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1.
Can J Cardiol ; 33(1): 17-32, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27865641

RESUMO

The Canadian Cardiovascular Society Guidelines Committee and key Canadian opinion leaders believed there was a need for up to date guidelines that used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system of evidence assessment for patients who undergo noncardiac surgery. Strong recommendations included: 1) measuring brain natriuretic peptide (BNP) or N-terminal fragment of proBNP (NT-proBNP) before surgery to enhance perioperative cardiac risk estimation in patients who are 65 years of age or older, are 45-64 years of age with significant cardiovascular disease, or have a Revised Cardiac Risk Index score ≥ 1; 2) against performing preoperative resting echocardiography, coronary computed tomography angiography, exercise or cardiopulmonary exercise testing, or pharmacological stress echocardiography or radionuclide imaging to enhance perioperative cardiac risk estimation; 3) against the initiation or continuation of acetylsalicylic acid for the prevention of perioperative cardiac events, except in patients with a recent coronary artery stent or who will undergo carotid endarterectomy; 4) against α2 agonist or ß-blocker initiation within 24 hours before surgery; 5) withholding angiotensin-converting enzyme inhibitor and angiotensin II receptor blocker starting 24 hours before surgery; 6) facilitating smoking cessation before surgery; 7) measuring daily troponin for 48 to 72 hours after surgery in patients with an elevated NT-proBNP/BNP measurement before surgery or if there is no NT-proBNP/BNP measurement before surgery, in those who have a Revised Cardiac Risk Index score ≥1, age 45-64 years with significant cardiovascular disease, or age 65 years or older; and 8) initiating of long-term acetylsalicylic acid and statin therapy in patients who suffer myocardial injury/infarction after surgery.


Assuntos
Cardiologia , Doenças Cardiovasculares/terapia , Gerenciamento Clínico , Guias de Prática Clínica como Assunto , Medição de Risco , Sociedades Médicas , Procedimentos Cirúrgicos Operatórios , Canadá , Humanos , Período Perioperatório
2.
Integr Biol (Camb) ; 8(4): 465-74, 2016 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-26906932

RESUMO

Phages or bacteriophages, viruses that infect and replicate inside bacteria, are the most abundant microorganisms on earth. The realization that antibiotic resistance poses a substantial risk to the world's health and global economy is revitalizing phage therapy as a potential solution. The increasing ease by which phage genomes can be modified, owing to the influx of new technologies, has led to an expansion of their natural capabilities, and a reduced dependence on phage isolation from environmental sources. This review will discuss the way synthetic biology has accelerated the construction of genetically modified phages and will describe the wide range of their applications. It will further provide insight into the societal and economic benefits that derive from the use of recombinant phages in various sectors, from health to biodetection, biocontrol and the food industry.


Assuntos
Bacteriófagos/genética , Engenharia Genética/métodos , Biologia Sintética/métodos , Animais , Bactérias/metabolismo , Bactérias/virologia , DNA/genética , Genoma , Humanos , Peptídeos/química , Controle Biológico de Vetores , Terapia por Fagos/métodos , Pseudomonas aeruginosa/virologia , Medicina Veterinária
3.
Methods Mol Biol ; 1316: 149-67, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25967060

RESUMO

RNA can self-assemble into complex structures through base pairing, as well as encode information and bind with proteins to induce enzymatic activity. Furthermore, RNA can possess intrinsic enzymatic-like (ribozymatic) activity, a property that, if necessary, can be activated only upon the binding of a small molecule or another RNA (as is the case in aptazymes). As such, RNA could be of use in nanotechnology as a programmable polymer capable of self-assembling into complex topological structures. In this chapter we describe a method for designing advanced topological structures using self-circulating RNA, exemplified by three tiers of topologically manipulated self-assembling synthetic RNA systems. The first tier of topological manipulation, the RNA knot is a physically locked structure, formed by circularizing one monomer of knotted single-stranded RNA left with loose ends (an "open" knot). The second tier, a two interlocking ring system, is made by interlocking two circular RNA components: a circular RNA target, and an RNA lasso designed to intercalate the target before circularizing. The third tier naturally extends this system into a string of topologically locked circular RNA molecules (an RNA chain). We detail the methodology used for designing such topologically complex RNAs, including computational predictions of secondary structure, and where appropriate, RNA-RNA interactions, illustrated by examples. We then describe the experimental methods used for characterizing such structures, and provide sequences of building blocks that can be used for topological manipulation of RNA.


