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2.
Fam Med ; 51(3): 227-233, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30676638

RESUMO

BACKGROUND AND OBJECTIVES: There are several trends compelling physicians to acquire team-based skills for interprofessional care. One underdeveloped area of team-based skills for physicians is integrated behavioral health (IBH) in primary care. We used a Delphi method to explore what skills were needed for residents to practice integrated behavioral health. METHODS: We conducted a literature review of IBH competencies and found 41 competencies across seven domains unique to physicians. Using a modified Delphi technique, we recruited family medicine educators to rate each competency as "essential," "compatible," or "irrelevant." We also shared findings from the Delphi study with a focus group for additional feedback. RESULTS: Twenty-one participants (12 physicians, nine behavioral health providers) completed all three rounds of the Delphi survey resulting in a list of 21 competencies. The focus group gave additional feedback. CONCLUSIONS: Participants chose skills that required physicians to share responsibilities across the entire care team, were not redundant with standard primary care, and necessitated strong communication ability. Many items were revised to reflect team-based care and a prescribed physician role as a team facilitator. Next steps include determining how these competencies fit with a variety of medical providers and creating effective training programs that develop competency in IBH.


Assuntos
Medicina do Comportamento , Prestação Integrada de Cuidados de Saúde/métodos , Técnica Delfos , Medicina de Família e Comunidade/educação , Internato e Residência , Grupos Focais , Humanos , Equipe de Assistência ao Paciente , Médicos
3.
Ecology ; 99(10): 2159-2166, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30039615

RESUMO

Eigenvector-mapping methods such as Moran's eigenvector maps (MEM) are derived from a spatial weighting matrix (SWM) that describes the relations among a set of sampled sites. The specification of the SWM is a crucial step, but the SWM is generally chosen arbitrarily, regardless of the sampling design characteristics. Here, we compare the statistical performances of different types of SWMs (distance-based or graph-based) in contrasted realistic simulation scenarios. Then, we present an optimization method and evaluate its performances compared to the arbitrary choice of the most-widely used distance-based SWM. Results showed that the distance-based SWMs generally had lower power and accuracy than other specifications, and strongly underestimated spatial signals. The optimization method, using a correction procedure for multiple tests, had a correct type I error rate, and had higher power and accuracy than an arbitrary choice of the SWM. Nevertheless, the power decreased when too many SWMs were compared, resulting in a trade-off between the gain of accuracy and the loss of power. We advocate that future studies should optimize the choice of the SWM using a small set of appropriate candidates. R functions to implement the optimization are available in the adespatial package and are detailed in a tutorial.


Assuntos
Ecologia , Modelos Teóricos , Software
4.
J Exp Biol ; 221(Pt 6)2018 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29444845

RESUMO

Bacteria can damage sperm and thus reduce the reproductive success of both males and females; selection should therefore favour the evolution of antimicrobial protection. Eusocial hymenopterans might be particularly affected by such bacterial infections because of their mating ecology. In both sexes, mating is restricted to a short window early in the adult stage; there are no further chances to mate later in life. Males die shortly after mating, but queens use the acquired sperm to fertilise their eggs for years, sometimes decades. The reproductive success of both sexes is, thus, ultimately sperm-limited, which maintains strong selection for high sperm viability before and after storage. We tested the antibacterial activity of the contents of the male and female sperm-storage organs - the accessory testes and the spermatheca, respectively. As our study species, we used the bacterium Escherichia coli and the garden ant Lasius niger, whose queens can live for several decades. Our results provide the first empirical evidence that male and female sperm-storage organs display different antibacterial activity. While the contents of the accessory testes actually enhanced bacterial growth, the contents of the spermatheca strongly inhibited it. Furthermore, mating appears to activate the general immune system in queens. However, antimicrobial activity in both the spermatheca and the control tissue (head-thorax homogenate) declined rapidly post-mating, consistent with a trade-off between immunity and reproduction. Overall, this study suggests that ejaculates undergo an immune 'flush' at the time of mating, allowing storage of sperm cells free of bacteria.


