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1.
Ann Oncol ; 26(6): 1118-1123, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25701452

RESUMO

BACKGROUND: We hypothesised that alternating inhibitors of the vascular endothelial growth factor receptor (VEGFR) and mammalian target of rapamycin pathways would delay the development of resistance in advanced renal cell carcinoma (aRCC). PATIENTS AND METHODS: A single-arm, two-stage, multicentre, phase 2 trial to determine the activity, feasibility, and safety of 12-week cycles of sunitinib 50 mg daily 4 weeks on / 2 weeks off, alternating with everolimus 10 mg daily for 5 weeks on / 1 week off, until disease progression or prohibitive toxicity in favourable or intermediate-risk aRCC. The primary end point was proportion alive and progression-free at 6 months (PFS6m). The secondary end points were feasibility, tumour response, overall survival (OS), and adverse events (AEs). The correlative objective was to assess biomarkers and correlate with clinical outcome. RESULTS: We recruited 55 eligible participants from September 2010 to August 2012. DEMOGRAPHICS: mean age 61, 71% male, favourable risk 16%, intermediate risk 84%. Cycle 2 commenced within 14 weeks for 80% of participants; 64% received ≥22 weeks of alternating therapy; 78% received ≥22 weeks of any treatment. PFS6m was 29/55 (53%; 95% confidence interval [CI] 40% to 66%). Tumour response rate was 7/55 (13%; 95% CI 4% to 22%, all partial responses). After median follow-up of 20 months, 47 of 55 (86%) had progressed with a median progression-free survival of 8 months (95% CI 5-10), and 30 of 55 (55%) had died with a median OS of 17 months (95% CI 12-undefined). AEs were consistent with those expected for each single agent. No convincing prognostic biomarkers were identified. CONCLUSIONS: The EVERSUN regimen was feasible and safe, but its activity did not meet pre-specified values to warrant further research. This supports the current approach of continuing anti-VEGF therapy until progression or prohibitive toxicity before changing treatment. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY: ACTRN12609000643279.


Assuntos
Inibidores da Angiogênese/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma de Células Renais/tratamento farmacológico , Everolimo/administração & dosagem , Indóis/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Pirróis/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Austrália , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Progressão da Doença , Intervalo Livre de Doença , Esquema de Medicação , Everolimo/efeitos adversos , Estudos de Viabilidade , Feminino , Humanos , Indóis/efeitos adversos , Neoplasias Renais/enzimologia , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Modelos de Riscos Proporcionais , Inibidores de Proteínas Quinases/efeitos adversos , Pirróis/efeitos adversos , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Risco , Sunitinibe , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Resultado do Tratamento
2.
Neuron ; 11(5): 801-15, 1993 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8240805

RESUMO

We have studied the spatial deployment of synapses arising from different axons that converge on the same developing neuromuscular junctions. Labeling the competing synaptic "cartels" with different dyes in mouse muscle showed that, perinatally, each axon adds similar terminal areas, whereas later, areas occupied by the competing cartels diverged by gradual elimination of one axon's synapses and ongoing addition of synaptic area by the other. Activity-dependent labeling of synapses capable of vesicle recycling in snake muscle also revealed a gradual change in territories occupied by competing inputs, implying that an axon maintained some functional synapses even as others in its cartel were being eliminated. Thus the process of synapse elimination is gradual, with loss of one viable synapse after another, until an axon is left with no synaptic territory and withdraws.


Assuntos
Axônios/fisiologia , Junção Neuromuscular/crescimento & desenvolvimento , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Carbocianinas , Corantes Fluorescentes , Camundongos , Camundongos Endogâmicos , Neurônios Motores/ultraestrutura , Terminações Nervosas/ultraestrutura , Junção Neuromuscular/ultraestrutura , Compostos de Piridínio , Ratos , Ratos Sprague-Dawley
3.
Oncogene ; 25(3): 329-37, 2006 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-16421604