Assuntos
Conformação de Ácido Nucleico , RNA/química , RNA/genética , Expressão Gênica , Técnicas In Vitro , RNA/síntese química , Transcrição Genética
4.
Arthritis Res Ther ; 14(1): R22, 2012 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-22284862

RESUMO

INTRODUCTION: The idiopathic inflammatory myopathies are rare diseases for which data regarding the natural history, response to therapies and factors affecting mortality are needed. We performed this study to examine the effects of treatment and clinical features on survival in polymyositis and dermatomyositis patients. METHODS: A total of 160 consecutive patients (77 with polymyositis and 83 with dermatomyositis) seen at the University of Michigan from 1997 to 2003 were included. Medical records were abstracted for clinical, laboratory and therapeutic data, including initial steroid regimen and immunosuppressive use. State vital records were utilized to derive mortality and cause of death data. Survival was modeled by left-truncated Kaplan-Meier estimation and Cox regression. RESULTS: The 5- and 10-year survival estimates were 77% (95% CI = 66 to 85), and 62% (95% CI = 48 to 73), respectively, and the rates were similar for polymyositis and dermatomyositis. Survival between the sexes was similar through 5 years and significantly lower thereafter for males (10-year survival: 18% male, 73% female; P = 0.002 for 5- to 10-year interval). The sex disparity was restricted to the polymyositis group. Increased age at diagnosis and non-Caucasian race were associated with lower survival. Intravenous versus oral corticosteroid use was associated with a higher risk of death among Caucasians (HR = 10.6, 95% CI = 2.1 to 52.8). Early survival between patients treated with methotrexate versus azathioprine was similar, but survival at 10 years was higher for the methotrexate-treated group (76% vs 52%, P = 0.046 for 5- to 10-year interval). CONCLUSIONS: Patients treated initially with intravenous corticosteroids had higher mortality, which was likely related to disease severity. Both methotrexate and azathioprine showed similar early survival benefits as first-line immunosuppressive drugs. Survival was higher between 5 and 10 years in the methotrexate-treated group, but could not be confirmed in multivariable modeling for the full follow-up period. Other important predictors of long-term survival included younger age, female sex and Caucasian race.


Assuntos
Corticosteroides/uso terapêutico , Dermatomiosite/tratamento farmacológico , Imunossupressores/uso terapêutico , Polimiosite/tratamento farmacológico , Administração Intravenosa , Administração Oral , Corticosteroides/administração & dosagem , Adulto , Azatioprina/administração & dosagem , Azatioprina/uso terapêutico , Estudos de Coortes , Dermatomiosite/patologia , Feminino , Seguimentos , Humanos , Imunossupressores/administração & dosagem , Estimativa de Kaplan-Meier , Masculino , Metotrexato/administração & dosagem , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Análise Multivariada , Avaliação de Resultados (Cuidados de Saúde)/estatística & dados numéricos , Polimiosite/patologia , Prednisona/administração & dosagem , Prednisona/uso terapêutico , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Tempo
5.
PLoS One ; 6(10): e26504, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22028889

RESUMO

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.


Assuntos
Regulação da Expressão Gênica , Osteoblastos/metabolismo , Receptores de Calcitriol/genética , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Sequência Rica em GC , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Fator de Transcrição Sp7 , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
6.
J Steroid Biochem Mol Biol ; 121(1-2): 317-21, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20362670

RESUMO

The vitamin D endocrine system plays important but poorly understood roles in the skin and in hair follicle cycling. Rare, human genetic disorders and knockout mouse models highlight essential roles and potentially novel mechanisms of the vitamin D endocrine system in the skin. Vitamin D receptor knockout mice express a hair follicle cycling defect and a hyperproliferative phenotype resulting in disordered skin structure, epidermal thickening, and alopecia. In contrast, ligand knockout mice (i.e., mice with a disrupted CYP27B1 gene that encodes the 25-hydroxyvitamin-D3 1alpha-hydroxylase) have normal hair follicle function and a comparatively modest skin phenotype. These disparate models indicate that VDR may function independently of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in regulating hair follicle cycling and skin biology. Recent studies highlight this concept and provide key support for this hypothesis. While VDR knockout mice are highly susceptible to chemically induced skin tumorigenesis, CYP27B1 knockouts are resistant. These studies reveal a second global physiological process in the skin that may be regulated by VDR in a 1,25(OH)2D3-independent fashion, namely, genoprotection against carcinogenic mutagens. Key cellular and molecular data supporting this mechanism were published recently showing a keratinocyte-selective transactivation activity mediated by VDR that is independent of the 1,25(OH)2D3 ligand. Thus, evidence is building to support a potentially novel, 1,25(OH)2D3-independent mechanism through which VDR functions in keratinocytes and perhaps within stem cell populations in the follicle to regulate genoprotection and other key developmental processes in the skin.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Calcitriol/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Calcitriol/metabolismo , Pele/metabolismo , Animais , Apoptose , Reparo do DNA , Humanos , Queratinócitos/citologia , Ligantes , Camundongos , Camundongos Knockout , Pele/patologia , Neoplasias Cutâneas/metabolismo , Transcrição Genética
7.
J Biol Chem ; 284(27): 18174-83, 2009 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-19386590