Assuntos
Antibacterianos/metabolismo , Formigas/fisiologia , Espermatozoides/fisiologia , Animais , Antibacterianos/isolamento & purificação , Feminino , Masculino
5.
J Clin Psychol Med Settings ; 25(2): 127-156, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-28975500

RESUMO

The Primary Care Behavioral Health (PCBH) model of service delivery is being used increasingly as an effective way to integrate behavioral health services into primary care. Despite its growing popularity, scientifically robust research on the model is lacking. In this article, we provide a qualitative review of published PCBH model research on patient and implementation outcomes. We review common barriers and potential solutions for improving the quantity and quality of PCBH model research, the vital data that need to be collected over the next 10 years, and how to collect those data.


Assuntos
Medicina do Comportamento/organização & administração , Prestação Integrada de Cuidados de Saúde/organização & administração , Pesquisa sobre Serviços de Saúde/organização & administração , Atenção Primária à Saúde/organização & administração , Medicina do Comportamento/tendências , Prestação Integrada de Cuidados de Saúde/tendências , Previsões , Necessidades e Demandas de Serviços de Saúde/organização & administração , Necessidades e Demandas de Serviços de Saúde/tendências , Pesquisa sobre Serviços de Saúde/tendências , Humanos , Comunicação Interdisciplinar , Colaboração Intersetorial , Equipe de Assistência ao Paciente/organização & administração , Equipe de Assistência ao Paciente/tendências , Atenção Primária à Saúde/tendências , Estados Unidos
6.
FEMS Microbiol Ecol ; 92(10)2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27402715

RESUMO

Ectomycorrhizal fungi (EMF) are highly diversified and dominant in a number of forest ecosystems. Nevertheless, their scales of spatial distribution and the underlying ecological processes remain poorly understood. Although most EMF are considered to be generalists regarding host identity, a preference toward functional strategies of host trees has never been tested. Here, the EMF community was characterised by DNA sequencing in a 10-ha tropical dry season forest-referred to as miombo-an understudied ecosystem from a mycorrhizal perspective. We used 36 soil parameters and 21 host functional traits (FTs) as candidate explanatory variables in spatial constrained ordinations for explaining the EMF community assemblage. Results highlighted that the community variability was explained by host FTs related to the 'leaf economics spectrum' (adjusted R(2) = 11%; SLA, leaf area, foliar Mg content), and by soil parameters (adjusted R(2) = 17%), notably total forms of micronutrients or correlated available elements (Al, N, K, P). Both FTs and soil generated patterns in the community at scales ranging from 75 to 375 m. Our results indicate that soil is more important than previously thought for EMF in miombo woodlands, and show that FTs of host species can be better predictors of symbiont distribution than taxonomical identity.


Assuntos
Florestas , Micorrizas/fisiologia , Árvores/microbiologia , Ecologia , Ecossistema , Micorrizas/genética , Fenótipo , Solo , Microbiologia do Solo
7.
Bioorg Med Chem Lett ; 23(12): 3650-3, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23659858

RESUMO

In an effort to understand the origin of blood-pressure lowering effects observed in recent clinical trials with 11ß-HSD1 inhibitors, we examined a set of 11ß-HSD1 inhibitors in a series of relevant in vitro and in vivo assays. Select 11ß-HSD1 inhibitors reduced blood pressure in our preclinical models but most or all of the blood pressure lowering may be mediated by a 11ß-HSD1 independent pathway.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Hipertensão/tratamento farmacológico , Hipertensão/enzimologia , Triazóis/farmacologia , Animais , Humanos , Camundongos , Camundongos Knockout , Ratos , Ratos Endogâmicos SHR
8.
Biochim Biophys Acta ; 1801(11): 1221-31, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20673851

