an example of how these findings on POCUS led the emergency physician to make the difficult diagnosis of pelvic DVT at the bedside within seconds.

4.
Clin Pract Cases Emerg Med ; 1(3): 221-224, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29849294

RESUMO

Blunt scrotal injury represents a diagnostic dilemma for emergency physicians (EP). Consequently, point-of-care ultrasound (POCUS) has emerged as a tool for early investigation of the acute scrotum in the emergency department. We describe a case where an EP used scrotal POCUS to immediately visualize the loss of testicular contour and underlying heterogeneous parenchyma to rapidly make the diagnosis of testicular rupture in a young male presenting with scrotal trauma. The use of POCUS in this case expedited therapy, likely improving the patient's outcome. To our knowledge, this is the first detailed description of testicular rupture diagnosed with POCUS by an EP.

5.
Clin Pract Cases Emerg Med ; 1(4): 395-398, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29849342

RESUMO

A 15-day-old male who was born at term presented with non-bilious projectile vomiting. He was nontoxic and his abdomen was benign without masses. Point-of-care ultrasound (POCUS) showed hypertrophic pyloric stenosis (HPS). Typical findings include target sign; pyloric muscle thickness greater than three millimeters (mm); channel length greater than 15-18 mm; and lack of gastric emptying. The patient was admitted; consultative ultrasound (US) was negative, but repeated 48 hours later for persistent vomiting. This second US was interpreted as HPS, which was confirmed surgically. Pyloromyotomy was successful. Few reports describe POCUS by general emergency physicians to diagnose HPS. Here, we emphasize the value in repeat US for patients with persistent symptoms.

6.
Birth ; 43(3): 269-70, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27534515
7.
Midwifery ; 39: 12-9, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27321715

RESUMO

OBJECTIVE: the primary objective for this study was to explore women's experiences of choosing to plan a birth at an out-of-hospital birth centre. We sought to understand how women make the choice to plan for an out-of-hospital birth and the meaning that women ascribe to this decision-making process. DESIGN, SETTING, AND PARTICIPANTS: a qualitative phenomenological study was conducted in Winnipeg, Canada with a sample of seventeen post partum women who represent the socio-demographic characteristics of the actual users of the Birth Centre in Winnipeg. The women participated in semistructured interviews. Through a feminist perspective and using interpretative phenomenological analysis (IPA), each participant's experience of birthplace decision-making was explored. FINDINGS: six themes emerged through the analysis: (1) Making the decision in the context of relationships; (2) Exercising personal agency; (3) An expression of one's ideology; (4) Really thinking it through; (5) Fitting into the eligibility criteria; and (6) The psychology of the space. The findings suggested that a woman's sense of safety was related to each of these themes. KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: the birth centre decision-making experience has many similarities to the homebirth decision-making process. The visceral impact of the physical design of the facility plays an important role and differentiates the birth centre decision from other birth setting options. The concept of relational autonomy was emphasised in this study, in that women make the decision in the context of their relationships with their midwives and partners. The study has implications for midwifery practice and health-care policy related to: client education on birth settings, design of birth environments, validation of the birth centre concept, and upholding the women-centred midwifery model of care. The study highlighted the importance of increasing access to out-of-hospital birth centres.


Assuntos
Centros de Assistência à Gravidez e ao Parto/normas , Comportamento de Escolha , Tomada de Decisões , Pais/psicologia , Adulto , Instituições de Assistência Ambulatorial/normas , Canadá , Feminino , Planejamento em Saúde/métodos , Humanos , Masculino , Gravidez , Pesquisa Qualitativa , Inquéritos e Questionários , Cobertura Universal do Seguro de Saúde
8.
BMC Health Serv Res ; 16: 92, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26976610