RESUMO

The vitamin D endocrine system is essential for calcium and phosphate homeostasis and skeletal mineralization. The 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) hormone binds to the vitamin D receptor (VDR) to regulate gene expression. These gene products in turn mediate the actions of 1,25(OH)(2)D(3) in mineral-regulating target cells such as the osteoblast. We showed previously that meningioma 1 (MN1) is a novel target of 1,25(OH)(2)D(3) in MG-63 osteoblastic cells and that it is a coactivator for VDR-mediated transcription (Sutton, A. L., Zhang, X., Ellison, T. I., and MacDonald, P. N. (2005) Mol. Endocrinol. 19, 2234-2244). However, the functional significance of MN1 in osteoblastic cell biology is largely unknown. Here, we demonstrate that MN1 expression is increased dramatically during differentiation of primary osteoblastic cells. Using calvarial osteoblasts derived from wild-type and MN1 knock-out mice, we provide data supporting an essential role of MN1 in maintaining appropriate osteoblast proliferation, differentiation, and function. MN1 knock-out osteoblasts displayed altered morphology, decreased growth rate, impaired motility, and attenuated 1,25(OH)(2)D(3)/VDR-mediated transcription as well as reduced alkaline phosphatase activity and mineralized nodule formation. MN1 null osteoblasts were also impaired in supporting osteoclastogenesis in co-culture studies presumably because of marked reduction in the RANKL:OPG ratio in the MN1 null cells. Mechanistic studies supported a transcriptional role for MN1 in controlling RANKL gene expression through activation of the RANKL promoter. Cumulatively, these studies indicate an important role for MN1 in maintaining the appropriate maturation and function of calvarial osteoblasts.


Assuntos
Movimento Celular/fisiologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Osteoblastos/citologia , Osteoblastos/fisiologia , Crânio/citologia , Células 3T3 , Fosfatase Alcalina/metabolismo , Animais , Calcitriol/farmacologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Genes Reporter , Camundongos , Camundongos Knockout , Osteoblastos/efeitos dos fármacos , Osteoclastos/citologia , Osteoclastos/fisiologia , Regiões Promotoras Genéticas/fisiologia , Ligante RANK/genética , RNA Mensageiro/metabolismo , Crânio/fisiologia , Vitaminas/farmacologia
8.
J Invest Dermatol ; 128(10): 2508-17, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18509362

RESUMO

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is the biologically active ligand for the vitamin D receptor (VDR). VDR(-/-) mice have a hair follicle-cycling defect resulting in alopecia. However, mice lacking 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1(-/-)), and having no circulating 1,25(OH)(2)D(3), have normal follicular function. These mouse models indicate that VDR functions independently of 1,25(OH)(2)D(3) in regulating hair-follicle cycling. Here, we show that VDR(-/-) mice rapidly develop chemically induced skin tumors, whereas CYP27B1(-/-) and wild-type mice do not, indicating that VDR, and not the 1,25(OH)(2)D(3) ligand, is essential for protection against skin tumorigenesis. Because the majority of human skin cancer results from exposure to UV, the susceptibility of VDR(-/-) mice to this carcinogen was also evaluated. VDR(-/-) mice developed UV-induced tumors more rapidly and with greater penetrance than did VDR(+/+) mice. p53 protein levels were upregulated at similar rates in UV-treated keratinocytes of VDR(-/-) and VDR(+/+) mice. However, rates of thymine-dimer repair and UV-induced apoptosis were significantly lower in VDR(-/-) epidermis compared with the wild type epidermis. UV-induced epidermal thickening was also attenuated in VDR(-/-) skin, indicating that VDR plays a critical role in the repair and removal of severely damaged keratinocytes and adaptation of the skin to chronic UV exposure.