RESUMO

Cerebral 3α-hydroxysteroid dehydrogenase (3α-HSD) activity was suggested to be responsible for the local directed formation of neuroactive 5α,3α-tetrahydrosteroids (5α,3α-THSs) from 5α-dihydrosteroids. We show for the first time that within human brain tissue 5α-dihydroprogesterone and 5α-dihydrotestosterone are converted via non-stereo-selective 3-ketosteroid reductase activity to produce the respective 5α,3α-THSs and 5α,3ß-THSs. Apart from this, we prove that within the human temporal lobe and limbic system cytochrome P450c17 and 3ß-HSD/Δ(5-4) ketosteroid isomerase are not expressed. Thus, it appears that these brain regions are unable to conduct de novo biosynthesis of Δ(4)-3-ketosteroids from Δ(5)-3ß-hydroxysteroids. Consequently, the local formation of THSs will depend on the uptake of circulating Δ(4)-3-ketosteroids such as progesterone and testosterone. 3α- and 3ß-HSD activity were (i) equally enriched in the cytosol, (ii) showed equal distribution between cerebral neocortex and subcortical white matter without sex- or age-dependency, (iii) demonstrated a strong and significant positive correlation when comparing 46 different specimens and (iv) exhibited similar sensitivities to different inhibitors of enzyme activity. These findings led to the assumption that cerebral 3-ketosteroid reductase activity might be catalyzed by a single enzyme and is possibly attributed to the expression of a soluble AKR1C aldo-keto reductase. AKR1Cs are known to act as non-stereo-selective 3-ketosteroid reductases; low AKR1C mRNA expression was detected. However, the cerebral 3-ketosteroid reductase was clearly refractory to inhibition by AKR1C inhibitors indicating the expression of a currently unidentified enzyme. Its lack of stereo-selectivity is of physiological significance, since only 5α,3α-THSs enhance the effect of GABA on the GABA(A) receptor, whereas 5α,3ß-THSs are antagonists.


Assuntos
20-Hidroxiesteroide Desidrogenases/genética , Encéfalo/metabolismo , Regulação Enzimológica da Expressão Gênica , 20-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/metabolismo , Adulto , Idoso , Encéfalo/patologia , Linhagem Celular Tumoral , Cromatografia em Camada Delgada/métodos , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Lactente , Pessoa de Meia-Idade , Esteroide 17-alfa-Hidroxilase/genética , Esteroides/química , Lobo Temporal/patologia
9.
Proc Natl Acad Sci U S A ; 106(39): 16764-9, 2009 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-19805370

RESUMO

25-Hydroxycholesterol is produced in mammalian tissues. The function of this oxysterol is unknown. Here we describe a central role for 25-hydroxycholesterol in regulating the immune system. In initial experiments, we found that stimulation of macrophage Toll-like receptors (TLR) induced expression of cholesterol 25-hydroxylase and the synthesis of 25-hydroxycholesterol. Treatment of naïve B cells with nanomolar concentrations of 25-hydroxycholesterol suppressed IL-2-mediated stimulation of B cell proliferation, repressed activation-induced cytidine deaminase (AID) expression, and blocked class switch recombination, leading to markedly decreased IgA production. Consistent with these findings, deletion of the mouse cholesterol 25-hydroxylase gene caused an increase in serum IgA. Conversely, inactivation of the CYP7B1 oxysterol 7alpha-hydroxylase, which degrades 25-hydroxycholesterol, decreased serum IgA. The suppression of IgA class switching in B cells by a macrophage-derived sterol in response to TLR activation provides a mechanism for local and systemic negative regulation of the adaptive immune response by the innate immune system.


Assuntos
Hidroxicolesteróis/metabolismo , Imunoglobulina A/biossíntese , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Animais , Linfócitos B/metabolismo , Citocinas/metabolismo , Camundongos , Camundongos Transgênicos , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo
10.
J Biol Chem ; 284(42): 28485-9, 2009 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-19687010

RESUMO

The CYP7B1 cytochrome P450 enzyme hydroxylates carbons 6 and 7 of the B ring of oxysterols and steroids. Hydroxylation reduces the biological activity of these substrates and facilitates their conversion to end products that are readily excreted from the body. CYP7B1 is expressed in the liver, reproductive tract, and brain and performs different physiological functions in each tissue. Hepatic CYP7B1 activity is crucial for the inactivation of oxysterols and their subsequent conversion into bile salts. Loss of CYP7B1 activity is associated with liver failure in children. In the reproductive tract, the enzyme metabolizes androgens that antagonize estrogen action; mice without CYP7B1 have abnormal prostates and ovaries. The role of CYP7B1 in brain is under investigation; recent studies show that spastic paraplegia type 5, a progressive neuropathy, is caused by loss-of-function mutations in the human gene.