RESUMO

Prostate cancer (PrCa) is characterized by progression from an androgen-dependent phenotype to one that is inevitably androgen independent (AI) and lethal. Recent evidence strongly suggests that the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) and androgen receptor (AR) signalling pathways provide prostatic epithelium with the necessary signalling events to escape the apoptotic response associated with androgen withdrawal therapy. Silencing of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and glycogen synthase kinase beta (GSK3beta) are frequently associated with advanced PrCa systems and likely serve critical roles in promoting AR and PI3K/Akt gain-of-function. That PTEN negatively regulates AR and is sufficient to promote metastatic PrCa in murine models strongly implies its role as a gatekeeper of progressive PrCa. In human PrCa, PTEN loss is correlated with substantial increases in Akt(Ser473) and integrin-linked kinase expression, both of which promote Ser(9) phospho-inhibition of GSK3beta and inactivation of apoptotic factors. Sufficient evidence also suggests that GSK3beta is not only a critical regulator of proproliferative signalling but also a promiscuous one as PI3K/Akt pools of GSK3beta are, at least in part, functionally interchangeable with those of the Wnt/beta-catenin pathway. Thus, GSK3beta may serve not only as a mediator of PI3K/Akt activation but may also regulate the potent transactivation and proproliferative effects that Wnt3a and beta-catenin confer upon AR. These data suggest that prostate-specific activation of GSK3beta may serve as a viable pharmacological option. Thus, in this review, we emphasize that temporal changes in GSK3beta and PTEN expression during progression to AI PrCa are important factors when considering the potential for therapies targeting the oncogenic contributions of PI3K/Akt and AR signalling pathways.


Assuntos
Androgênios/fisiologia , Quinase 3 da Glicogênio Sintase/fisiologia , PTEN Fosfo-Hidrolase/fisiologia , Neoplasias da Próstata/fisiopatologia , Progressão da Doença , Glicogênio Sintase Quinase 3 beta , Humanos , Masculino
4.
Oncogene ; 36(1): 24-34, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27270433

RESUMO

MicroRNA-375 (miR-375) is frequently elevated in prostate tumors and cell-free fractions of patient blood, but its role in genesis and progression of prostate cancer is poorly understood. In this study, we demonstrated that miR-375 is inversely correlated with epithelial-mesenchymal transition signatures (EMT) in clinical samples and can drive mesenchymal-epithelial transition (MET) in model systems. Indeed, miR-375 potently inhibited invasion and migration of multiple prostate cancer lines. The transcription factor YAP1 was found to be a direct target of miR-375 in prostate cancer. Knockdown of YAP1 phenocopied miR-375 overexpression, and overexpression of YAP1 rescued anti-invasive effects mediated by miR-375. Furthermore, transcription of the miR-375 gene was shown to be directly repressed by the EMT transcription factor, ZEB1. Analysis of multiple patient cohorts provided evidence for this ZEB1-miR-375-YAP1 regulatory circuit in clinical samples. Despite its anti-invasive and anti-EMT capacities, plasma miR-375 was found to be correlated with circulating tumor cells in men with metastatic disease. Collectively, this study provides new insight into the function of miR-375 in prostate cancer, and more broadly identifies a novel pathway controlling epithelial plasticity and tumor cell invasion in this disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Fosfoproteínas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Transdução de Sinais , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Epitélio/metabolismo , Epitélio/patologia , Expressão Gênica , Humanos , Masculino , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Fenótipo , Fosfoproteínas/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Interferência de RNA , Fatores de Transcrição , Proteínas de Sinalização YAP , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
5.
Oncogene ; 36(24): 3417-3427, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28092670

RESUMO

Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.


Assuntos
Neuropilina-1/genética , Neuropilina-1/metabolismo , Neoplasias de Próstata Resistentes à Castração/mortalidade , Neoplasias da Próstata/tratamento farmacológico , Regulação para Cima , Antagonistas de Androgênios/uso terapêutico , Linhagem Celular Tumoral , Progressão da Doença , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Gradação de Tumores , Metástase Neoplásica , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Análise de Sobrevida
6.
Nucleic Acids Res ; 28(18): 3615-24, 2000 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-10982884

RESUMO

To expand the genetic code for specification of multiple non-natural amino acids, unique codons for these novel amino acids are needed. As part of a study of the potential of quadruplets as codons, the decoding of tandem UAGA quadruplets by an engineered tRNA(Leu) with an eight-base anticodon loop, has been investigated. When GCC is the codon immediately 5' of the first UAGA quadruplet, and release factor 1 is partially inactivated, the tandem UAGAs specify two leucines with an overall efficiency of at least 10%. The presence of a purine at anticodon loop position 32 of the tRNA decoding the codon 5' to the first UAGA seems to influence translation of the following codon. Another finding is intraribosomal dissociation of anticodons from codons and their re-pairing to mRNA at overlapping or nearby codons. In one case where GCC is replaced by CGG, only a single Watson-Crick base pair can form upon re-pairing when decoding is resumed. This has implications for the mechanism of some cases of programmed frameshifting.