Assuntos
Neoplasias Induzidas por Radiação , Receptores de Calcitriol/deficiência , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/metabolismo , Raios Ultravioleta , Adaptação Fisiológica , Animais , Apoptose , Suscetibilidade a Doenças , Epiderme/fisiopatologia , Epiderme/efeitos da radiação , Queratinócitos/patologia , Camundongos , Camundongos Knockout , Penetrância , Dímeros de Pirimidina/química , Dímeros de Pirimidina/metabolismo , Receptores de Calcitriol/metabolismo , Pele/patologia , Pele/fisiopatologia , Pele/efeitos da radiação , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/fisiopatologia , Vitamina D/análogos & derivados , Vitamina D/genética
9.
J Hist Behav Sci ; 43(3): 285-303, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17623872

RESUMO

Francis Bacon offers two accounts of the nature and function of the human mind: one is a medical-physical account of the composition and operation of spirits specific to human beings, the other is a behavioral account of the character and activities of individual persons. The medical-physical account is a run-of-the-mill version of the late Renaissance model of elemental constituents and humoral temperaments. The other, less well-known, behavioral account represents an unusual position in early modern philosophy. This theory espouses a form of behavioral psychology according to which (a) supposed mental properties are "hidden forms" best described in dispositional terms, (b) the true character of an individual can be discovered in his observable behavior, and (c) an "informed" understanding of these properties permits the prediction and control of human behavior. Both of Bacon's theories of human nature fall under his general notion of systematic science: his medical-physical theory of vital spirits is theoretical natural philosophy and his behavioral theory of disposition and expression is operative natural philosophy. Because natural philosophy as a whole is "the inquiry of causes and the production of effects," knowledge of human nature falls under the same two-part definition. It is an inquisition of forms that pertains to the patterns of minute motions in the vital spirits and the production of effects that pertains both to the way these hidden motions produce behavioral effects and to the way in which a skillful agent is able to produce desired effects in other persons' behavior.


Assuntos
Pesquisa Comportamental/história , Características Humanas , Psicologia Social/história , Ciências do Comportamento/história , História do Século XVI , História do Século XVII , Humanos , Religião , Espiritualidade
10.
J Telemed Telecare ; 13(3): 136-41, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17519055

RESUMO

We studied four cardiology grand rounds transmitted by videoconference from a tertiary care centre to four other hospitals in eastern Canada. We collected data with evaluation questionnaires and by video recording participants at the receiving-site and the presentation they received from the presenting site. Receiving-site participants (n=20) rated their satisfaction with all domains significantly lower than did presenting-site participants (n=49). The greatest difference in ratings was in the ability to see slides (receiving sites 2.6/5, presenting site 4.6/5). Review of video recordings revealed that slides showing material copied from print sources was difficult to read at receiving sites. While 56% of discussion time was devoted to questions from receiving sites, participants there rated their ability to participate in discussion lower than those at the presenting site (3.9/5 vs 4.4/5). This may be because some parts of the discussion from the presenting site were not transmitted. Receiving-site participants rated the educational value of the rounds lower than their presenting-site colleagues (3.9/5 vs 4.4/5) but answered a similar percentage of knowledge questions correctly (85 vs 89%).


Assuntos
Cardiologia/educação , Gravação em Vídeo/normas , Videoconferência/normas , Cardiologia/economia , Feminino , Humanos , Masculino , Determinação de Necessidades de Cuidados de Saúde/organização & administração , Nova Escócia , Gravação em Vídeo/métodos , Videoconferência/estatística & dados numéricos
11.
J Biol Chem ; 282(15): 10953-62, 2007 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-17310066