Assuntos
Falência Hepática/genética , Doenças do Sistema Nervoso/genética , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/fisiologia , Animais , Aspergillus niger/metabolismo , Criança , Família 7 do Citocromo P450 , Humanos , Imunoglobulinas/metabolismo , Ligantes , Falência Hepática/diagnóstico , Camundongos , Modelos Químicos , Doenças do Sistema Nervoso/diagnóstico , RNA Mensageiro/metabolismo , Ratos , Esteroides/metabolismo
11.
Mol Cell Endocrinol ; 281(1-2): 1-8, 2008 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-18060684

RESUMO

The human androgen receptor (AR) is a ligand-activated nuclear transcription factor and mediates the induction of genes involved in the development of the male phenotype and male secondary sex characteristics, as well as the normal and abnormal growth of the prostate. We have identified the pair of hydroxysteroid dehydrogenases (HSDs) that regulate ligand access to the AR in human prostate. We find that type 3 3alpha-HSD (aldo-keto reductase (AKR)1C2) catalyzes the NADPH dependent reduction of the potent androgen 5alpha-dihydrotestosterone (5alpha-DHT) to yield the inactive androgen 3alpha-androstanediol (3alpha-diol). We also find that RoDH like 3alpha-HSD (RL-HSD) catalyzes the NAD(+) dependent oxidation of 3alpha-diol to yield 5alpha-DHT. Together these enzymes are involved in the pre-receptor regulation of androgen action. Inhibition of AKR1C2 would be desirable in cases of androgen insufficiency and inhibition of RL-HSD might be desirable in benign prostatic hyperplasia.


Assuntos
Regulação da Expressão Gênica , Receptores Androgênicos/genética , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/fisiologia , Aldeído Redutase , Aldo-Ceto Redutases , Androgênios/biossíntese , Androgênios/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Hidroxiesteroide Desidrogenases/genética , Hidroxiesteroide Desidrogenases/metabolismo , Hidroxiesteroide Desidrogenases/fisiologia , Ligantes , Masculino , Modelos Biológicos , Modelos Moleculares , Próstata/metabolismo
12.
Mol Cell Endocrinol ; 265-266: 77-82, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17223255

RESUMO

Pairs of hydroxysteroid dehydrogenases (HSDs) govern ligand access to steroid receptors in target tissues and act as molecular switches. By acting as reductases or oxidases, HSDs convert potent ligands into their cognate inactive metabolites or vice versa. This pre-receptor regulation of steroid hormone action may have profound effects on hormonal response. We have identified the HSDs responsible for regulating ligand access to the androgen receptor (AR) in human prostate. Type 3 3alpha-hydroxysteroid dehydrogenase (aldo-keto reductase 1C2) acts solely as a reductase to convert 5alpha-dihydrotestosterone (DHT), a potent ligand for the AR (K(d)=10(-11)M for the AR), to the inactive androgen 3alpha-androstanediol (K(d)=10(-6)M for the AR); while RoDH like 3alpha-HSD (a short-chain dehydrogenase/reductase (SDR)) acts solely as an oxidase to convert 3alpha-androstanediol back to 5alpha-DHT. Our studies suggest that aldo-keto reductase (AKRs) and SDRs function as reductases and oxidases, respectively, to control ligand access to nuclear receptors.


Assuntos
Di-Hidrotestosterona/metabolismo , Hidroxiesteroide Desidrogenases/metabolismo , Receptores Androgênicos/metabolismo , Androgênios/metabolismo , Animais , Humanos , Isoenzimas/metabolismo , Masculino , Próstata/metabolismo
13.
Endocrinology ; 147(12): 5806-16, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16959841