RESUMO

BACKGROUND: In 2000, midwifery was regulated in the Canadian Province of Manitoba. Since the establishment of the midwifery program, little formal research has analyzed the utilization of regulated midwifery services. In Manitoba, the demand for midwifery services has exceeded the number of midwives in practice. The specific objective of this study was to explore factors influencing the implementation and utilization of regulated midwifery services in Manitoba. METHODS: The case study design incorporated qualitative exploratory descriptive methods, using data derived from two sources: interviews and public documents. Twenty-four key informants were purposefully selected to participate in semi-structured in-depth interviews. All documents analyzed were in the public domain. Content analysis was employed to analyze the documents and transcripts of the interviews. RESULTS: The results of the study were informed by the Behavioral Model of Health Services Use. Three main topic areas were explored: facilitators, barriers, and future strategies and recommendations. The most common themes arising under facilitators were funding of midwifery services and strategies to integrate the profession. Power and conflict, and lack of a productive education program emerged as the most prominent themes under barriers. Finally, future strategies for sustaining the midwifery profession focused on ensuring avenues for registration and education, improving management strategies and accountability frameworks within the employment model, enhancing the work environment, and evaluating both the practice and employment models. Results of the document analysis supported the themes arising from the interviews. CONCLUSION: These findings on factors that influenced the implementation and integration of midwifery in Manitoba may provide useful information to key stakeholders in Manitoba, as well as other provinces as they work toward successful implementation of regulated midwifery practice. Funding for new positions and programs was consistently noted as a successful strategy. While barriers such as structures of power within Regional Health Authorities and inter and intra-professional conflict were identified, the lack of a productive midwifery education program emerged as the most prominent barrier. This new knowledge highlights issues that impact the ongoing growth and capacity of the midwifery profession and suggests directions for ensuring its sustainability.


Assuntos
Difusão de Inovações , Regulamentação Governamental , Tocologia/legislação & jurisprudência , Tocologia/normas , Feminino , Política de Saúde , Humanos , Entrevistas como Assunto , Manitoba , Estudos de Casos Organizacionais , Gravidez , Pesquisa Qualitativa
9.
Birth ; 43(2): 108-15, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26889889

RESUMO

BACKGROUND: Registered midwives, obstetricians/gynecologists, and general or family practice physicians (GPs) provide maternity care across Canada. Few North American studies have assessed whether maternity outcomes differ across these three groups. This study compared maternal and neonatal outcomes of low-risk pregnant women whose birth was attended by registered midwives, obstetricians/gynecologists, and family practice physicians in Winnipeg, Manitoba from 2001/02 to 2012/13. METHODS: Descriptive statistics and logistic regression were used to examine differences in types of intervention, mode of delivery, and outcomes by provider type among low-risk women. Logistic regression models controlled for socio-demographic and birth-related covariates. RESULTS: Low-risk births comprised 83,774 (48.7%) of total births (n = 171,910). The adjusted odds ratio (aOR), (95% confidence interval) for midwife vs OB/GYN showed women who had a midwife attend the birth had reduced odds of having an episiotomy 0.47 (0.40-0.54), epidural 0.25 (0.23-0.27), and cesarean delivery 0.13 (0.10-0.16) and their infants had less Neonatal Intensive Care Unit admissions 0.28 (0.18-0.43). The aOR for GP versus OB/GYN showed women who had a GP had reduced odds of having an epidural/spinal 0.83 (0.79-0.88) and cesarean delivery 0.44 (0.40-0.48). CONCLUSIONS: The effectiveness of Manitoba maternity services can be improved with increased use of integrated midwifery services. Future research should examine how midwifery and physician-led models of care differ, and the influence of these differences on birth outcomes and cost-effectiveness to the health care system. Improvement of data tracking systems is also needed.


Assuntos
Nascido Vivo/epidemiologia , Serviços de Saúde Materna , Enfermeiras Obstétricas , Obstetrícia , Médicos de Família , Adolescente , Adulto , Cesárea/estatística & dados numéricos , Episiotomia/estatística & dados numéricos , Feminino , Humanos , Modelos Logísticos , Manitoba , Gravidez , Recursos Humanos , Adulto Jovem
10.
J Obstet Gynaecol Can ; 37(8): 707-714, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26474227

RESUMO

OBJECTIVE: To describe the trends in numbers of midwives and midwifery-attended births and the characteristics of women who used midwifery health care services in Manitoba from 2001-2002 to 2009-2010. METHODS: We conducted a quantitative descriptive analysis using population-based, de-identified administrative data from the Population Health Research Data Repository at the Manitoba Centre for Health Policy in Winnipeg, Manitoba to study the use of midwifery care. Trends in the numbers of practising and non-practising midwives were based on data from the College of Midwives of Manitoba registries and its annual reports. RESULTS: There were 132,123 births in Manitoba during this time frame. Of those births, 6326 (4.8%) were midwife-attended births. There was modest growth in the overall proportion of midwife-attended births and in the number of midwives over the 10-year time period. The number of midwife-attended hospital births increased from 308 to 612 between 2001-2002 and 2009-2010, while the number of home births increased from 97 to 127. Most women who received midwifery care were in the 20- to 34-year age group and were multiparous. CONCLUSION: The volume and distribution of midwifery services in Manitoba has slowly increased. The proportion of births attended by midwives continues to fall short of the goals set by the original human resource strategy, which projected that by 2005, 14% of births would be attended by midwives. Further research is needed to analyze the factors that have influenced the growth and sustainability of the midwifery profession in this province.