RESUMO

The vitamin D endocrine system plays critical although poorly understood roles in skin. Vitamin D receptor (VDR) knock-out (VDRKO) mice have defects in hair follicle cycling and keratinocyte proliferation leading to epidermal thickening, dermal cyst formation, and alopecia. Surprisingly, skin defects are not apparent in mice lacking 25-hydroxyvitamin D 1alpha-hydroxylase, the enzyme required for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone biosynthesis. These disparate phenotypes indicate that VDR effects in skin are independent of the 1,25(OH)2D3 ligand. However, cellular or molecular data supporting this hypothesis are lacking. Here, we show transcriptional activation of the vitamin D-responsive 24-hydroxylase promoter by VDR in primary keratinocytes that is independent of the 1,25(OH)2D3 ligand. This activity required functional vitamin D-responsive promoter elements as well as an intact VDR DNA binding domain and thus could not be distinguished from 1,25(OH)2D3-dependent VDR transactivation. The 1,25(OH)2D3-independent activation of VDR was also observed in keratinocytes from 1alpha-hydroxylase knock-out mice, indicating that it is not due to endogenous 1,25(OH)2D3 production. Mammalian two-hybrid studies showed strong, 1,25(OH)2D3-independent interaction between VDR and retinoid X receptors in primary keratinocytes, indicating that enhanced heterodimerization of these receptors was involved. Indeed, this 1,25(OH)2D3-independent VDR-RXR heterodimerization was sufficient to drive transactivation by VDR(L233S), an inactive ligand binding mutant of VDR that was previously shown to rescue the skin phenotype of VDR null mice. Cumulatively, these studies support the concept that transactivation by VDR in keratinocytes may be uncoupled from the 1,25(OH)2D3 ligand.


Assuntos
Calcitriol/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Receptores de Calcitriol/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Animais , Sítios de Ligação , Calcitriol/biossíntese , Células Cultivadas , DNA/genética , DNA/metabolismo , Dimerização , Regulação Enzimológica da Expressão Gênica , Humanos , Ligantes , Camundongos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores de Calcitriol/genética , Elementos de Resposta , Receptores X Retinoide/metabolismo , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Transcrição Genética/genética , Vitamina D3 24-Hidroxilase
12.
J Steroid Biochem Mol Biol ; 103(3-5): 344-6, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17223342

RESUMO

The vitamin D receptor (VDR) is expressed in numerous cells and tissues, including the skin. The critical requirement for cutaneous expression of the VDR has been proven by investigations in mice and humans lacking functional receptors. These studies demonstrate that absence of the VDR leads to the development of alopecia. The hair follicle is formed by reciprocal interactions between an epidermal placode, which gives rise to the hair follicle keratinocytes and the underlying mesoderm which gives rise to the dermal papilla. Hair follicle morphogenesis ends the second week of life in mice. Studies in VDR null mice have failed to demonstrate a cutaneous abnormality during this period of hair follicle morphogenesis. However, VDR null mice are unable to initiate a new hair cycle after the period of morphogenesis is complete, therefore, do not grow new hair. Investigations in transgenic mice have demonstrated that restricted expression of the VDR to keratinocytes is capable of preventing alopecia in the VDR null mice, thus demonstrating that the epidermal component of the hair follicle requires VDR expression to maintain normal hair follicle homeostasis. Studies were then performed to determine which regions of the VDR were required for these actions. Investigations in mice lacking the first zinc finger of the VDR have demonstrated that they express a truncated receptor containing an intact ligand binding and AF2 domain. These mice are a phenocopy of mice lacking the VDR, thus demonstrate the critical requirement of the DNA binding domain for hair follicle homeostasis. Transgenic mice expressing VDRs with mutations in either the ligand-binding domain or the AF2 domain were generated. These investigations demonstrated that mutant VDRs incapable of ligand-dependent transactivation were able to prevent alopecia. Investigations are currently underway to define the mechanism by which the unliganded VDR maintains hair follicle homeostasis.


Assuntos
Folículo Piloso/metabolismo , Receptores de Calcitriol/metabolismo , Animais , Humanos , Receptores de Calcitriol/genética
13.
Cancer Lett ; 229(2): 205-15, 2005 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-16115727

RESUMO

1,25-Dihydroxyvitamin D3 and several of its analogs, such as EB1089, induce growth arrest and apoptosis of breast cancer cells in culture. EB1089 has also been shown to limit growth of xenografts in nude mice and carcinogen-induced mammary tumors in rats. Coupled with the fact that the vitamin D receptor is highly expressed in a large proportion of breast tumors, these data suggest that it may be a broad spectrum therapeutic target. We utilized a transgenic model of hormone-induced mammary cancer, the LH-overexpressing mouse, to assess, for the first time, the efficacy of EB1089 in a spontaneous tumor model. Similar to human breast cancers, the pre-neoplastic mammary glands and mammary tumors in these mice express high levels of vitamin D receptor. Treatment with EB1089 decreased proliferation of mammary epithelial cells in pre-neoplastic glands by 35%. Moreover, half of hormone-induced mammary tumors treated with EB1089 demonstrated a decreased rate of growth, with a subset of these tumors even regressing, suggesting that 1,25-dihydroxyvitamin D3 analogs may be effective chemopreventive and chemotherapeutic agents for breast cancer.


