RESUMO
Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that possesses an epoxide hydrolase and lipid phosphatase activity (sEH-P) at two distinct catalytic domains. While the physiological role of the epoxide hydrolase domain is well understood, the consequences of the phosphatase activity remain unclear. Herein we describe the bacterial expression of the recombinant N-terminal domain of sEH-P and the development of a high-throughput screening protocol using a sensitive and commercially available substrate fluorescein diphosphate. The usability of the assay system was demonstrated and novel inhibitors of sEH-P were identified.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Epóxido Hidrolases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Animais , Domínio Catalítico/genética , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Camundongos , Monoéster Fosfórico Hidrolases/genética , SolubilidadeRESUMO
Profile hidden Markov models (profile HMMs) are known to efficiently predict whether an amino acid (AA) sequence belongs to a specific protein family. Profile HMMs can also be used to search for protein domains in genome sequences. In this case, HMMs are typically learned from AA sequences and then used to search on the six-frame translation of nucleotide (NT) sequences. However, this approach demands additional processing of the original data and search results. Here, we propose an alternative and more direct method which converts an AA alignment into an NT one, after which an NT-based HMM is trained to be applied directly on a genome.
Assuntos
Genômica/métodos , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Animais , Bactérias/enzimologia , Bactérias/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Cadeias de Markov , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Estrutura Terciária de Proteína , Ribonuclease H/químicaRESUMO
Studies in cell culture and mouse models of cancer have indicated that the soluble sphingolipid metabolite sphingosine 1-phosphate (S1P) promotes cancer cell proliferation, survival, invasiveness, and tumor angiogenesis. In contrast, its metabolic precursor ceramide is prodifferentiative and proapoptotic. To determine whether sphingolipid balance plays a significant role in glioma malignancy, we undertook a comprehensive analysis of sphingolipid metabolites in human glioma and normal gray matter tissue specimens. We demonstrate, for the first time, a systematic shift in sphingolipid metabolism favoring S1P over ceramide, which increases with increasing cancer grade. S1P content was, on average, 9-fold higher in glioblastoma tissues compared with normal gray matter, whereas the most abundant form of ceramide in the brain, C18 ceramide, was on average 5-fold lower. Increased S1P content in the tumors was significantly correlated with increased sphingosine kinase 1 (SPHK1) and decreased sphingosine phosphate phosphatase 2 (SGPP2) expression. Inhibition of S1P production by cultured glioblastoma cells, using a highly potent and selective SPHK1 inhibitor, blocked angiogenesis in cocultured endothelial cells without affecting VEGF secretion. Our findings validate the hypothesis that an altered ceramide/S1P balance is an important feature of human cancers and support the development of SPHK1 inhibitors as antiangiogenic agents for cancer therapy.
Assuntos
Neoplasias Encefálicas/metabolismo , Ceramidas/biossíntese , Glioblastoma/metabolismo , Metabolismo dos Lipídeos , Lisofosfolipídeos/biossíntese , Neovascularização Patológica/metabolismo , Esfingosina/análogos & derivados , Inibidores da Angiogênese/uso terapêutico , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Ceramidas/genética , Inibidores Enzimáticos/uso terapêutico , Seguimentos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Lisofosfolipídeos/genética , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/biossíntese , Esfingosina/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: The purpose of the study was to determine if the tumor suppressor gene phosphate and tensin homolog (PTEN) (mutated in multiple advanced cancers 1) in combination with Gleason scoring and serum prostate specific antigen (PSA) could be employed to better predict the progression of prostate carcinoma. MATERIALS AND METHODS: The study group consisted of 43 patients with benign prostate hyperplasia (BPH), 15 with organ confined prostate carcinoma (OCPCa), and 18 with advanced prostate carcinoma (APCa). Prostate tissue samples were obtained from radical prostatectomy, transurethral resection, and TRUS guided trans-rectal needle biopsy and then evaluated for biomarker expression. The clinical stage was assessed according to tumor node metastasis classification and grade according to Gleason system. Serum PSA was measured by conventional techniques and Western blotting analysis was used to determine PTEN expression in the primary tissue. Multivariate analysis was performed to analyze whether these markers could individually predict the progression of prostate carcinoma. RESULTS: APCa patients displayed higher Gleason scores and serum PSA levels. But much lower PTEN expression was detected in prostate of APCa patients compared to patients with BPH or OCPCa. Hormone refractory (HR) and hormone sensitive (HS) APCa cases did not yield any significant differences in terms of Gleason scoring, serum PSA and PTEN expression. PSA levels were significantly higher in patients with OCPCa or APCa compared to patients with BPH. CONCLUSION: Our results suggested that both PTEN and serum PSA appeared to be useful as independent markers to depict the nature of tumor behavior as benign or malign. In addition, PTEN also appeared to be useful as an independent marker to predict the progression of prostate carcinoma.