Assuntos
Enfermeiras Obstétricas/estatística & dados numéricos , Enfermeiras Obstétricas/tendências , Adulto , Feminino , Humanos , Manitoba , Paridade , Gravidez , Adulto Jovem
11.
Dis Model Mech ; 4(5): 634-48, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21596710

RESUMO

A growing body of evidence supports the 'calcium hypothesis' of Alzheimer's disease (AD), which postulates that a variety of insults might disrupt the homeostatic regulation of neuronal calcium (Ca(2+)) in the brain, resulting in the progressive symptoms that typify the disease. However, despite ongoing efforts to develop new methods for testing therapeutic compounds that might be beneficial in AD, no single bioassay permits both rapid screening and in vivo validation of candidate drugs that target specific components of the Ca(2+) regulatory machinery. To address this issue, we have integrated four distinct model systems that provide complementary information about a trial compound: the human neuroblastoma MC65 line, which provides an in vitro model of amyloid toxicity; a transgenic Drosophila model, which develops age-dependent pathologies associated with AD; the 3×TgAD transgenic mouse, which recapitulates many of the neuropathological features that typify AD; and the embryonic nervous system of Manduca, which provides a novel in vivo assay for the acute effects of amyloid peptides on neuronal motility. To demonstrate the value of this 'translational suite' of bioassays, we focused on a set of clinically approved dihydropyridines (DHPs), a class of well-defined inhibitors of L-type calcium channels that have been suggested to be neuroprotective in AD. Among the DHPs tested in this study, we found that isradipine reduced the neurotoxic consequences of ß-amyloid accumulation in all four model systems without inducing deleterious side effects. Our results provide new evidence in support of the Ca(2+) hypothesis of AD, and indicate that isradipine represents a promising drug for translation into clinical trials. In addition, these studies also demonstrate that this continuum of bioassays (representing different levels of complexity) provides an effective means of evaluating other candidate compounds that target specific components of the Ca(2+) regulatory machinery and that therefore might be beneficial in the treatment of AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Modelos Animais de Doenças , Isradipino/uso terapêutico , Pesquisa Médica Translacional , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Animais , Bioensaio , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Drosophila/efeitos dos fármacos , Humanos , Isradipino/administração & dosagem , Isradipino/farmacologia , Manduca/efeitos dos fármacos , Manduca/embriologia , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/ultraestrutura , Substâncias Protetoras/farmacologia
12.
Mol Imaging Biol ; 12(3): 240-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19949987

RESUMO

PURPOSE: The purpose of this study is to detect myelin-specific T cells, key pathological mediators in early multiple sclerosis, and the corresponding animal model, experimental autoimmune encephalomyelitis (EAE), in the mouse spinal cord. PROCEDURES: T cells were labeled with the iron-based, magnetic resonance (MR) contrast reagent, Feridex, and the transfection reagent, protamine sulfate, resulting in approximately 100% iron-labeling efficiency. Feridex-labeling did not alter the induction of EAE by T cells, and recipients were imaged by a 12-T MR instrument. RESULTS: Focal hypointense lesions were resolvable to gray or white matter of the lumbar spinal cord in T(2)-weighted images of the recipients of Feridex-labeled T cells. Lesions corresponded to histological evidence of inflammatory lesions and iron-labeled cells in eight-of-eight mice. In contrast, hypointense lesions were not observed eight-of-eight recipients of unlabeled T cells. CONCLUSIONS: These results demonstrate and provide methodologies for labeling, detecting, and extracting MRI-detectable foci of iron-labeled cells.