Assuntos
Antineoplásicos/farmacologia , Calcitriol/análogos & derivados , Neoplasias Mamárias Experimentais/tratamento farmacológico , Lesões Pré-Cancerosas/tratamento farmacológico , Receptores de Calcitriol/agonistas , Animais , Calcitriol/farmacologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Imuno-Histoquímica , Hormônio Luteinizante/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos
14.
Mol Endocrinol ; 19(9): 2309-19, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15919723

RESUMO

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] promotes intestinal absorption of calcium primarily by binding to the vitamin D receptor (VDR) and regulating gene expression. 1,25-(OH)2D3 also exerts rapid actions at the cell membrane that include increasing intracellular calcium levels and activating protein kinase cascades. To explore potential cross talk between calcium signaling elicited by the nongenomic actions of 1,25-(OH)2D3 and the genomic pathway mediated by VDR, we examined the effects of activated Ca2+/calmodulin-dependent kinases (CaMKs) on 1,25-(OH)2D3/VDR-mediated transcription. Expression of a constitutively active form of CaMKIV dramatically stimulated 1,25-(OH)2D3-activated reporter gene expression in COS-7, HeLa, and ROS17/2.8 cell lines. Metabolic labeling studies indicated that CaMKIV increased VDR phosphorylation levels. In addition, CaMKIV increased the independent transcription activity of the VDR coactivator SRC (steroid receptor coactivator) 1, and promoted ligand-dependent interaction between VDR and SRC coactivator proteins in mammalian two-hybrid studies. The functional consequences of this multifaceted mechanism of CaMKIV action were revealed by reporter gene studies, which showed that CaMKIV and select SRC coactivators synergistically enhanced VDR-mediated transcription. These studies support a model in which CaMKIV signaling stimulates VDR-mediated transcription by increasing phosphorylation levels of VDR and enhancing autonomous SRC activity, resulting in higher 1,25-(OH)2D3-dependent interaction between VDR and SRC coactivators.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Receptores de Calcitriol/metabolismo , Ativação Transcricional , Animais , Células COS , Calcitriol/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Cercopithecus aethiops , Células HeLa , Histona Acetiltransferases , Humanos , Coativador 1 de Receptor Nuclear , Fosforilação , Regiões Promotoras Genéticas , Ratos , Fatores de Transcrição/metabolismo
15.
Mol Endocrinol ; 19(4): 855-62, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15591533

RESUMO

Alopecia is a feature of vitamin D receptor (VDR) mutations in humans and in VDR null mice. This alopecia results from an inability to initiate the anagen phase of the hair cycle after follicle morphogenesis is complete. Thus, once the initial hair is shed it does not regrow. VDR expression in the epidermal component of the hair follicle, the keratinocyte, is critical for maintenance of the hair cycle. To determine which functional domains of the VDR are required for hair cycling, mutant VDR transgenes were targeted to the keratinocytes of VDR null mice. Keratinocyte-specific expression of a VDR transgene with a mutation in the hormone-binding domain that abolishes ligand binding restores normal hair cycling in VDR null mice, whereas a VDR transgene with a mutation in the activation function 2 domain that impairs nuclear receptor coactivator recruitment results in a partial rescue. Mutations in the nuclear receptor corepressor Hairless are also associated with alopecia in humans and mice. Hairless binds the VDR, resulting in transcriptional repression. Neither VDR mutation affects Hairless interactions or its ability to repress transcription. These studies demonstrate that the effects of the VDR on the hair follicle are ligand independent and point to novel molecular and cellular actions of this nuclear receptor.