Assuntos
Estadiamento de Neoplasias/métodos , Monoéster Fosfórico Hidrolases/genética , Antígeno Prostático Específico/análise , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Supressoras de Tumor/genética , Western Blotting , Progressão da Doença , Genes Supressores de Tumor , Humanos , Masculino , PTEN Fosfo-Hidrolase , Valor Preditivo dos Testes , Prognóstico , Hiperplasia Prostática/genética , Hiperplasia Prostática/patologia , Sensibilidade e EspecificidadeAssuntos
Neoplasias do Colo/genética , Polipose Adenomatosa do Colo/diagnóstico , Neoplasias do Colo/economia , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Síndrome do Hamartoma Múltiplo/diagnóstico , Síndrome do Hamartoma Múltiplo/genética , Humanos , Seguro Saúde , PTEN Fosfo-Hidrolase , Síndrome de Peutz-Jeghers/diagnóstico , Monoéster Fosfórico Hidrolases/genética , Medição de Risco , Proteínas Supressoras de Tumor/genéticaRESUMO
In the de novo synthesis of inositol, the conversion of D-glucose-6-phosphate to L-myo-inositol-1-phosphate (MIP) is catalyzed by MIP synthase. Little is known about mammalian MIP synthase and nothing is known about its regulation. The second step in inositol biosynthesis is the conversion of MIP to inositol by inositol-monophosphatase (IMPase), a common step to inositol production via the de novo pathway and its recycling from inositol phosphates. Because lithium inhibits IMPase both in yeast and in mammals, and the drug upregulates yeast MIP synthase (INO1) and downregulates IMPase (INM1), the present study was undertaken to determine whether chronic in vivo therapeutic lithium concentrations affect MIP synthase and IMPase expression in mouse frontal cortex and hippocampus. Mice were treated with food containing LiCl (2.5 g/kg) for 10 days. RNA was purified from the brain areas and mRNA amplified using RT-PCR. Expression of MIP synthase and IMPA1 (one of the genes coding for IMPase) but not IMPA2 was upregulated in mouse hippocampus. None of the genes were affected in the frontal cortex. In yeast, when inositol is limiting, the heterodimeric transcriptional activator Ino2p/Ino4p derepresses expression of INO1 by binding to the upstream activation sequence UAS(INO). Using the TFSEARCH program, we found that the promoter of the virtual human MIP synthase gene contains upstream stimulating factor (USF) elements with a similar core binding sequence. The fact that lithium treatment upregulates both MIP synthase and IMPA1 mRNA levels in mouse hippocampus may reflect a compensatory response of both genes to inositol depletion.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Inositol/biossíntese , Cloreto de Lítio/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Antimaníacos/farmacologia , Primers do DNA/metabolismo , Densitometria/instrumentação , Densitometria/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/enzimologia , Hipocampo/enzimologia , Cloreto de Lítio/sangue , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Apoio ao Desenvolvimento de Recursos Humanos , LevedurasRESUMO
OBJECT: The nature and origin of multinucleated giant cells in glioma have not been made clear. To investigate the phosphorylation of intermediate filaments, the authors studied multinucleated giant cells in vitro and in vivo by using mitosis-specific phosphorylated antibodies. METHODS: Cultured human glioma cells were immunostained with monoclonal antibodies (mAbs) 4A4, KT13, and TM71, which recognized the phosphorylation of vimentin at Ser55, glial fibrillary acidic protein at Serl3, and vimentin at Ser71, respectively. Subsequently, the nature of multinucleated giant cells was investigated using laser scanning confocal microscopy. In addition, paraffin-embedded tissue sections obtained in three patients with giant cell glioblastoma were also investigated. Multinucleated giant cells were immunoreacted with the mAb 4A4 and not with KT13 and TM71 in vitro and in vivo. In addition, the authors obtained these results in multinucleated giant cells under natural conditions, without drug treatments. CONCLUSIONS: Findings in this investigation indicated that multinucleated giant cells are those remaining in mitosis between metaphase and telophase, undergoing neither fusion nor degeneration.