Assuntos
Autoimunidade/imunologia , Encefalomielite Autoimune Experimental/imunologia , Inflamação/imunologia , Imageamento por Ressonância Magnética , Bainha de Mielina/imunologia , Medula Espinal/imunologia , Linfócitos T/imunologia , Animais , Encefalomielite Autoimune Experimental/patologia , Inflamação/diagnóstico , Ferro/metabolismo , Camundongos , Bainha de Mielina/patologia , Protaminas/metabolismo , Medula Espinal/patologia , Coloração e Rotulagem
13.
J Neuroimmunol ; 204(1-2): 110-7, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18722022

RESUMO

Xenotransplantation of rat bone marrow cells (BMC) into immunodeficient (SCID) mice generates chimeric mice susceptible to paralytic autoimmune CNS inflammation. Herein, we identified a disease relevant subset of transplantable BMC lacking expression of CD11b/c and CD49d. Moreover, disease susceptibility was enhanced in the presence of non-myelin specific T-cells. Only the CD11b/c negative population of BM retained the capability to populate the blood, spleen and spinal cord of recipients and matured after transplant to express CD11b/c. These results indicate non-myelin T cells in combination with integrin negative BM represent pre-pathogenic determinants of an enhanced disease susceptibility to myelin reactive T cells.


Assuntos
Transplante de Medula Óssea/métodos , Encefalomielite Autoimune Experimental/cirurgia , Integrinas/deficiência , Transplante Heterólogo/métodos , Animais , Células da Medula Óssea , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Transplante de Células/métodos , Quimera , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Imunofenotipagem/métodos , Integrina alfa1/metabolismo , Camundongos , Camundongos SCID , Ratos , Ratos Endogâmicos Lew , Índice de Gravidade de Doença
14.
Nat Protoc ; 3(6): 941-7, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18536642

RESUMO

A general protocol is described to improve the specificity for imaging superoxide formation in live cells via fluorescence microscopy with either hydroethidine (HE) or its mitochondrially targeted derivative Mito-HE (MitoSOX Red). Two different excitation wavelengths are used to distinguish the superoxide-dependent hydroxylation of Mito-HE (385-405 nm) from the nonspecific formation of ethidium (480-520 nm). Furthermore, the dual wavelength imaging in live cells can be combined with immunocolocalization, which allows superoxide formation to be compared simultaneously in cocultures of two types of genetically manipulated cells in the same microscopic field. The combination of these approaches can greatly improve the specificity for imaging superoxide formation in cultured cells and tissues.


Assuntos
Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Superóxidos/metabolismo , Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/genética , Animais , Animais Geneticamente Modificados , Astrócitos/metabolismo , Técnicas de Cocultura , Corantes Fluorescentes/metabolismo , Humanos , Hidroxilação , Mutação , Oxirredução , Fenantridinas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
15.
J Neurosci ; 28(16): 4115-22, 2008 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-18417691

RESUMO

Mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Recent reports indicate that astrocytes expressing the mutations of superoxide dismutase-1 (SOD1) may contribute to motor neuron injury in ALS. Here, we provide evidence that mitochondrial dysfunction in SOD1(G93A) rat astrocytes causes astrocytes to induce apoptosis of motor neurons. Mitochondria from SOD1(G93A) rat astrocytes displayed a defective respiratory function, including decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential. Protein 3-nitrotyrosine was detected immunochemically in mitochondrial proteins from SOD1(G93A) astrocytes, suggesting that mitochondrial defects were associated with nitroxidative damage. Furthermore, superoxide radical formation in mitochondria was increased in SOD1(G93A) astrocytes. Similar defects were found in mitochondria isolated from the spinal cord of SOD1(G93A) rats, and pretreatment of animals with the spin trap 5,5-dimethyl-1-pyrroline N-oxide restored mitochondrial function, forming adducts with mitochondrial proteins in vivo. As shown previously, SOD1(G93A) astrocytes induced death of motor neurons in cocultures, compared with nontransgenic ones. This behavior was recapitulated when nontransgenic astrocytes were treated with mitochondrial inhibitors. Remarkably, motor neuron loss was prevented by preincubation of SOD1(G93A) astrocytes with antioxidants and nitric oxide synthase inhibitors. In particular, low concentrations (approximately 10 nm) of two mitochondrial-targeted antioxidants, ubiquinone and carboxy-proxyl nitroxide, each covalently coupled to a triphenylphosphonium cation (Mito-Q and Mito-CP, respectively), prevented mitochondrial dysfunction, reduced superoxide production in SOD1(G93A) astrocytes, and restored motor neuron survival. Together, our results indicate that mitochondrial dysfunction in astrocytes critically influences motor neuron survival and support the potential pharmacological utility of mitochondrial-targeted antioxidants in ALS treatment.


