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1.
BMC Med Educ ; 20(1): 271, 2020 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807145

RESUMO

BACKGROUND: An experiential curriculum exposing medical students to the clinic early has many benefits but comes with the emotional stress this environment engenders. Schwartz rounds (SR) are an effective means to combat emotional stress and increasingly used in UK and USA hospitals. Recent studies show that the SR format may also provide benefits for medical students. This study aimed to investigate whether the guidance of SR in second year medical students provides the same benefits as to healthcare professionals. METHODS: SR assessment involved 83 s year MBChB students in facilitated groupwork sessions. Topics discussed were "change and resilience" and "duty of candour". Students completed a Likert Scale questionnaire evaluating outcomes proffered by the Point of Care Foundation in collaboration with the Schwartz Foundation, with freeform feedback. RESULTS: There was an 86% completion rate with 25% providing written feedback. Participants were more likely to agree than disagree that SR were beneficial. SR effectiveness in enhancing students' working relationship awareness and skills was strongly correlated with understanding the purpose of, and engagement with, the SR (P < 0.001). Similarly, engagement with the SR was strongly correlated with self-reporting of enhanced patient-centredness (P < 0.001). Freeform feedback could be grouped into five themes that revolved around understanding of the SR and engagement with the process. Many positive comments regarded the SR as a forum not only to "learn experientially" but to so in a "safe environment". Many negative comments stemmed from students not seeing any benefits of engagement with the SR, in that sharing experiences was "unbeneficial", "empathy is inherent and not learnt", or that sharing emotional problems is simply "moaning". CONCLUSION: SRs are an effective way of fostering empathy and understanding towards patients and colleagues. However, for the students to benefit fully from the SR it is necessary for them to engage and understand the process. Therefore, for the successful implementation of SR into pre-clinical medical education, it is important to help students realise that SR are not merely a "facilitated whinge".


Assuntos
Educação de Graduação em Medicina , Educação Médica , Estudantes de Medicina , Visitas de Preceptoria , Currículo , Empatia , Humanos
2.
Ir Med J ; 113(1): 7, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-32298567

RESUMO

Aims To determine the completeness of polyp resection (i.e. achieving an R0 margin) and its relation with Endoscopists, histopathologist, size, location and technique of polypectomy in an NSS cohort. The definition of R0 margin is complete macroscopic resection with a negative microscopic margin at polypectomy. Method NCCS (National Colon Cancer Screening) colonoscopies are offered to bowel cancer screening patients after a positive faecal immunochemical test (FIT) test in a Joint Advisory Group (JAG) accredited Gastrointestinal Endoscopy centre. We histologically evaluated the polyp margins for complete resection, which was defined as the absence of adenomatous or hyperplastic tissue in the resected polyp margins in a cohort of faecal immunochemical test positive patients. Results A total of 186 consecutive NCCS colonoscopies out of a total of 542 performed between 2013 and 2017 were included in this study. Of the polyps excised 152(27%) had a R0 margin histologically, and 30(5%) had involvement of the margin. Surprisingly in 373(67%) of polyps pathologists were unable to assess the margin. Conclusion Achieving an R0 margin should be a key performance indicator for endoscopists performing polypectomy. At the same time more studies on polyp margins are recommended.


Assuntos
Pólipos Intestinais/cirurgia , Margens de Excisão , Estudos de Coortes , Neoplasias Colorretais/prevenção & controle , Endoscopia Gastrointestinal , Humanos
3.
Gene Ther ; 24(10): 674-679, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28880020

RESUMO

Lentiviral vectors pseudotyped with the baculovirus envelope protein GP64 transduce primary cultures of human airway epithelia (HAE) at their apical surface. Our goal in this study was to harness a directed evolution approach to develop a novel envelope glycoprotein with increased transduction properties for HAE. Using error-prone PCR, a library of GP64 mutants was generated and used to prepare a diverse pool of lentiviral virions pseudotyped with GP64 variants. The library was serially passaged on HAE and three GP64 mutations were recovered. Single-, double- and the triple-combination mutant envelope glycoproteins were compared with wild-type GP64 for their ability to transduce HAE. Our results suggest that lentiviral vectors pseudotyped with evolved GP64 transduced HAE with greater efficiency than wild-type GP64. This effect was not observed in primary cultures of porcine airway epithelial cells, suggesting that the directed evolution protocol was species specific. In summary, our studies indicate that serial passage of a GP64 mutant library yielded specific variants with improved HAE cell tropism, yielding tools with the potential to improve the success of gene therapy for airway diseases.