RESUMO

Human prostate adenocarcinoma (CaP) and benign prostatic hyperplasia (BPH) have epithelial and stromal cell origins, respectively. To determine whether the androgen signal is processed differently in these cell types the expression of transcripts for enzymes that control ligand access to the androgen receptor (AR) were measured. Transcripts for type 2 5alpha-reductase, ketosteroid reductases [aldo-keto reductase (AKR)1C1-AKR1C4], the major oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) retinol dehydrogenase (RODH)-like 3alpha-HSD (RL-HSD) and nuclear receptors [AR, estrogen receptor (ER)alpha, and ERbeta] were determined in whole human prostate and in cultures of primary epithelial cells (PEC) and primary stromal cells (PSC) from normal prostate, CaP and BPH by real-time RT-PCR. Normal PEC (n=14) had higher levels of AKR1C1 (10-fold, P<0.001), AKR1C2 (115-fold, P<0.001) and AKR1C3 (6-fold, P<0.001) than normal PSC (n=15), suggesting that reductive androgen metabolism occurs. By contrast, normal PSC had higher levels of AR (8-fold, P<0.001) and RL-HSD (21-fold, P<0.001) than normal PEC, suggesting that 3alpha-androstanediol is converted to 5alpha-dihydrotestosterone to activate AR. In CaP PEC (n=14), no significant changes in transcript levels vs. normal PEC were observed. In BPH PSC (n=21) transcripts for AR (2-fold, P<0.001), AKR1C1 (4-fold, P<0.001), AKR1C2 (10-fold P<0.001), AKR1C3 (4-fold, P<0.001) and RL-HSD (3-fold, P<0.003) were elevated to increase androgen response. Differences in the AR:ERbeta transcript ratios (eight in normal PEC vs. 280 in normal PSC) were maintained in PEC and PSC in diseased prostate. These data suggest that CaP may be more responsive to an ERbeta agonist and BPH may be more responsive to androgen ablation.


Assuntos
Androgênios/metabolismo , Carcinoma/metabolismo , Perfilação da Expressão Gênica/métodos , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , 20-Hidroxiesteroide Desidrogenases/metabolismo , Células Epiteliais/metabolismo , Humanos , Hidroxiesteroide Desidrogenases/metabolismo , Masculino , Modelos Biológicos , Especificidade de Órgãos , Receptores Androgênicos/metabolismo , Receptores Estrogênicos/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas
14.
Proc Natl Acad Sci U S A ; 103(36): 13304-9, 2006 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-16938874

RESUMO

The current arsenal of tools and methods for the continuous monitoring and imaging of redox metabolic pathways in the context of intact cells is limited. Fluorogenic substrates allow for direct measurement of enzyme activity in situ; however, in contrast to proteases and exo-glycosidases, there are no simple guidelines for the design of selective probes for redox metabolic enzymes. Here, we introduce redox probe 1 and demonstrate its high selectivity in living cells for human hydroxysteroid dehydrogenases (HSDs) of the aldo-keto reductase (AKR) superfamily. AKR1C isoforms perform multiple functions among which the metabolism of potent steroid hormones is well documented. Moreover, expression of these enzymes is responsive to cellular stress and pathogenesis, including cancer. Our probe design is based on redox-sensitive optical switches, which couple a ketone-alcohol redox event to a profound change in fluorescence. The high selectivity of phenyl ketone 1 for AKR1C2 over the many endogenous reductases present in mammalian cells was established by a quantitative comparison of the metabolic rates between null control cells (COS-1) and AKR1C2-transfected cells. Phenyl ketone 1 is a cell-permeable fluorogenic probe that permits a direct, real-time, and operationally simple readout of AKR1C2 enzyme activity in intact mammalian cells. Furthermore, it was demonstrated that probe 1 enables the quantitative examination of physiological substrate 5alpha-dihydrotestosterone ("dark substrate") in situ by means of a two-substrate competitive assay. Similarly, inhibitor potency of physiological (ursodeoxycholate) and synthetic inhibitors (flufenamic acid, ibuprofen, and naproxen) was also readily evaluated.