Assuntos
Anticódon , Código Genético , RNA de Transferência de Leucina/genética , Códon , Escherichia coli/genética , Conformação de Ácido Nucleico , Proteínas/genética , RNA Mensageiro/genética , RNA de Transferência de Leucina/química
7.
Oncogene ; 6(5): 781-7, 1991 May.
Artigo em Inglês | MEDLINE | ID: mdl-1828873

RESUMO

We show using mild extraction procedures that the p53 proto-oncogene forms a complex with adenovirus 5 E1b-58 kD during infection. These complexes are detected as coimmunoprecipitates from radiolabeled extracts of adenovirus infected cells on SDS-PAGE. Furthermore, adenovirus mutants with defects in E1b-58 kD fail to form complexes, whereas mutants in other early region genes still show evidence of complex. Using a panel of monoclonal antibodies to mouse p53, we show that antibodies reacting with N-terminal epitopes on p53, displace E1b-58 kD. This result suggests that E1b-58 kD binds to an N-terminal region of mouse p53. In addition, in a transient transfection assay in monkey COS cells, we show that an N-terminal deletion mutant of mouse p53 does not bind to E1b-58 kD but wild-type mouse p53 does bind. This result again suggests that E1b-58 kD binds an N-terminal determinant on p53.


Assuntos
Adenoviridae/genética , Proteínas Oncogênicas Virais/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Precoces de Adenovirus , Animais , Anticorpos Monoclonais , Sítios de Ligação , Linhagem Celular , Deleção Cromossômica , Células HeLa/fisiologia , Humanos , Peso Molecular , Proteínas Oncogênicas Virais/genética , Ligação Proteica , Proto-Oncogene Mas , Transfecção , Proteína Supressora de Tumor p53/genética
8.
J Mol Biol ; 298(2): 195-209, 2000 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-10764591

RESUMO

One of the requirements for engineering expansion of the genetic code is a unique codon which is available for specifying the new amino acid. The potential of the quadruplet UAGA in Escherichia coli to specify a single amino acid residue in the presence of a mutant tRNA(Leu) molecule containing the extra nucleotide, U, at position 33.5 of its anticodon loop has been examined. With this mRNA-tRNA combination and at least partial inactivation of release factor 1, the UAGA quadruplet specifies a leucine residue with an efficiency of 13 to 26 %. The decoding properties of tRNA(Leu) with U at position 33.5 of its eight-membered anticodon loop, and a counterpart with A at position 33.5, strongly suggest that in both cases their anticodon loop bases stack in alternative conformations. The identity of the codon immediately 5' of the UAGA quadruplet influences the efficiency of quadruplet translation via the properties of its cognate tRNA. When there is the potential for the anticodon of this tRNA to dissociate from pairing with its codon and to re-pair to mRNA at a nearby 3' closely matched codon, the efficiency of quadruplet translation at UAGA is reduced. Evidence is presented which suggests that when there is a purine base at position 32 of this 5' flanking tRNA, it influences decoding of the UAGA quadruplet.


Assuntos
Anticódon/genética , Códon/genética , Código Genético/genética , Biossíntese de Proteínas/genética , Sequência de Aminoácidos , Anticódon/química , Anticódon/metabolismo , Sequência de Bases , Códon/química , Códon/metabolismo , Códon de Terminação/genética , Evolução Molecular , Mudança da Fase de Leitura do Gene Ribossômico/genética , Genes Reporter/genética , Espectrometria de Massas , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas/química , Proteínas/genética , Sondas RNA/química , Sondas RNA/genética , Sondas RNA/metabolismo , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/genética , RNA de Transferência de Leucina/metabolismo , Análise de Sequência de Proteína , Supressão Genética/genética
9.
J Mol Biol ; 309(5): 1029-48, 2001 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-11399077

RESUMO

A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames. In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame. Mutations affecting ribosomal protein L9 or tRNA(Gly)(2), the tRNA that decodes GGA, alter the efficiency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing signals and mutant translational components on -1 frameshifting at G GGA and hopping over a stop codon immediately flanked by two GGA glycine codons (stop-hopping). Mutant variants of tRNA(Gly)(2) that impair bypassing mediate stop-hopping with unexpected landing specificities, suggesting that these variants are defective in ribosomal P-site codon-anticodon pairing. In a direct competition between -1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of -1 frameshifting without substantially impairing the ability of mutant tRNA(Gly)(2) variants to re-pair with the mRNA by sub-optimal pairing. These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing. Two of the bypassing signals, a cis-acting nascent peptide encoded by the first open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing.