Assuntos
Técnicas de Transferência de Genes , Mucosa Respiratória/metabolismo , Proteínas do Envelope Viral/genética , Animais , Baculoviridae/genética , Células Cultivadas , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Mucosa Respiratória/citologia , Proteínas do Envelope Viral/metabolismo
4.
Gene Ther ; 24(11): 742-748, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28905885

RESUMO

Current therapies for hemophilia A include frequent prophylactic or on-demand intravenous factor treatments which are costly, inconvenient and may lead to inhibitor formation. Viral vector delivery of factor VIII (FVIII) cDNA has the potential to alleviate the debilitating clotting defects. Lentiviral-based vectors delivered to murine models of hemophilia A mediate phenotypic correction. However, a limitation of lentiviral-mediated FVIII delivery is inefficient transduction of target cells. Here, we engineer a feline immunodeficiency virus (FIV) -based lentiviral vector pseudotyped with the baculovirus GP64 envelope glycoprotein to mediate efficient gene transfer to mouse hepatocytes. In anticipation of future studies in FVIII-deficient dogs, we investigated the efficacy of FIV-delivered canine FVIII (cFVIII). Codon-optimization of the cFVIII sequence increased activity and decreased blood loss as compared to the native sequence. Further, we compared a standard B-domain deleted FVIII cDNA to a cDNA including 256 amino acids of the B-domain with 11 potential asparagine-linked oligosaccharide linkages. Restoring a partial B-domain resulted in modest reduction of endoplasmic reticulum (ER) stress markers. Importantly, our optimized vectors achieved wild-type levels of phenotypic correction with minimal inhibitor formation. These studies provide insights into optimal design of a therapeutically relevant gene therapy vector for a devastating bleeding disorder.


Assuntos
Fator VIII/genética , Fator VIII/uso terapêutico , Hemofilia A/terapia , Animais , DNA Complementar/genética , Cães , Fator VIII/fisiologia , Terapia Genética/métodos , Vetores Genéticos , Hemofilia A/genética , Hepatócitos , Lentivirus/genética , Infecções por Lentivirus , Fígado/metabolismo , Camundongos , Fenótipo
5.
Mol Cell Endocrinol ; 444: 26-37, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28137614

RESUMO

Theca cell-selective Pten mutation (tPtenMT) in mice resulted in increases in PDK1 and Akt phosphorylation, indicating an over-activation of PI3K signaling in the ovaries. These mice displayed elevated androgen levels, ovary enlargement, antral follicle accumulation, early fertility loss and increased expression of Lhcgr and genes that are crucial to androgenesis. These abnormalities were partially reversed by treatments of PI3K or Akt inhibitor. LH actions in Pten deficient theca cells were potentiated. The phosphorylation of Foxo1 was increased, while the binding of Foxo1 to forkhead response elements in the Lhcgr promoter was reduced in tPtenMT theca cells, implying a mechanism by which PI3K/Akt-induced upregulation of Lhcgr in theca cells might be mediated by reducing the inhibitory effect of Foxo1 on the Lhcgr promoter. The phenotype of tPtenMT females is reminiscent of human PCOS and suggests that dysregulated PI3K cascade in theca cells may be involved in certain types of PCOS pathogenesis.


Assuntos
Androgênios/metabolismo , Deleção de Genes , Ovário/fisiopatologia , PTEN Fosfo-Hidrolase/genética , Células Tecais/metabolismo , Envelhecimento/metabolismo , Animais , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Feminino , Fertilidade , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Modelos Biológicos , Ovário/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Receptores do LH/genética , Receptores do LH/metabolismo , Esteroides/biossíntese , Testosterona/sangue , Células Tecais/efeitos dos fármacos
6.
J R Nav Med Serv ; 100(2): 157-60, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25335310