Assuntos
Alopecia/genética , Folículo Piloso/metabolismo , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células COS , Células Cultivadas , Cercopithecus aethiops , Regulação para Baixo , Homeostase , Queratinócitos/metabolismo , Ligantes , Camundongos , Camundongos Transgênicos , Mutação , Receptores de Calcitriol/genética , Fatores de Transcrição/genética , Transcrição Genética
16.
Mol Cell Biol ; 25(1): 472-87, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15601867

RESUMO

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces the synthesis of 25-hydroxyvitamin D(3) 24-hydroxylase [24(OH)ase], an enzyme involved in its catabolism, thereby regulating its own metabolism. Here we demonstrate that CCAAT enhancer binding protein beta (C/EBPbeta) is induced by 1,25(OH)(2)D(3) in kidney and in osteoblastic cells and is a potent enhancer of vitamin D receptor (VDR)-mediated 24(OH)ase transcription. Transfection studies indicate that 1,25(OH)(2)D(3) induction of 24(OH)ase transcription is enhanced a maximum of 10-fold by C/EBPbeta. Suppression of 1,25(OH)(2)D(3)-induced 24(OH)ase transcription was observed with dominant negative C/EBP or osteoblastic cells from C/EBPbeta(-/-) mice. A C/EBP site was identified at positions -395 to -388 (-395/-388) in the rat 24(OH)ase promoter. Mutation of this site inhibited C/EBPbeta binding and markedly attenuated the transcriptional response to C/EBPbeta. We also report the cooperation of CBP/p300 with C/EBPbeta in regulating VDR-mediated 24(OH)ase transcription. We found that not only 1,25(OH)(2)D(3) but also parathyroid hormone (PTH) can induce C/EBPbeta expression in osteoblastic cells. PTH potentiated the induction of C/EBPbeta and 24(OH)ase expression in response to 1,25(OH)(2)D(3) in osteoblastic cells. Data with the human VDR promoter (which contains two putative C/EBP sites) indicate a role for C/EBPbeta in the protein kinase A-mediated induction of VDR transcription. From this study a fundamental role has been established for the first time for cooperative effects and cross talk between the C/EBP family of transcription factors and VDR in 1,25(OH)(2)D(3)-induced transcription. These findings also indicate a novel role for C/EBPbeta in the cross talk between PTH and 1,25(OH)(2)D(3) that involves the regulation of VDR transcription.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Sistema Enzimático do Citocromo P-450/química , Regulação Enzimológica da Expressão Gênica , Receptores de Calcitriol/metabolismo , Esteroide Hidroxilases/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Northern Blotting , Células COS , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Imunoprecipitação , Rim/metabolismo , Luciferases/metabolismo , Camundongos , Modelos Biológicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Hormônio Paratireóideo/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Fatores de Tempo , Transcrição Genética , Transfecção , Vitamina D3 24-Hidroxilase
17.
J Steroid Biochem Mol Biol ; 89-90(1-5): 179-86, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15225769

RESUMO

NCoA62/SKIP was discovered as a nuclear protein that interacts with the Vitamin D receptor (VDR) and the SKI oncoprotein. NCoA62/SKIP expresses properties consistent with other nuclear receptor transcriptional coactivator proteins. For example, NCoA62/SKIP interacts selectively with the VDR-RXR heterodimer, it forms a ternary complex with liganded VDR and steroid receptor coactivator (SRC) proteins, and it synergizes with SRCs to augment 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]- and VDR-activated transcription. Chromatin immunoprecipitation studies show that NCoA62/SKIP is recruited in a 1,25-(OH)(2)D(3)-dependent manner to native Vitamin D responsive gene promoters and it enters these promoter complexes after VDR and SRC entry. This suggests that NCoA62/SKIP functions at a distal step in the transactivation process. Recent studies indicate that NCoA62/SKIP is a component of the spliceosome machinery and interacts with important splicing factors such as prp8 and the U5 200kDa helicase. Functional studies also support an involvement of NCoA62/SKIP in mRNA splicing. Collectively, these data suggest a pivotal role for NCoA62/SKIP in coupling transcriptional regulation by VDR to RNA splicing. They further solidify an important role for VDR/NR-interactors downstream of the transcription process in determining the overall response of Vitamin D and steroid hormone regulated genes.


Assuntos
Proteínas Nucleares/fisiologia , Vitamina D/farmacologia , Animais , Humanos , Coativadores de Receptor Nuclear , Fatores de Transcrição , Transcrição Genética
18.
J Biol Chem ; 278(37): 35325-36, 2003 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-12840015

RESUMO

Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDR-activated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D3-dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D3-induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing.


