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1.
Eur Radiol ; 33(4): 2809-2820, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36562786

RESUMO

OBJECTIVE: To develop a prognostic model for post-transjugular intrahepatic portosystemic shunt (TIPS) patients with hepatocellular carcinoma (HCC) beyond the Milan criteria treated by transarterial chemoembolization (TACE). DESIGN: Between January 2013 and January 2020, 512 patients with HCC beyond the Milan criteria who underwent TACE after TIPS were retrospectively recruited from 15 tertiary centers. Patients were randomly sorted into a training set (n = 382) and a validation set (n = 130). Medical data and overall survival were assessed. A prediction model was developed using multivariate Cox regression analyses. Predictive performance and discrimination were evaluated and compared with other prognostic models. RESULTS: Vascular invasion, log10(AFP), 1/creatinine, extrahepatic spread, and log10(ALT) were the most significant prognostic factors of survival. These five parameters were included in a new VACEA score. This score was able to stratify patients in the training set into four distinct risk grades whose median overall survival were 25.2, 15.1, 8.9, and 6.2 months, respectively. The 6-month, 1-year, 2-year, and 3-year AUROC values and C-index of the VACEA model were 0.819, 0.806, 0.779, 0.825, and 0.735, respectively, and higher than those of other seven currently available models in both the training and validation sets, as well as in different subgroups. CONCLUSION: The VACEA score could stratify post-TIPS patients with HCC beyond the Milan criteria treated by TACE and help to identify candidates who benefit from this treatment. KEY POINTS: • Vascular invasion, AFP, creatinine, extrahepatic spread, and ALT were independent significant prognostic factors of survival for HCC patients who underwent TACE after TIPS. • Our new model, named VACEA score, can accurately predict prognosis at the individual level and stratify patients into four distinct risk grades. • The VACEA model showed better prognostic discrimination and calibration than other current TACE-/TIPS-specific models Graphical abstract.


Assuntos
Carcinoma Hepatocelular , Quimioembolização Terapêutica , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , alfa-Fetoproteínas , Estudos Retrospectivos , Creatinina , Prognóstico , Resultado do Tratamento
2.
Neurobiol Dis ; 125: 1-13, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659984

RESUMO

Maternal infection during pregnancy is an important factor involved in the pathogenesis of brain disorders in the offspring. Mounting evidence from maternal immune activation (MIA) animals indicates that microglial priming may contribute to neurodevelopmental abnormalities in the offspring. Because peroxisome proliferator-activated receptor gamma (PPARγ) activation exerts neuroprotective effects by regulating neuroinflammatory response, it is a pharmacological target for treating neurogenic disorders. We investigated the effect of PPARγ-dependent microglial activation on neurogenesis and consequent behavioral outcomes in male MIA-offspring. Pregnant dams on gestation day 18 received Poly(I:C) (1, 5, or 10 mg/kg; i.p.) or the vehicle. The MIA model that received 10 mg/kg Poly(I:C) showed significantly increased inflammatory responses in the maternal serum and fetal hippocampus, followed by cognitive deficits, which were highly correlated with hippocampal neurogenesis impairment in prepubertal male offspring. The microglial population in hippocampus increased, displayed decreased processes and larger soma, and had a higher expression of the CD11b, which is indicative of the M1 phenotype (classical activation). Activation of the PPARγ pathway by pioglitazone in the MIA offspring rescued the imbalance of the microglial activation and ameliorated the MIA-induced suppressed neurogenesis and cognitive impairments and anxiety behaviors. In an in vitro experiment, PPARγ-induced M2 microglia (alternative activation) promoted the proliferation and differentiation of neural precursor cells. These results indicated that the MIA-induced long-term changes in microglia phenotypes were associated with hippocampal neurogenesis and neurobehavioral abnormalities in offspring. Modulation of the microglial phenotypes was associated with a PPARγ-mediated neuroprotective mechanism in the MIA offspring and may serve as a potential therapeutic approach for prenatal immune activation-induced neuropsychiatric disorders.