RESUMO

AIMS: The purpose of this study was to determine the accuracy of Magnetic Resonance Imaging (MRI) scanning in the detection of meniscal pathology in a district general hospital. METHODS: We retrospectively analysed a single-surgeon series of 240 knee arthroscopic investigations for all indications. The arthroscopic reports included an outline diagram of the meniscus upon which the surgeon could record his operative findings. 112 of these patients had also had a recent MRI scan. We compared the MRI findings with the arthroscopy findings. RESULTS: 66 patients had a positive MRI scan. 64 of these were found to have a meniscal tear at surgery. 37 MRI scans were reported as "no tear", of which four were found to have a meniscal tear at surgery. Nine MRI scans were descriptive, e.g. "signal change, possible tear", or "tear cannot be ruled out." These tended to correspond with equivocal arthroscopic findings of "degeneration" or "fibrillation". In our series of 112 patients with meniscal pathology, MRI scanning was 90.5% sensitive, 89.5% specific and 90.1% accurate. CONCLUSIONS: False positive MRI scans may lead to unnecessary surgery. Patients with negative MRI scans had a mean delay to surgery of 33 weeks compared to 18 weeks for patients with positive MRI scans. Patients with false negative MRI results may wait longer for their surgery. Two of the false negative MRI scan reports clearly showed meniscus tears, which were not identified by the reporting radiologist. In our series, the MRI scan itself was more accurate than the reporting. It is important to have an experienced musculoskeletal radiologist to minimise the number of missed meniscal tears. It is also important for the surgeon to review the MRI scan itself, as well as the report.


Assuntos
Traumatismos do Joelho/diagnóstico , Imageamento por Ressonância Magnética , Lesões do Menisco Tibial , Adolescente , Adulto , Idoso , Artroscopia , Humanos , Traumatismos do Joelho/cirurgia , Meniscos Tibiais/cirurgia , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
8.
Knee Surg Sports Traumatol Arthrosc ; 20(12): 2399-404, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22278658

RESUMO

PURPOSE: The quadriceps angle (Q-angle) represents the angle between the vector of action of the quadriceps and the patellar tendon. An increased Q-angle has been associated with an increased risk of patellar instability, although there is disagreement on its reliability and validity as it is affected by the position of the limb and contraction of the quadriceps. Tibial tuberosity-trochlear groove distance (TT-TG) is ascertained by axial CT scanning, with an increased value associated with patellar instability. This study aimed to determine whether the Q-angle correlates with the TT-TG distance in patients with patellar instability. METHODS: Q-angles were measured in 34 knees that had previously undergone CT scanning for assessment of patellar instability. Measurements were made with the patient supine, the knee extended and the lower limbs in neutral rotation with the quadriceps relaxed and contracted. TT-TG distance was measured on CT scanning in an identical position. RESULTS: Of the 34 knees measured, 24 had symptoms of patellar instability, and 10 were normal. A significant negative correlation between relaxed Q-angle and TT-TG in all knees was demonstrated (p = 0.028). In symptomatic knees, contracted Q-angle also demonstrated a significant negative correlation with TT-TG (p = 0.037). CONCLUSIONS: If TT-TG distance is regarded as the gold standard measurement, Q-angle is not a reliable indicator of patellar instability. There is a clear need to develop methods to more fully characterise the knee and factors contributing to patellar instability. LEVEL OF EVIDENCE: II.


Assuntos
Instabilidade Articular/diagnóstico por imagem , Articulação do Joelho/anatomia & histologia , Luxação Patelar/diagnóstico por imagem , Músculo Quadríceps/anatomia & histologia , Tíbia/anatomia & histologia , Feminino , Humanos , Articulação do Joelho/diagnóstico por imagem , Masculino , Músculo Quadríceps/diagnóstico por imagem , Tíbia/diagnóstico por imagem , Tomografia Computadorizada por Raios X
9.
Spinal Cord ; 49(2): 215-8, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20697421