Assuntos
Proteínas Nucleares/metabolismo , Processamento de RNA , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Animais , Sequência de Bases , Células COS , Calcitriol/farmacologia , Núcleo Celular/metabolismo , Cercopithecus aethiops , Primers do DNA , Células HeLa , Humanos , Coativadores de Receptor Nuclear , Plasmídeos , Processamento de RNA/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismo , Transcrição Genética/efeitos dos fármacos , Transfecção
19.
Mol Endocrinol ; 17(5): 777-91, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12637589

RESUMO

The vitamin D endocrine system is critical for the proper development and maintenance of mineral ion homeostasis and skeletal integrity. Beyond these classical roles, recent evidence suggests that the bioactive metabolite of vitamin D, 1,25-dihydroxyvitamin D3, functions in diverse physiological processes, such as hair follicle cycling, blood pressure regulation, and mammary gland development. This minireview explores the current progress in unraveling the complexities of the vitamin D endocrine system by focusing on four main areas of research: the resolution of the vitamin D receptor crystal structure, the molecular details of 1,25-dihydroxyvitamin D3-mediated transcription, murine knockout models of key genes in the endocrine system, and alternative vitamin D receptors and ligands.


Assuntos
Sistema Endócrino/fisiologia , Receptores de Calcitriol/metabolismo , Vitamina D/fisiologia , Animais , Calcitriol/metabolismo , Membrana Celular/metabolismo , Colestanotriol 26-Mono-Oxigenase , Humanos , Camundongos , Camundongos Knockout , Receptores de Calcitriol/química , Receptores de Calcitriol/genética , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X Retinoide , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
J Cell Biochem ; 85(2): 435-57, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-11948698

RESUMO

Two controversial aspects in the mechanism of human vitamin D receptor (hVDR) action are the possible significance of VDR homodimers and the functional role of receptor phosphorylation. To address these issues, milligram quantities of baculovirus-expressed hVDR were purified to 97% homogeneity, and then tested for binding to the rat osteocalcin vitamin D responsive element (VDRE) via electrophoretic mobility shift and half-site competition assays in the presence or absence of a CV-1 nuclear extract containing retinoid X receptor (RXR). Methylation interference analysis revealed that both the hVDR homodimer and the VDR-RXR heterodimer display similar patterns of VDRE G-base protection. However, in competition studies, the relative dissociation of the homodimeric hVDR complex from the VDRE was extremely rapid (t1/2 < 30 s) compared to the dissociation of the heteromeric complex (t1/2 > 5 min), thus illustrating the relative instability and low affinity of homodimeric VDR binding to DNA. These results indicate that VDR-RXR heterodimers are the preferred VDRE binding species. Further, two dimensional gel electrophoresis of hVDR demonstrated several isoelectric forms of the receptor, suggesting that it is subject to multiple phosphorylation events. In vitro kinase assays confirmed that purified hVDR is an efficient substrate for protein kinases A and Cbeta, as well as casein kinase II. In vivo studies of the expressed receptor in intact cells, namely baculovirus vector infected Sf9 insect cells and transfected mammalian COS-7 cells, demonstrated that hVDR was phosphorylated in a hormone-enhanced fashion. Functional consequences of hVDR phosphorylation were suggested by the observations that: (i) potato acid phosphatase (PAP)-treated hVDR no longer interacted with the VDRE as either a homodimer or a heteromeric complex with RXR, and (ii) treatment of transfected COS-7 cells with a phosphatase inhibitor (okadaic acid) along with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) resulted in a synergistic enhancement of both hVDR phosphorylation and transactivation of a VDRE-linked reporter gene, compared to the effect of treatment with either agent alone. These studies point to a significant role for phosphorylation of VDR in regulating high-affinity VDR-RXR interactions with VDREs, and also in modulating 1,25(OH)2D3-elicited transcriptional activation in target cells.


Assuntos
Baculoviridae/genética , DNA/metabolismo , Genes Reguladores/genética , Regiões Promotoras Genéticas/genética , Receptores de Calcitriol/genética , Elementos de Resposta/genética , Tretinoína/fisiologia , Fosfatase Ácida/metabolismo , Animais , Sítios de Ligação , Western Blotting , Células COS , Primers do DNA/química , Dimerização , Ensaio de Desvio de Mobilidade Eletroforética , Vetores Genéticos , Humanos , Ácido Okadáico/farmacologia , Osteocalcina/genética , Osteocalcina/metabolismo , Fosforilação , Ratos , Receptores de Calcitriol/isolamento & purificação , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores X Retinoide , Fatores de Transcrição/genética , Vitamina D/metabolismo
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