Assuntos
Genes Virais/genética , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Anticódon/genética , Bacteriófago T4/genética , Pareamento de Bases , Sequência de Bases , Ligação Competitiva , Códon/genética , Escherichia coli/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Genótipo , Óperon Lac/genética , Espectrometria de Massas , Peso Molecular , Mutação/genética , Fases de Leitura Aberta/genética , Sinais Direcionadores de Proteínas/fisiologia , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Ribossomos/química , Ribossomos/genética , Salmonella typhimurium/genética
10.
J Mol Biol ; 311(3): 445-52, 2001 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-11492998

RESUMO

Ribosomes bypass a 50 nucleotide non-coding segment of mRNA between the two open reading frames of bacteriophage T4 gene 60 in order to synthesize a topoisomerase subunit. While nearly all ribosomes appear to initiate bypassing, only 50 % resume translation in the second open reading frame. Failure to bypass is shown here to be independent of the stop codon at the end of the first open reading frame and to be amplified by mutant variants of tRNA(Gly)(2) known to diminish bypassing efficiency. Unproductive bypassing may result from premature dissociation of peptidyl-tRNAs from ribosomes (drop-off) or resumption of translation at inappropriate sites. Assessment of the influence of factors known to induce drop-off reveals that ribosome recycling factor accounts for a small fraction of unproductive bypassing products, but none of the other known factors appear to play a significant role. Resumption of translation at inappropriate sites appears to be minimal, which suggests that spontaneous release of the peptidyl-tRNA may account for the remaining unproductive bypassing products and may be inherent to the gene 60 bypassing mechanism.


Assuntos
Regulação Viral da Expressão Gênica , Biossíntese de Proteínas , Ribossomos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriófago T4/genética , Sequência de Bases , Proteínas de Transporte/biossíntese , Proteínas de Transporte/química , Proteínas de Transporte/genética , Códon/genética , Genes Virais/genética , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Fases de Leitura Aberta/genética , Ligação Proteica , RNA Bacteriano/genética , RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/genética , RNA de Transferência de Glicina/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Tiorredoxinas/biossíntese , Tiorredoxinas/química , Tiorredoxinas/genética
11.
Mol Endocrinol ; 7(9): 1185-95, 1993 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8247021

RESUMO

The roles in DNA binding and transcriptional activation of individual amino acids in the putative recognition alpha-helix of the first zinc finger of the beta-isoform of the human thyroid hormone receptor (hT3R beta) have been probed by site-directed mutagenesis. Alanine substitutions of highly conserved residues involved in the folding of this zinc finger abolished the binding of hT3R beta to various thyroid response elements. A similar effect was observed for alanine substitutions of those conserved residues in hT3R beta that were expected to make specific contacts to DNA bases common to all hormone response elements. The three P-box amino acids have previously been shown to be essential for discrimination of the base pairs that differ between the DNA binding sites for related steroid/thyroid hormone receptors. In hT3R beta, the P-box residues are E, G, and G; the results of this study show that alanine substitution of the glutamic acid dramatically reduces DNA binding activity by hT3R beta, while the substitution of either glycine has little or no effect on DNA binding. The effects of alanine substitutions on hT3R beta transcriptional activation properties were consistent with the effect of these substitutions on DNA binding properties, with the exception of the second P-box amino acid. T3R beta substituted with alanine at this position is substantially more defective in transcriptional activation than it is in specific DNA binding. These results indicate that there are two separate mechanisms of response element discrimination by P-box amino acids of steroid/thyroid hormone receptors, one which operates at the level of DNA recognition and a second which operates at the level of transcriptional activation.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Estrutura Secundária de Proteína , Receptores dos Hormônios Tireóideos/química , Receptores dos Hormônios Tireóideos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Transcrição Gênica , Transfecção , Dedos de Zinco
12.
Mol Endocrinol ; 8(7): 829-40, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7984145