RESUMO

STUDY DESIGN: Retrospective cohort study. OBJECTIVE: To describe functional outcome and discharge destination of elderly patients with traumatic spinal cord injuries. SETTING: National Spinal Injuries Unit, Glasgow, UK. METHODS: We collected data for 5 years on all patients >65 years old with a traumatic spinal cord injury treated at the National Spinal Injuries Unit. RESULTS: We identified 39 patients. Of these, nine patients died during admission; all had cervical spine injuries. The mean age of the 30 survivors was 73 years (range 65-88). The most common cause of injury was a fall: 26 patients (87%). In addition, 21 (70%) sustained injury to cervical cord, 3 (10%) had thoracic and 6 (20%) had lumbar spine fractures. In all, 23 patients (77%) were treated by orthosis and 7 (23%) underwent surgical intervention. Twelve (40%) patients showed an improvement in American Spinal Injury Association impairment scale. The median hospital stay was 136 days. Thus, 11 patients (37%), all with incomplete injuries, were discharged home, 10 (33%) were transferred to nursing homes/community hospitals and 9 patients (30%) were discharged back to the referring hospital, while they were awaiting adjustments at home. Patients who were discharged home had significantly higher Functional Independence Measure scores, both at the onset of rehabilitation and at discharge, than those who were discharged to a nursing home or other hospitals (P<0.01 and <0.001, respectively). DISCUSSION AND CONCLUSION: Although the elderly patients may benefit from the services of a dedicated spinal injuries centre, they should be carefully selected. The patient, relatives as well as the referring doctors should be alerted to the likely long-term outcomes early in the course of the injury. Elderly patients with complete lesions of the spinal cord will almost certainly remain institutionalized. Early endeavour should be made to find alternate rehabilitation settings with a lower-intensity treatment.


Assuntos
Avaliação de Resultados em Cuidados de Saúde/métodos , Traumatismos da Medula Espinal/reabilitação , Traumatismos da Medula Espinal/terapia , Acidentes por Quedas/mortalidade , Idoso , Idoso de 80 Anos ou mais , Vértebras Cervicais/lesões , Estudos de Coortes , Humanos , Vértebras Lombares/lesões , Estudos Retrospectivos , Traumatismos da Medula Espinal/mortalidade , Vértebras Torácicas/lesões , Reino Unido/epidemiologia
10.
Injury ; 37(8): 763-7, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16504193

RESUMO

OBJECTIVE: To determine if postoperative check X-rays influence the management of patients who have undergone hip fracture fixation using image intensifier guidance and the possible economic impact. A fractured neck of femur is the commonest fracture sustained in the UK amongst the elderly population, with internal fixation commonly carried out under image intensifier guidance, producing high quality intraoperative images. In the postoperative period, patients often receive a subsequent check X-ray, although recent evidence has suggested little additional information is gained. METHODS: We examined the X-rays of 100 patients who underwent internal fixation (using image intensifier guidance) for a fractured neck of femur. RESULTS: Twenty-four patients received a postoperative check X-ray, although none of these patients had their management altered by this subsequent X-ray. CONCLUSION: If these check X-rays are avoided, there is a potential saving of over 2000 sterling pound in our hospital and 38,800 sterling pound in Scotland per annum.


Assuntos
Fraturas do Colo Femoral/diagnóstico por imagem , Fraturas do Colo Femoral/cirurgia , Fixação Interna de Fraturas , Cuidados Pós-Operatórios , Radiografia , Cirurgia Assistida por Computador , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação das Necessidades , Avaliação de Processos e Resultados em Cuidados de Saúde
11.
Colorectal Dis ; 6(6): 418-27, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15521929

RESUMO

OBJECTIVE: To evaluate the immediate impact of national evidence-based guidelines about colorectal cancer on Australian surgeons' self-reported practice and their deficits in awareness of scientific evidence underpinning clinical management practices. DESIGN: Pre/post evaluation, comprising preguidelines survey (November 1998) and postguidelines survey (February 2001). METHODS: One hundred and fourteen Australian surgeons returned postguidelines surveys, of whom 103 (90%) agreed to matching of their pre- and postguidelines responses. National distribution of the CRC guidelines occurred in November 1999. Over the ensuing year, dissemination strategies included seminars, presentations at conferences and journal articles. The main outcome measures used were changes in awareness of evidence for each of 23 clinical recommendations, changes in overall awareness score (maximum possible 23), changes in subscore for nine items for which evidence was compelling and predictors of change. RESULTS: Of those surgeons followed up, 95% were aware of the guidelines and 32% had read them in detail. Only 47% recalled the consumer version. The three most highly rated topics in the guidelines were: high-risk familial syndromes (45%); screening based on family history of colorectal cancer (40%); population screening for colorectal cancer (25%). Compared with baseline, there was a modest improvement in the mean overall awareness score (P = 0.02). Paired analyses of awareness of the evidence for each of 23 individual topics revealed significant improvement only in five. For two, awareness significantly decreased. CONCLUSIONS: Our pre/post findings are not inconsistent with the expectation that dissemination of the CRC guidelines has had some short-term impact. However, definitive evidence acquired through more rigorously designed controlled trials will be needed to determine first, whether surgical practice has changed and, second, whether implementation of the CRC guidelines or some other secular event caused such change.