Assuntos
Corantes Fluorescentes/metabolismo , Hidroxiesteroide Desidrogenases/análise , Animais , Células COS , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Hidroxiesteroide Desidrogenases/química , Hidroxiesteroide Desidrogenases/metabolismo , Cinética , Estrutura Molecular , Oxirredução , Sensibilidade e Especificidade , Espectrometria de Fluorescência , Especificidade por Substrato
15.
Mol Cell Endocrinol ; 248(1-2): 182-91, 2006 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-16417966

RESUMO

Human aldo-keto reductases (AKR) of the 1A, 1B, 1C and 1D subfamilies are involved in the pre-receptor regulation of nuclear (steroid hormone and orphan) receptors by regulating the local concentrations of their lipophilic ligands. AKR1C3 is one of the most interesting isoforms. It was cloned from human prostate and the recombinant protein was found to function as a 3-, 17- and 20-ketosteroid reductase with a preference for the conversion of Delta4-androstene-3,17-dione to testosterone implicating this enzyme in the local production of active androgens within the prostate. Using a validated isoform specific real-time RT-PCR procedure the AKR1C3 transcript was shown to be more abundant in primary cultures of epithelial cells than stromal cells, and its expression in stromal cells increased with benign and malignant disease. Using a validated isoform specific monoclonal Ab, AKR1C3 protein expression was also detected in prostate epithelial cells by immunoblot analysis. Immunohistochemical staining of prostate tissue showed that AKR1C3 was expressed in adenocarcinoma and surprisingly high expression was observed in the endothelial cells. These cells are a rich source of prostaglandin G/H synthase 2 (COX-2) and vasoactive prostaglandins (PG) and thus the ability of recombinant AKR1C enzymes to act as PGF synthases was compared. AKR1C3 had the highest catalytic efficiency (kcat/Km) for the 11-ketoreduction of PGD2 to yield 9alpha,11beta-PGF2 raising the prospect that AKR1C3 may govern ligand access to peroxisome proliferator activated receptor (PPARgamma). Activation of PPARgamma is often a pro-apoptotic signal and/or leads to terminal differentiation, while 9alpha,11beta-PGF2 is a pro-proliferative signal. AKR1C3 is potently inhibited by non-steroidal anti-inflammatory drugs suggesting that the cancer chemopreventive properties of these agents may be mediated either by inhibition of AKR1C3 or COX. To discriminate between these effects we developed potent AKR1C inhibitors based on N-phenylanthranilic acids that do not inhibit COX-1 or COX-2. These compounds can now be used to determine the role of AKR1C3 in producing two proliferative signals in the prostate namely testosterone and 9alpha,11beta-PGF2.


Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/farmacologia , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Doenças Prostáticas/enzimologia , 3-Hidroxiesteroide Desidrogenases/análise , 3-Hidroxiesteroide Desidrogenases/fisiologia , Membro C3 da Família 1 de alfa-Ceto Redutase , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Dinoprosta/biossíntese , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Hormônios Esteroides Gonadais/biossíntese , Hormônios Esteroides Gonadais/metabolismo , Humanos , Hidroxiprostaglandina Desidrogenases/análise , Hidroxiprostaglandina Desidrogenases/fisiologia , Masculino , Próstata/enzimologia , Doenças Prostáticas/genética , Relação Estrutura-Atividade , Testosterona/biossíntese , Transcrição Genética
16.
Mol Endocrinol ; 20(2): 444-58, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16179381

RESUMO

Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.


Assuntos
3-alfa-Hidroxiesteroide Desidrogenase (B-Específica)/metabolismo , Androgênios/metabolismo , Androstano-3,17-diol/metabolismo , Di-Hidrotestosterona/metabolismo , Próstata/enzimologia , Doenças Prostáticas/enzimologia , 3-alfa-Hidroxiesteroide Desidrogenase (B-Específica)/antagonistas & inibidores , 3-alfa-Hidroxiesteroide Desidrogenase (B-Específica)/genética , Animais , Células Cultivadas , Ácido Graxo Sintases/genética , Humanos , Masculino , NADH NADPH Oxirredutases/genética , Próstata/metabolismo , Doenças Prostáticas/tratamento farmacológico , Doenças Prostáticas/metabolismo , Receptores Androgênicos/genética , Ativação Transcricional , Transfecção
17.
Mol Pharmacol ; 67(1): 60-8, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15475569