RESUMO

Three "P-box" amino acids within the DNA recognition alpha-helix of members of the steroid hormone and thyroid hormone families of nuclear receptors are known to determine the identity of two of the six base pairs within the half-sites of cognate DNA elements. We introduced P-box substitutions derived from different members of the thyroid hormone/estrogen receptor (T3R/ER) family into the beta-isoform of human thyroid hormone receptor (hT3R beta) and tested the DNA binding and transactivation activities of these mutants using thyroid hormone response elements (TREs) with half-sites composed of different sequences and arranged in different orientations. Different P-box sequences derived from the T3R/ER family resulted in distinct DNA binding specificities determined by the fourth base pair of the half-site. Thyroid hormone receptor mutants containing EGA, EAA, EGS substitutions for the wild type EGG P-box bound with wild type affinity to consensus AGGTCA half-sites, regardless of orientation. TREs composed of AGGACA half-sites bound hT3R beta s with an EGG or EAA P-box sequence, but not those with EGA or EGS P-box sequence. A reversal of this specificity was observed on a direct repeat TRE with AGGGCA half-sites. Additionally, an ESG P-box substitution in hT3R beta prevented the receptor from binding to a direct repeat as a homodimer, but this mutant could bind as a heterodimer with retinoid X receptor or to the everted repeat TRE from the chicken lysozyme promoter.


Assuntos
DNA/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Chlorocebus aethiops , Sequência Consenso , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Ligação Proteica , Coelhos , Receptores de Estrogênio/metabolismo , Transcrição Gênica
13.
Mol Endocrinol ; 9(11): 1522-32, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8584030

RESUMO

In Xenopus oocytes, the rat thyroid hormone receptor alpha (rTR alpha), but not its oncogenic homolog v-ErbA, constitutively activated thyroid hormone (T3)-responsive reporter genes at four positive thyroid hormone-responsive elements (TREs). At a subset of the positive TREs tested, the addition of T3 resulted in a further enhancement of reporter gene activation. In contrast, both rTR alpha and v-ErbA functioned as constitutive activators when bound to the clone 122 TREs, which are induced by unliganded TR in mammalian cells. Therefore, the responses of the ligand-independent activation domains of TR and v-ErbA to cell-specific and TRE-mediated induction are not equivalent. Coexpression of the human retinoid X receptor alpha (hRXR alpha) enhanced both ligand-dependent and ligand-independent activation functions of rTR alpha and human TR beta (hTR beta) at a palindromic TRE (TREp). An endogenous TR activity mediated T3 induction of TREp, while being repressed by an in vitro-generated dominant negative mutant of TR. T3-mediated gene activation, by exogenous or endogenous TR, was repressed by v-ErbA at three positive TREs, but not at the TRE from the third intron of the rat GH gene (rGH3 TRE). Interestingly, preinjection of nuclear protein extract from anterior pituitary cells converted v-ErbA into a constitutive activator at rGH3 TRE. The pituitary-specific factor Pit-1/GHF-1 or hRXR alpha did not induce activation by v-ErbA at rGH3 TRE, suggesting that the dominant negative phenotype of v-ErbA can be abolished by direct or indirect interactions with other nuclear factors.


Assuntos
Proteínas Oncogênicas v-erbA/fisiologia , Receptores dos Hormônios Tireóideos/fisiologia , Ativação Transcricional , Animais , Sequência de Bases , Ensaio de Imunoadsorção Enzimática , Feminino , Genes Reporter , Humanos , Ligantes , Microinjeções , Dados de Sequência Molecular , Oócitos , Especificidade de Órgãos , Adeno-Hipófise/metabolismo , Ratos , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , Receptor alfa de Ácido Retinoico , Ovinos , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia , Xenopus laevis
14.
Mol Endocrinol ; 13(12): 2090-107, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10598584

RESUMO

While androgen, progesterone, and glucocorticoid receptors perform distinct physiological functions by regulating unique sets of genes, in vitro they can transactivate a common high-affinity DNA-binding target. Naturally occurring steroid response elements display nucleotide divergence that lowers binding affinity in comparison to the optimal binding element, but enhances receptor-type specificity. We investigated the role of nucleotide deviations within the DNA-binding site for contribution to steroid receptor specificity. We hypothesized that receptor specificity drives the evolution of binding site sequence, rather than strictly receptor-binding affinity. Receptor-selective targets can evolve by some nucleotides selected on the basis of additional bond energy, and others may be selected by differential tolerance to discourage binding from inappropriate receptors. To identify receptor-specific binding sites, we mimicked these dual selection pressures in a receptor-competitive environment in which DNA binding sites for the androgen or progesterone receptors were selected in the presence of the glucocorticoid receptor. These analyses also demonstrated that steroid receptors strongly select nucleotides in the spacer and flanking regions of the half-site and do so in an asymmetric fashion, indicating that steroid receptors interact with DNA in an allosteric manner that affects the transcriptional activation potential.