Assuntos
Competência Clínica , Neoplasias Colorretais/cirurgia , Fidelidade a Diretrizes , Guias de Prática Clínica como Assunto , Adulto , Austrália , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Medicina Baseada em Evidências , Feminino , Pesquisas sobre Atenção à Saúde , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Padrões de Prática Médica , Probabilidade , Qualidade da Assistência à Saúde , Inquéritos e Questionários
13.
Dev Cell ; 1(3): 377-87, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11702949

RESUMO

The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/fisiologia , Células Germinativas/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem da Célula , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Fatores de Transcrição Fushi Tarazu , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Reporter , Proteínas de Homeodomínio , Hibridização In Situ , Substâncias Macromoleculares , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Correpressor 1 de Receptor Nuclear , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares , Fator 3 de Transcrição de Octâmero , Regiões Promotoras Genéticas/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Fator Esteroidogênico 1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transgenes/genética , Tretinoína/farmacologia , Técnicas do Sistema de Duplo-Híbrido
14.
Front Biosci ; 6: D1186-91, 2001 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-11578963

RESUMO

GCNF is an orphan member of the nuclear receptor superfamily. The nuclear receptor superfamily is a large superfamily of transcription factors, the majority of which are designated as orphan receptors because their ligands and functions are currently unknown. GCNF (Germ Cell Nuclear Factor) is so named because of its restricted expression pattern in the adult. In the testis, GCNF is expressed only in the post meiotic round spermatids. Likewise in the ovary, GCNF's expression is restricted to the growing oocyte. To date nothing is known of GCNF's putative ligand; however, much is known about its physiological function through the use of gene targeting. Inactivation of the GCNF gene showed that it was essential for normal embryonic development. In addition to being expressed in the germ cells of the adult, it is expressed widely throughout the embryo after gastrulation. Significant strides have also been made in understanding GCNF's mechanism of action using molecular biology. The DNA binding properties of GCNF have been investigated and its response element identified. GCNF binds as a homodimer to a direct repeat element with zero nucleotides between the reiterated sequence AGGTCA. GCNF target genes have been identified that contain this DR0 element in their promoters. Such genes as Protamines 1 and 2 and Oct4 are regulated by GCNF through this element. GCNF has been shown to be a repressor of the protamine and Oct4 genes. GCNF's repression function has been shown to be mediated by interaction with the co-repressors N-CoR and SMRT in the absence of ligand. Our current efforts are to explore GCNF function in the adult germ cells using tissue specific gene targeting to specifically knock out the GCNF gene in oocytes and spermatogenic cells. In addition, efforts are being made to identify the endogenous ligand that regulates GCNF's transcriptional properties.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Animais , Desenvolvimento Embrionário e Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares , Transcrição Gênica
15.
Int J Biochem Cell Biol ; 33(12): 1141-6, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11606249

RESUMO

Germ cell nuclear factor (GCNF) is an orphan nuclear receptor for which a ligand has yet to be identified. However, we do know that GCNF binds to a novel response element as a homodimer and regulates expression of genes, such as the protamines, through this element. In the absence of a ligand, GCNF is a transcriptional repressor that interacts with co-repressors. During embryonic development, GCNF is expressed between the gastrula and neurula stages. Loss of GCNF causes embryonic lethality, disrupts normal somitogenesis, as well as neural tube and axis formation, suggesting that GCNF is a critical factor for normal embryonic development. In adult vertebrates, GCNF expression is predominantly found in the germ cells of gonads. GCNF expression in germ cells suggests that understanding its function in adults will yield greater insight into the regulation of gametogenesis, leading to new contraceptive targets.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Núcleo Celular/metabolismo , Dimerização , Embrião de Mamíferos/metabolismo , Éxons , Humanos , Ligantes , Modelos Biológicos , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Tempo , Transcrição Gênica
16.
Endocrinology ; 142(9): 3791-9, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11517155