RESUMO

Human aldo-keto reductases (AKRs) regulate nuclear receptors by controlling ligand availability. Enzymes implicated in regulating ligand occupancy and trans-activation of the nuclear receptors belong to the AKR1C family (AKR1C1-AKR1C3). Nuclear receptors regulated by AKR1C members include the steroid hormone receptors (androgen, estrogen, and progesterone receptors) and the orphan peroxisome proliferator-activated receptor (PPARgamma). In human myeloid leukemia (HL-60) cells, ligand access to PPARgamma is regulated by AKR1C3, which diverts PGD(2) metabolism away from J-series prostanoids (Desmond et al., 2003). Inhibition of AKR1C3 by indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), caused PPARgamma-mediated terminal differentiation of the HL-60 cells. To discriminate between antineoplastic effects of NSAIDs that are mediated by either AKR1C or cyclooxygenase (COX) isozymes, selective inhibitors are required. We report a structural series of N-phenylanthranilic acid derivatives and steroid carboxylates that selectively inhibit recombinant AKR1C isoforms but do not inhibit recombinant COX-1 or COX-2. The inhibition constants, IC(50), K(I) values, and inhibition patterns were determined for the NSAID analogs and steroid carboxylates against AKR1C and COX isozymes. Lead compounds, 4-chloro-N-phenylanthranilic acid and 4-benzoyl-benzoic acid for the N-phenylanthranilic acid analogs and most steroid carboxylates, exhibited IC(50) values that had greater than 500-fold selectivity for AKR1C isozymes compared with COX-1 and COX-2. Crystallographic and molecular modeling studies showed that the carboxylic acid of the inhibitor ligand was tethered by the catalytic Tyr55-OH(2)(+) and explained why A-ring substituted N-phenylanthranilates inhibited only AKR1C enzymes. These compounds can be used to dissect the role of the AKR1C isozymes in neoplastic diseases and may have cancer chemopreventive roles independent of COX inhibition.


Assuntos
Oxirredutases do Álcool/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Ácidos Carboxílicos/farmacologia , Isoenzimas/metabolismo , Oxirredutases do Álcool/efeitos dos fármacos , Aldeído Redutase , Aldo-Ceto Redutases , Anti-Inflamatórios não Esteroides/síntese química , Sítios de Ligação , Ácidos Carboxílicos/síntese química , Inibidores de Ciclo-Oxigenase/farmacologia , Ácido Flufenâmico/química , Ácido Flufenâmico/farmacologia , Células HL-60 , Humanos , Isoenzimas/efeitos dos fármacos , Cinética , Ligantes , Modelos Moleculares , Prostaglandina-Endoperóxido Sintases/metabolismo , Esteroides/síntese química , Esteroides/farmacologia
18.
Drug News Perspect ; 17(9): 563-78, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15645014

RESUMO

The human aldo-keto reductase 1C (AKR1C) isozymes are implicated in the pre-receptor regulation of steroid receptors, nuclear orphan receptors and membrane-bound ligand-gated ion channels. Human AKR members that may regulate the local concentration of steroid hormones include: AKR1C1, AKR1C2, AKR1C3, AKR1C4 and AKR1D1. Since, these enzymes are pluripotent, the physiological role for the human AKR1C isozymes is determined by their tissue-specific expression patterns and their substrate availability in target tissues. AKRs work in concert with short-chain dehydrogenases/reductases as switches to control ligand access to nuclear receptors. Consequently, they are potential targets in treating prostate cancer, breast cancer, endometriosis and endometrial cancer.


Assuntos
Oxirredutases do Álcool/fisiologia , Hormônios Esteroides Gonadais/farmacologia , Oxirredutases do Álcool/efeitos dos fármacos , Oxirredutases do Álcool/farmacologia , Aldeído Redutase , Aldo-Ceto Redutases , Animais , Hormônios Esteroides Gonadais/fisiologia , Humanos , Isoenzimas/química , Isoenzimas/farmacologia , Isoenzimas/fisiologia
19.
Proc Natl Acad Sci U S A ; 100(21): 12033-8, 2003 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-14530408