Assuntos
Antioxidantes/administração & dosagem , Astrócitos/enzimologia , Mitocôndrias/enzimologia , Neurônios Motores/enzimologia , Degeneração Neural/enzimologia , Superóxido Dismutase/genética , Substituição de Aminoácidos/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Esclerose Amiotrófica Lateral/prevenção & controle , Animais , Animais Geneticamente Modificados , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Degeneração Neural/genética , Degeneração Neural/prevenção & controle , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/fisiologia
16.
J Neurosci ; 27(29): 7777-85, 2007 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-17634371

RESUMO

Nerve growth factor (NGF) can induce apoptosis by signaling through the p75 neurotrophin receptor (p75(NTR)) in several nerve cell populations. Cultured embryonic motor neurons expressing p75(NTR) are not vulnerable to NGF unless they are exposed to an exogenous flux of nitric oxide (*NO). In the present study, we show that p75(NTR)-mediated apoptosis in motor neurons involved neutral sphingomyelinase activation, increased mitochondrial superoxide production, and cytochrome c release to the cytosol. The mitochondria-targeted antioxidants mitoQ and mitoCP prevented neuronal loss, further evidencing the role of mitochondria in NGF-induced apoptosis. In motor neurons overexpressing the amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1(G93A) (SOD1(G93A)) mutation, NGF induced apoptosis even in the absence of an external source of *NO. The increased susceptibility of SOD1(G93A) motor neurons to NGF was associated to decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and downregulation of the enzymes involved in glutathione biosynthesis. In agreement, depletion of glutathione in nontransgenic motor neurons reproduced the effect of SOD1(G93A) expression, increasing their sensitivity to NGF. In contrast, rising antioxidant defenses by Nrf2 activation prevented NGF-induced apoptosis. Together, our data indicate that p75(NTR)-mediated motor neuron apoptosis involves ceramide-dependent increased mitochondrial superoxide production. This apoptotic pathway is facilitated by the expression of ALS-linked SOD1 mutations and critically modulated by Nrf2 activity.


Assuntos
Apoptose/fisiologia , Mitocôndrias/metabolismo , Neurônios Motores/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Medula Espinal/citologia , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Células Cultivadas , Citocromos c/metabolismo , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Mitocôndrias/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/farmacologia
17.
J Biol Chem ; 282(9): 6324-37, 2007 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-17200124

RESUMO

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radical-mediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.


Assuntos
Apoptose/efeitos dos fármacos , Neurônios Motores/patologia , Peptídeos/farmacologia , Ácido Peroxinitroso/farmacologia , Tirosina , Animais , Sequestradores de Radicais Livres/farmacologia , Radicais Livres , Células PC12 , Ratos , Tirosina/análogos & derivados
18.
Free Radic Biol Med ; 41(11): 1632-44, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17145551

RESUMO

Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo posttranslational oxidative or nitrative modifications that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed an exceptional ability to induce p75(NTR)-dependent motor neuron apoptosis at physiologically relevant concentrations. Whereas native NGF requires an external source of nitric oxide (NO) to induce motor neuron death, peroxynitrite-treated NGF induced motor neuron apoptosis in the absence of exogenous NO. Nevertheless, NO potentiated the apoptotic activity of peroxynitrite-modified NGF. Blocking antibodies to p75(NTR) or downregulation of p75(NTR) expression by antisense treatment prevented motor neuron apoptosis induced by peroxynitrite-treated NGF. We investigated what oxidative modifications were responsible for inducing a toxic gain of function and found that peroxynitrite induced tyrosine nitration in a dose-dependent manner. Moreover, peroxynitrite triggered the formation of stable high-molecular-weight oligomers of NGF. Preventing tyrosine nitration by urate abolished the effect of peroxynitrite on NGF apoptotic activity. These results indicate that the oxidation of NGF by peroxynitrite enhances NGF apoptotic activity through p75(NTR) 10,000-fold. To our knowledge, this is the first known posttranslational modification that transforms a neurotrophin into an apoptotic agent.