RESUMO

It has previously been demonstrated that 19-nor contraceptive progestins undergo in vivo and in vitro enzyme-mediated A-ring double bond hydrogenation. Bioconversion of 19-nor progestins to their corresponding tetrahydro derivatives results in the loss of progestational activity and acquisition of estrogenic activities and binding to the ER. Herein, we report subtype-selective differences in ligand binding and transcriptional potency of nonphenolic synthetic 19-nor derivatives between ER alpha and ER beta. In this study, we have examined both ER- and PR-mediated transcriptional activity of a number of A-ring chemically reduced derivatives of norethisterone and Gestodene. Double bond hydrogenation decreased the transcriptional potency of norethisterone and Gestodene through both PR isoforms with a 100- to 1,000-fold difference, respectively. In terms of the effects of norethisterone and Gestodene and their corresponding 5 alpha-dihydro (5 alpha-norethisterone and 5 alpha-Gestodene), or 3 alpha,5 alpha-tetrahydro or 3 beta,5 alpha-tetrahydro derivatives (3 alpha,5 alpha-norethisterone/3 alpha,5 alpha-Gestodene and 3 beta,5 alpha-norethisterone/3beta,5 alpha-Gestodene, respectively) on estrogen-mediated transcriptional regulation, the 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene showed the highest induction when HeLa cells were transiently transfected with an expression vector for ER alpha. This activity could be inhibited with tamoxifen. These compounds did not activate gene transcription via ER beta, and none of them showed antagonistic activities through either ER subtype. The 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene were active in other cells in addition to HeLa cells and activated reporter expression through the oxytocin promoter. In summary, two ER alpha selective agonists have been identified. These compounds, with ER alpha vs. ER beta selective agonist activity, may be useful in evaluating the distinct role of these receptors as well as in providing useful insights into ER action.


Assuntos
Progestinas/metabolismo , Receptores de Estrogênio/agonistas , Animais , Células CHO , Cricetinae , Receptor alfa de Estrogênio , Células HeLa , Humanos , Estrutura Molecular , Noretindrona/metabolismo , Noretindrona/farmacologia , Norpregnenos/metabolismo , Norpregnenos/farmacologia , Oxirredução , Receptores de Estrogênio/classificação , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/fisiologia , Ativação Transcricional/fisiologia
17.
Trends Endocrinol Metab ; 12(6): 247-51, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11445441

RESUMO

Orphan nuclear receptors are members of the nuclear receptor superfamily of ligand-activated transcription factors for which ligands and functions have not been identified. Since the cloning of the original orphans, ligands have been identified for several orphan receptors that heterodimerize with the retinoid X receptor and are no longer classified as orphan receptors. Considering the central role that nuclear receptors play in differentiation, development, metabolic regulation, homeostasis and disease, it is crucial that we understand the roles of the remaining orphans. However, the identification of ligands for those orphans that form homodimers has proven more difficult. Thus, to gain greater insight into the functions of orphan receptors, gene targeting has been used to knock out these factors and study mouse development in their absence. Here we will review the progress made in understanding the roles of the orphans GCNF and the COUP-TFs with the use of gene knockouts.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Fatores de Transcrição COUP , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Camundongos Knockout , Mutagênese , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides , Proteínas Repressoras , Fatores de Transcrição/genética
18.
J Biol Chem ; 276(29): 27584-90, 2001 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-11349133

RESUMO

ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células CHO , Compartimento Celular , Cricetinae , Cicloeximida/farmacologia , Endocitose , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Imuno-Histoquímica , Proteínas Luminescentes/genética , Monensin/farmacologia , Proteínas Recombinantes de Fusão/genética , Esteróis/metabolismo , Transfecção
19.
Proc Natl Acad Sci U S A ; 98(8): 4466-71, 2001 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-11296289

RESUMO

Niemann-Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.


Assuntos
Endossomos/metabolismo , Doenças de Niemann-Pick/metabolismo , Animais , Western Blotting , Células CHO , Proteínas de Transporte/metabolismo , Compartimento Celular , Colesterol/metabolismo , Cricetinae , Endocitose , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência , Proteína C1 de Niemann-Pick
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