RESUMO

The functional capacity of genetically encoded histone proteins can be powerfully expanded by posttranslational modification. A growing body of biochemical and genetic evidence clearly links the unique combinatorial patterning of side chain acetylation, methylation, and phosphorylation mainly within the highly conserved N termini of histones H2A, H2B, H3, and H4 with the regulation of gene expression and chromatin assembly and remodeling, in effect constituting a "histone code" for epigenetic signaling. Deconvoluting this code has proved challenging given the inherent posttranslational heterogeneity of histone proteins isolated from biological sources. Here we describe the application of native chemical ligation to the preparation of full-length histone proteins containing site-specific acetylation and methylation modifications. Peptide thioesters corresponding to histone N termini were prepared by solid phase peptide synthesis using an acid labile Boc/HF assembly strategy, then subsequently ligated to recombinantly produced histone C-terminal globular domains containing an engineered N-terminal cysteine residue. The ligation site is then rendered traceless by hydrogenolytic desulfurization, generating a native histone protein sequence. Synthetic histones generated by this method are fully functional, as evidenced by their self-assembly into a higher order H3/H4 heterotetramer, their deposition into nucleosomes by human ISWI-containing (Imitation of Switch) factor RSF (Remodeling and Spacing Factor), and by enzymatic modification by human Sirt1 deacetylase and G9a methyltransferase. Site-specifically modified histone proteins generated by this method will prove invaluable as novel reagents for the evaluation of the histone code hypothesis and analysis of epigenetic signaling mechanisms.


Assuntos
Histonas/biossíntese , Histonas/genética , Acetilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatina/metabolismo , Histonas/química , Humanos , Técnicas In Vitro , Indicadores e Reagentes , Metilação , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Molecular , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Xenopus laevis
20.
Endocrinology ; 144(7): 2922-32, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12810547

RESUMO

Human aldo-keto reductases (AKRs) of the AKR1C subfamily function in vitro as 3-keto-, 17-keto-, and 20-ketosteroid reductases or as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidases. These AKRs can convert potent sex hormones (androgens, estrogens, and progestins) into their cognate inactive metabolites or vice versa. By controlling local ligand concentration AKRs may regulate steroid hormone action at the prereceptor level. AKR1C2 is expressed in prostate, and in vitro it will catalyze the nicotinamide adenine dinucleotide (NAD(+))-dependent oxidation of 3alpha-androstanediol (3alpha-diol) to 5alpha-dihydrotestosterone (5alpha-DHT). This reaction is potently inhibited by reduced NAD phosphate (NADPH), indicating that the NAD(+): NADPH ratio in cells will determine whether AKR1C2 makes 5alpha-DHT. In transient COS-1-AKR1C2 and in stable PC-3-AKR1C2 transfectants, 5alpha-DHT was reduced by AKR1C2. However, the transfected AKR1C2 oxidase activity was insufficient to surmount the endogenous 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity, which eliminated 3alpha-diol as androsterone. PC-3 cells expressed retinol dehydrogenase/3alpha-HSD and 11-cis-retinol dehydrogenase, but these endogenous enzymes did not oxidize 3alpha-diol to 5alpha-DHT. In stable LNCaP-AKR1C2 transfectants, AKR1C2 did not alter androgen metabolism due to a high rate of glucuronidation. In primary cultures of epithelial cells, high levels of AKR1C2 transcripts were detected in prostate cancer, but not in cells from normal prostate. Thus, in prostate cells AKR1C2 acts as a 3-ketosteroid reductase to eliminate 5alpha-DHT and prevents activation of the androgen receptor. AKR1C2 does not act as an oxidase due to either potent product inhibition by NADPH or because it cannot surmount the oxidative 17beta-HSD present. Neither AKR1C2, retinol dehydrogenase/3alpha-HSD nor 11-cis-retinol dehydrogenase is a source of 5alpha-DHT in PC-3 cells.


Assuntos
Hidroxiesteroide Desidrogenases/metabolismo , Próstata/enzimologia , Testosterona/metabolismo , 17-Hidroxiesteroide Desidrogenases/metabolismo , Adenocarcinoma , Androstano-3,17-diol/química , Androstano-3,17-diol/metabolismo , Animais , Neoplasias Ósseas , Células COS , Di-Hidrotestosterona/química , Di-Hidrotestosterona/metabolismo , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Regulação Enzimológica da Expressão Gênica , Humanos , Hidroxiesteroide Desidrogenases/genética , Técnicas In Vitro , Isoenzimas/metabolismo , Masculino , Próstata/citologia , Neoplasias da Próstata , Testosterona/química , Células Tumorais Cultivadas
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