Assuntos
Apoptose/fisiologia , Neurônios Motores/metabolismo , Fator de Crescimento Neural/química , Ácido Peroxinitroso/farmacologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Espectrometria de Massas , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Fator de Crescimento Neural/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ratos , Receptor de Fator de Crescimento Neural/antagonistas & inibidores , Receptor de Fator de Crescimento Neural/efeitos dos fármacos , Receptor de Fator de Crescimento Neural/metabolismo , Tirosina/metabolismo
19.
Proc Natl Acad Sci U S A ; 103(41): 15038-43, 2006 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-17015830

RESUMO

The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd). Recently, superoxide was shown to react with HE to generate 2-hydroxyethidium [Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J. & Kalyanaraman, B. (2003) Free Radic. Biol. Med. 34, 1359-1368]. However, 2-hydroxyethidium is difficult to distinguish from Etd by conventional fluorescence techniques exciting at 510 nm. While investigating the oxidation of Mito-HE by superoxide, we found that the superoxide product of both HE and Mito-HE could be selectively excited at 396 nm with minimal interference from other nonspecific oxidation products. The oxidation of Mito-HE monitored at 396 nm by antimycin-stimulated mitochondria was 30% slower than at 510 nm, indicating that superoxide production may be overestimated at 510 nm by even a traditional superoxide-stimulating mitochondrial inhibitor. The rate-limiting step for oxidation by superoxide was 4x10(6) M-1.s-1, which is proposed to involve the formation of a radical from Mito-HE. The rapid reaction with a second superoxide anion through radical-radical coupling may explain how Mito-HE and HE can compete for superoxide in vivo with intracellular superoxide dismutases. Monitoring oxidation at both 396 and 510 nm of excitation wavelengths can facilitate the more selective detection of superoxide in vivo.


Assuntos
Etídio , Corantes Fluorescentes , Fenantridinas , Superóxidos/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Microscopia Confocal , Mitocôndrias/química , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Superóxidos/química
20.
J Surg Res ; 136(1): 25-30, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16978650

RESUMO

BACKGROUND: Cytokine activation in the pancreatitis induces local and systemic cellular damage. Transcription factors interferon regulatory factor-1 (IRF-1) and the tumor suppressor gene p53 collaborate to enhance p21 related cell cycle regulation during pathological disease progression. However, little is known about their role in the pancreas after cytokine challenge. Our laboratory has previously shown that TNF-alpha induces the binding of many transcription factors, including NF-kappa B, and treatment with the gut hormone, Peptide YY (PYY), ameliorates the effects. We hypothesized that TNF-alpha would induce IRF-1 and p53 protein binding in pancreatic acinar cells and that PYY would attenuate the effect. MATERIALS AND METHODS: Rat pancreatic acinar AR42J cells were treated with rat recombinant TNF-alpha (200 ng/ml). To verify that our model was inducing pancreatitis, alpha-amylase activity was measured in the cell culture supernatant by fluorescence spectroscopy. PYY [3-36] was added at 500 pM 30 min post-TNF treatment; cells were harvested at 2 h for extraction of nuclear protein. Transcription factor binding of IRF-1 and p53 were determined by protein/DNA array analysis using chemiluminescence detection, and relative spot densities were measured by densitometry. A two-fold increase or decrease in density was considered significant. RESULTS: Amylase enzyme activity was significantly (P < 0.05) elevated in the TNF-alpha-treated cells by 2 h. Protein/DNA array analysis revealed significant up-regulation of both IRF-1 and p53 protein in nuclear extracts. Induction by TNF-alpha increased IRF-1 protein binding 3.5-fold, while binding levels of p53 protein increased six-fold. The addition of PYY to TNF-treated cells reduced IRF-1 and p53 binding to control levels. CONCLUSIONS: We have shown for the first time that short-term exposure to TNF-alpha induces the binding activity of transcription factors IRF-1 and p53 in rat pancreatic acinar cells, and that addition of PYY reduces it. Regulation of transcription factor activity by PYY may have therapeutic potential in altering the progression of pancreatitis.


Assuntos
Fator Regulador 1 de Interferon/metabolismo , Pâncreas Exócrino/metabolismo , Peptídeo YY/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Doença Aguda , Amilases/metabolismo , Animais , Linhagem Celular , Fator Regulador 1 de Interferon/genética , Análise de Sequência com Séries de Oligonucleotídeos , Pâncreas Exócrino/citologia , Pâncreas Exócrino/efeitos dos fármacos , Pancreatite/metabolismo , Peptídeo YY/farmacologia , Ligação Proteica/efeitos dos fármacos , Ratos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/genética
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