Assuntos
DNA/química , DNA/metabolismo , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Elementos de Resposta , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Citosina , Metilação de DNA , Guanina/metabolismo , Humanos , Dados de Sequência Molecular , Ratos , Receptores Androgênicos/química , Receptores de Glucocorticoides/química , Receptores de Progesterona/química , Relação Estrutura-Atividade , Transcrição Gênica , Ativação Transcricional
15.
J Med Genet ; 38(11): 761-6, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11694548

RESUMO

BACKGROUND: Hereditary lymphoedema-distichiasis (LD) is an autosomal dominant disorder that classically presents as lymphoedema of the limbs, with variable age of onset, and extra aberrant growth of eyelashes from the Meibomian gland (distichiasis). Other major reported complications include cardiac defects, cleft palate, and extradural cysts. Photophobia, exotropia, ptosis, congenital ectropion, and congenital cataracts are additional eye findings. Recently, we reported that truncating mutations in the forkhead transcription family member FOXC2 resulted in LD in two families. METHODS: The clinical findings in seven additional families with LD, including the original family described by Falls and Kertesz, were determined and mutational analyses were performed. RESULTS: Distichiasis was the most common clinical feature followed by age dependent lymphoedema. There is a wide variation of associated secondary features including tetralogy of Fallot and cleft palate. The mutational analyses identified truncating mutations in all of the families studied (two nonsense, one deletion, three insertion, and one insertion-deletion), which most likely result in haploinsufficiency of FOXC2. CONCLUSIONS: FOXC2 mutations are highly penetrant with variable expressivity which is not explicable by the pattern of mutations.


Assuntos
Proteínas de Ligação a DNA/genética , Pestanas/anormalidades , Linfedema/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Fatores de Transcrição Forkhead , Heterogeneidade Genética , Humanos , Linfedema/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo
16.
J Clin Endocrinol Metab ; 77(2): 433-8, 1993 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8345048

RESUMO

A recent report has identified a new autoantigen called D1 that appears to be associated with Graves' ophthalmopathy and is expressed in the thyroid and eye muscle. To better characterize the tissue specificity and disease relevance of this antigen, we evaluated the expression of D1 RNA in various human tissues using a reverse transcriptase polymerase chain reaction assay. These studies indicate a wide tissue distribution of the messenger RNA for this antigen, including the thyroid, eye muscle, parathyroid, spleen, skeletal muscle, and uterus. There were variations in the relative amounts of specific message for D1 in the different tissues, with the uterus, thyroid, and eye muscle having the greatest amount of product per microgram of total RNA. A maltose binding protein-D1 fusion protein was expressed in Escherichia coli, purified, and used to assess serologic reactivity to D1 by Western blot. Autoantibodies to this antigen were noted in 19 of 24 (78%) of Hashimoto's disease patients, 26 of 41 (63%) of Graves' disease patients, and in 9 of 17 (53%) of normal controls. Sixty percent of Graves' disease patients with clinical ophthalmopathy had antibodies to D1, as did 63% of Graves' patients without signs or symptoms of clinical ophthalmopathy. There was no correlation between reactivity to D1 and either clinical measures of hyperthyroidism or antibody titers to thyroid peroxidase or thyroglobulin. The presence of autoantibodies to this antigen in patients with Hashimoto's disease, in Graves' disease patients without ophthalmopathy and in normal controls indicate that serologic recognition of this antigen is not restricted to patients with ophthalmopathy. In addition, the expression of messenger RNA for this antigen in multiple types of cells questions the tissue specificity of this autoantigen.


Assuntos
Autoantígenos/biossíntese , Doenças Autoimunes/imunologia , Doença de Graves/imunologia , Tireoidite Autoimune/imunologia , Adulto , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Autoantígenos/genética , Autoantígenos/imunologia , Sequência de Bases , Western Blotting , Oftalmopatias/etiologia , Oftalmopatias/imunologia , Feminino , Doença de Graves/complicações , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão , Glândula Tireoide/imunologia
17.
Invest Ophthalmol Vis Sci ; 32(3): 646-54, 1991 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1848212

RESUMO

This is the first study devoted to the histologic and histochemical characteristics of the orbicularis oculi muscle in children to the authors' knowledge. The orbicularis muscle was compared with extraocular, facial, and limb striated muscle. Light microscopy showed the orbicularis oculi muscle to be much smaller and more loosely packed than skeletal limb muscles. It further showed these muscle fibers to have greater variation in fiber size and shape and more endomysial and perimysial connective tissue. Finally, analysis of the histochemical reactions showed the orbicularis oculi had a higher percentage of fast-contracting fibers (Type II). This study establishes the histologic and histochemical standard characteristics for the orbicularis oculi muscle in children. It was found that orbicularis oculi muscles have some histologic and histochemical features in common with other facial muscles and other features in common with extraocular muscles.


Assuntos
Pálpebras/anatomia & histologia , Músculos/anatomia & histologia , AMP Desaminase/metabolismo , Adenosina Trifosfatases/metabolismo , Adolescente , Criança , Pré-Escolar , Esterases/metabolismo , Pálpebras/enzimologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Lactente , Masculino , Músculos/enzimologia , NADH Tetrazólio Redutase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
18.
J Am Soc Mass Spectrom ; 5(5): 339-49, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-24222588

RESUMO

The collision-induced dissociation of protonated uracil has been studied by tandem mass spectrometry using models extensively labeled with stable isotopes, and derivatives of the kinds found in nucleic acids. Following collisional activation at 30 eV translational energy, protonated uracil dissociates through two principal pathways which do not occur in electron ionization mass spectra: (1) elimination of NH3 almost entirely from N-3, followed by loss of CO from C-4, 0(4); (2) loss of H2O, equally from 0(2) and 0(4). Elimination of HNCO, also the principal dissociation process from odd-electron molecular ions, proceeds primarily by loss of N-3, C-Z, O(2) and 10% from N-l, C-Z, 0(2). Several secondary dissociation products are formed with quantitative site specificity of skeletal atoms: C,HO+ (4-C0, C-5, C-6); H2CN+ (N-l, C-6); C2H2+ (N-l, C-5, C-6). First-step dissociation reactions are interpreted in terms of pyrimidine ring opening at likely sites of protonation after collisional activation of MH+. Collision-induced dissociation mass spectra of uracils with structural themes common to nucleic acids (methylation, replacement of 0 by S, C-5 substitution) follow analogous reaction paths which permit assignment of sites of substitution, and exhibit ion abundance changes attributed to differences in substituent basicity and electron density.

19.
Arch Ophthalmol ; 112(2): 272-3, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8311782

RESUMO

A drill guide to facilitate the second drilling of hydroxyapatite orbital implants is presented. The drill guide is adaptable to various drill types. It is nontraumatic and provides excellent, one-handed fixation, eliminating the need for an assistant. It allows for consistent, straight drilling to a predetermined depth and, because it is encased, it protects the patient from injury. The drill guide has been used on 30 patients and is without untoward effects.


Assuntos
Durapatita , Oftalmologia/instrumentação , Órbita/cirurgia , Próteses e Implantes , Humanos
20.
Arch Ophthalmol ; 107(3): 402-7, 1989 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2923565

RESUMO

Xeroderma pigmentosum is an autosomal recessive, precancerous dermatosis caused by defective repair of ultraviolet-damaged DNA. Characterized clinically by progressive cutaneous pigmentary alterations and tumorigenesis, it serves as a model for ultraviolet carcinogenesis. We describe the clinical and histopathologic findings in a 31-year-old woman with xeroderma pigmentosum and a massive iris melanoma of the left eye. Histologic examination following enucleation revealed diffuse iris replacement by spindle and epithelioid cells with extension into the trabecular meshwork. Evidence of direct extraocular extension was absent, and a metastatic evaluation showed no abnormalities. To our knowledge, this is the first reported case of xeroderma pigmentosum complicated by melanoma of the iris. It provides further evidence suggesting a role for sunlight exposure in the pathogenesis of uveal melanoma.


Assuntos
Doenças da Íris/etiologia , Melanoma/etiologia , Dermatopatias/complicações , Neoplasias Uveais/etiologia , Xeroderma Pigmentoso/complicações , Adulto , Feminino , Humanos , Iris/patologia , Doenças da Íris/patologia , Melanoma/patologia , Pele/patologia , Dermatopatias/patologia , Neoplasias Uveais/patologia , Xeroderma Pigmentoso/patologia
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