Assuntos
Comportamento Animal/efeitos dos fármacos , Microglia/imunologia , Neurogênese/imunologia , Complicações Infecciosas na Gravidez/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Animais , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , PPAR gama/imunologia , Pioglitazona/farmacologia , Gravidez , Ratos , Ratos Wistar
3.
Arch Biochem Biophys ; 657: 41-55, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30217511

RESUMO

The uncoupling protein (UCP1) is a proton (H+) transporter in the mitochondrial inner membrane. By dissipating the electrochemical H+ gradient, UCP1 uncouples respiration from ATP synthesis, which drives an increase in substrate oxidation via the TCA cycle flux that generates more heat. The mitochondrial uncoupling-mediated non-shivering thermogenesis in brown adipose tissue is vital primarily to mammals, such as rodents and new-born humans, but more recently additional functions in adult humans have been described. UCP1 is regulated by ß-adrenergic receptors through the sympathetic nervous system and at the molecular activity level by nucleotides and fatty acid to meet thermogenesis needs. The discovery of novel UCP homologs has greatly contributed to the understanding of human diseases, such as obesity and diabetes. In this article, we review the progress made towards the molecular mechanism and function of the UCPs, in particular focusing on the influential contributions from Martin Klingenberg's laboratory. Because all members of the UCP family are potentially promising drug targets, we also present and discuss possible approaches and methods for UCP-related drug discovery.


Assuntos
Proteínas de Desacoplamento Mitocondrial/química , Proteínas de Desacoplamento Mitocondrial/metabolismo , Trifosfato de Adenosina/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Sítios de Ligação , Ácidos Graxos não Esterificados/metabolismo , Humanos , Ligação Proteica , Termogênese/fisiologia
4.
Biochem Cell Biol ; 87(3): 541-4, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19448747

RESUMO

Urea is the major nitrogenous end product of protein metabolism in mammals. Here, we describe a quantitative, sensitive method for urea determination using a modified Jung reagent. This assay is specific for urea and is unaffected by ammonia, a common interferent in tissue and cell cultures. We demonstrate that this convenient colorimetric microplate-based, room temperature assay can be applied to determine urea synthesis in cell culture.


Assuntos
Meios de Cultura/química , Ureia/análise , Animais , Calibragem , Plasmodium falciparum/citologia , Padrões de Referência , Ureia/normas
5.
J Biomol Screen ; 10(4): 383-90, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15964940

RESUMO

Solubility and permeability are intimately linked in drug absorption processes. They have, however, been traditionally assayed separately. To support this linkage, a combined solubility/permeability assay was developed for determining absorption properties of chemical entities. First, solubility is determined at 4 pH values by comparing the concentration of a saturated compound solution to its dilute, known concentration. The filtered, saturated solution from the solubility assay is then used as input material for the membrane permeability determination. The permeability assay is a parallel artificial membrane technique whereby a membrane is created on a solid support parallel artificial membrane permeation assay (PAMPA). The 2 artificial membranes presented here model the gastrointestinal tract and the blood-brain barrier (BBB). Data are presented for control compounds, which are well documented in the literature and exemplify a range of solubility and membrane permeability. The advantages of the combination method are 1) reduction of sample usage and preparation time, 2) elimination of interference from compound precipitation in membrane permeability determination, 3) maximization of input concentration to permeability assay for improved reproducibility, and 4) optimization of sample tracking by streamlining data entry and calculations. BBB permeability ranking of compounds correlates well with literature CNS activity.


Assuntos
Farmacocinética , Barreira Hematoencefálica , Trato Gastrointestinal/metabolismo , Permeabilidade , Reprodutibilidade dos Testes , Solubilidade
6.
J Biomol Screen ; 9(7): 614-20, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15475481

RESUMO

Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.


Assuntos
Inibidores Enzimáticos/farmacologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Animais , Bacitracina/farmacologia , Bioensaio , Catálise , Bovinos , Dimetil Sulfóxido/farmacologia , Peróxido de Hidrogênio/farmacologia , Insulina/metabolismo , Análise dos Mínimos Quadrados , Isomerases de Dissulfetos de Proteínas/metabolismo , Fatores de Tempo
7.
Arch Biochem Biophys ; 420(1): 40-5, 2003 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-14622973

RESUMO

Limited proteolytic digestion of uncoupling protein-1 (UCP1) from hamster brown adipose tissue mitochondria was studied. Under optimal conditions, trypsin and chymotrypsin cleave at Lys-292 and at Phe-102, yielding major products 31-kDa T1 and 22-kDa Ch1. Both T1 and Ch1 remained dimers, as in UCP1. Using fluorescent nucleotide derivative 2'-O-dansyl GTP, it is shown that T1 retains the nucleotide binding affinity (K(D)=1 microM for dansyl GTP) while Ch1 does not bind nucleotide. Previously kinetic binding and H(+) transport studies [Biochemistry 35 (1996) 7846] have shown that UCP1 forms tight complexes to varying degrees with nucleotides and their derivatives. Nucleotides strongly protect against tryptic digestion but less against chymotryptic digestion, because the chymotryptic product Ch1 does not bind nucleotide. The nucleotides and derivatives show the same potency profile in protecting against both trypsinolysis and chymotryptic digestion, suggesting that UCP1 undergoes a major conformational change upon nucleotide binding from an initial loose complex into a tight complex, in which the cleavage sites become masked from proteolysis.


Assuntos
Proteínas de Transporte/química , Quimotripsina/química , Proteínas de Membrana/química , Mitocôndrias/química , Nucleotídeos/química , Tripsina/química , Adipócitos/química , Adipócitos/metabolismo , Tecido Adiposo Marrom , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Células Cultivadas , Cricetinae , Hidrólise , Canais Iônicos , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Químicos , Peptídeo Hidrolases/química , Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteína Desacopladora 1
8.
J Biomol Screen ; 8(2): 198-204, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12844441

RESUMO

A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated (33)P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5- to 9-fold and a Z' factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors.


Assuntos
Bioensaio/métodos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Avidina/metabolismo , Biotina/metabolismo , Bovinos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Dimetil Sulfóxido/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Polilisina/metabolismo , Reprodutibilidade dos Testes , Contagem de Cintilação
9.
Arch Biochem Biophys ; 412(1): 142-6, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12646277

RESUMO

The uncoupling protein 1 (UCP1) is a H(+) carrier which plays a key role in heat generation in brown adipose tissue. The H(+) transport activity of UCP1 is activated by long-chain fatty acids and inhibited by purine nucleotides. While nucleotide binding has been well characterized, the interaction of fatty acid with UCP1 remains unknown. Here I demonstrate the binding of fatty acids by competition with a fluorescent nucleotide probe 2(')-O-dansyl guanosine 5(')-triphosphate (GTP), which has been shown previously to bind at the nucleotide binding site in UCP1. Fatty acids but not their esters competitively inhibit the binding of 2(')-O-dansyl GTP to UCP1. The fatty acid effect was enhanced at higher pH, suggesting the binding of fatty acid anion to UCP1. The inhibition constants K(i) were determined by fluorescence titrations for various fatty acids. Short-chain (C<8) fatty acids display no affinity, whereas medium-chain (C10-14) and unsaturated C18 fatty acids exhibit stronger affinity (K(i)=65 microM, for elaidic acid). This specificity profile agrees with previous functional data obtained in both proteoliposomes and mitochondria, suggesting a possible physiological role of this fatty acid binding site.


Assuntos
Tecido Adiposo/metabolismo , Proteínas de Transporte/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Relação Dose-Resposta a Droga , Guanosina Trifosfato/metabolismo , Hidrogênio , Concentração de Íons de Hidrogênio , Canais Iônicos , Cinética , Proteínas Mitocondriais , Oxigênio/metabolismo , Fosforilação , Ligação Proteica , Espectrometria de Fluorescência , Proteína Desacopladora 1
10.
Assay Drug Dev Technol ; 1(1 Pt 2): 175-80, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15090144

RESUMO

Interleukin 1 receptor activation innervates a cascade of signal transduction events that ultimately lead to the activation of inflammatory and immune response genes. TRAF6 is a Ub ligase (E3) involved in this pathway, and inhibition of this critical enzyme may provide a means for treating inflammatory and immune diseases. A TR-FRET assay has been developed and evaluated for HTS for TRAF6 inhibitors. Bio-Ub and Eu-Ub were polymerized in the presence of Ub activating enzyme E1, conjugating enzyme E2, and TRAF6. Following a 2-h incubation, the reaction was stopped with a buffer containing 10 m M EDTA and the fluorescence donor SA-APC. Fluorescence energy transfer from Eu to APC was measured as a ratio of fluorescence intensity at 655 nm to that at 615 nm (excitation at 340 nm). This homogeneous assay has been optimized and validated in a 384-well format. A window of five- to eightfold and Z' factor of 0.6-0.8 suggests that this assay can be applied to screen for inhibitors of the polyubiquitination activity of TRAF6.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Enzimas Ativadoras de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Ácido Edético/química , Transferência Ressonante de Energia de Fluorescência/instrumentação , Polímeros , Reprodutibilidade dos Testes , Ubiquitina-Proteína Ligases/antagonistas & inibidores
11.
J Biomol Screen ; 7(4): 383-9, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12230893

RESUMO

The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC(50) values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Membranas Intracelulares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Bioensaio/métodos , Células CHO , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Permeabilidade da Membrana Celular , Cricetinae , Dinitrofenóis/farmacologia , Corantes Fluorescentes , Fluorometria/métodos , Humanos , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/fisiologia , Rodaminas/farmacologia , Células Tumorais Cultivadas
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