Assuntos
Anticorpos Antineoplásicos/imunologia , Proteína Glial Fibrilar Ácida/imunologia , Glioblastoma/imunologia , Glioblastoma/ultraestrutura , Mitose/imunologia , Fosfotransferases/imunologia , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/metabolismo , Divisão Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Genes erbB-1/genética , Genes erbB-1/imunologia , Genes p53/genética , Genes p53/imunologia , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Microscopia de Fluorescência/métodos , Mitose/genética , Técnicas de Amplificação de Ácido Nucleico , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Mutação Puntual/genética , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/metabolismo , Vimentina/imunologia , Vimentina/metabolismoRESUMO
The quality and frequency of mutations in PTEN gene were assessed in 59 carcinomas and 6 hyperplasias of the endometrium in women. Screening for mutations was done in all exons of PTEN gene by the PCR-SSCP analysis and DNA sequencing. Results were correlated with histological status and clinical features of endometrial carcinomas. In 45.8% (27/59) of carcinomas, 36 somatic mutations were detected in PTEN gene. In seven carcinomas, two mutations and in one carcinoma three mutations coexisted simultaneously. Moreover in 33.3% (2/6) of hyperplasia cases mutations were shown. Most identified mutations (57.9%) were present in exons 5 and 8, less frequently in exons 2 (15.8%) and 7 (13.2%) and they were least frequent in exons 1 and 3 (5.3% each). No mutations were found in exons 4, 6 and 9. Of all identified mutations, 73.7% of those resulting in truncated protein were present due to deletions, insertions and nonsense mutations. Missense mutations accounted for 13.2% of mutations and they were present only in exon 5. One point mutation (2.5%) was in intronic splice site. The remaining 10.5% of mutations were neutral polymorphisms. No statistically significant correlation were found between the frequency of PTEN gene mutations and the clinical stage of endometrial carcinomas. However, evident statistically significant, reverse correlation were observed between the frequency of mutations and the grade of morphological differentiation of the diseases (chi(2)=7.2393, alpha=0.0071). In conclusion, our data support the view that PTEN gene mutations are frequent events involved in development of endometrial carcinomas in women.
Assuntos
Adenocarcinoma/genética , Hiperplasia Endometrial/genética , Neoplasias do Endométrio/genética , Mutação/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/patologia , Idoso , Análise Mutacional de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/patologia , Éxons/genética , Feminino , Genes Supressores de Tumor , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , PTEN Fosfo-Hidrolase , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , PrognósticoAssuntos
Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Animais , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Avaliação Pré-Clínica de Medicamentos , Indústria Farmacêutica/organização & administração , Genes Supressores de Tumor , Humanos , Imunossupressores/farmacologia , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/fisiologia , Fosforilação , Inibidores de Proteínas Quinases , Proteínas Quinases/fisiologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Previous studies have demonstrated that the F isoform of
Assuntos
Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Quinases S6 Ribossômicas/fisiologia , Transcrição Gênica , Animais , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Genes Dominantes , Immunoblotting , Morfolinas/farmacologia , Fosfofrutoquinase-2 , Inibidores de Fosfoinositídeo-3 Quinase , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , TransfecçãoRESUMO
A potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents was developed by modifying a pH electrode with an immobilized layer of Escherichia coli cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosporus pesticides to release protons, the concentration of which is proportional to the amount of hydrolyzed substrate. The sensor signal and response time were optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and weight of cells immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 2.5 mg of cells and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, methyl parathion, and diazinon. The biosensor had very good storage and multiple use stability. The use of cells with the metabolic enzyme expressed on cell surface as a biological transducer provides advantages of no resistances to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively.