RESUMO
Molecular probes for imaging of live cells are of great interest for studying biological and pathological processes. The anionic luminescent conjugated polythiophene (LCP) polythiophene acetic acid (PTAA), has previously been used for vital staining of cultured fibroblasts as well as transformed cells with results indicating differential staining due to cell phenotype. Herein, we investigated the behavior of PTAA in two normal and five transformed cells lines. PTAA fluorescence in normal cells appeared in a peripheral punctated pattern whereas the probe was more concentrated in a one-sided perinuclear localization in the five transformed cell lines. In fibroblasts, PTAA fluorescence was initially associated with fibronectin and after 24 h partially localized to lysosomes. The uptake and intracellular target in malignant melanoma cells was more ambiguous and the intracellular target of PTAA in melanoma cells is still elusive. PTAA was well tolerated by both fibroblasts and melanoma cells, and microscopic analysis as well as viability assays showed no signs of negative influence on growth. Stained cells maintained their proliferation rate for at least 12 generations. Although the probe itself was nontoxic, photoinduced cellular toxicity was observed in both cell lines upon irradiation directly after staining. However, no cytotoxicity was detected when the cells were irradiated 24 h after staining, indicating that the photoinduced toxicity is dependent on the cellular location of the probe. Overall, these studies certified PTAA as a useful agent for vital staining of cells, and that PTAA can potentially be used to study cancer-related biological and pathological processes.
Assuntos
Ácido Acético/análise , Fibroblastos/química , Melanoma/patologia , Polímeros/análise , Coloração e Rotulagem/métodos , Tiofenos/análise , Linhagem Celular Transformada , Células HeLa , Humanos , Sondas Moleculares/análiseRESUMO
AIM: It is generally agreed that traditional alcohol biomarkers lack in sensitivity to detect hazardous alcohol consumption. The present study was undertaken to evaluate the ability of phosphatidylethanol (PEth) and traditional alcohol markers to detect moderate alcohol consumption and to distinguish between moderate alcohol consumption and abstinence. METHODS: Forty-four subjects, 32 females and 12 males, were included in the study. They were randomized to alcohol abstention or to alcohol consumption. Female participants consumed 150 ml of red wine (equivalent to 16 g of alcohol) per 24 h and the male participants double the amount. The study lasted for 3 months. Blood samples were drawn at the start and at the end of the study period. Blood samples were analysed for PEth, carbohydrate-deficient transferrin (CDT), mean corpuscular volume (MCV), γ-glutamyltransferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). RESULTS: ROC curves for the various biochemical markers were plotted in order to assess their ability to discriminate between abstention and moderate daily consumption of alcohol. PEth and CDT were the only markers with AUROCs significantly higher than 0.5, and PEth was detected in all participants randomized to alcohol consumption. CONCLUSION: PEth was the only marker that could detect moderate intake and the present results also indicate that PEth probably can distinguish moderate alcohol consumption from abstinence.
Assuntos
Alanina Transaminase/sangue , Consumo de Bebidas Alcoólicas/sangue , Aspartato Aminotransferases/sangue , Índices de Eritrócitos , Glicerofosfolipídeos/sangue , Transferrina/análogos & derivados , gama-Glutamiltransferase/sangue , Adulto , Abstinência de Álcool , Biomarcadores/sangue , Feminino , Voluntários Saudáveis , Humanos , Masculino , Curva ROC , Sensibilidade e Especificidade , Detecção do Abuso de Substâncias/métodosRESUMO
BACKGROUND: There are several shortcomings in present methods for estimation of GFR from plasma clearance. The aim of the present study was therefore to develop a physiologically based method for calculation of plasma clearance of iohexol. METHODS: A mechanistic model founded on classical biochemical engineering principles where in- and outgoing molecular flows of iohexol between plasma and surrounding tissues were balanced over time. After intravenous injections of iohexol, plasma samples were taken from the investigated subjects until complete elimination of iohexol. After tuning of the model parameters, the clearance value was calculated from the injected dose and the integral of the iohexol concentrations over the investigated period. RESULTS: The mass balance model was able to predict the time course of iohexol distribution and elimination after parameterization of mass balance and kinetic equations. Four model structures were evaluated, all based on model parameters derived from published data and from internal tests, each complied at varying physiological conditions. Iohexol clearance was assessed through the model and compared with calculations from previously practiced methods. When testing the mass balance model on ten healthy subjects, clearance was estimated accurately. CONCLUSIONS: The physiological and mechanistic character of the mass balance model may suggest that its derived clearance comes closer to actual in vivo conditions than data derived from previously practiced calculation methods. Although here, only verified with the clearance marker iohexol, the mass balance model should be applicable also to other renal clearance markers.
Assuntos
Iohexol , Rim , Modelos Biológicos , Iohexol/farmacocinética , Iohexol/metabolismo , Iohexol/análise , Humanos , Rim/metabolismo , Testes de Função Renal , Taxa de Filtração Glomerular , Adulto , Masculino , Feminino , Meios de Contraste/farmacocinéticaRESUMO
PURPOSE: Neuroblastoma is the most common extracranial solid tumour in childhood. It accounts for 15% of all paediatric oncology deaths. In the last few decades, improvement in treatment outcome for high-risk patients has not occurred, with an overall survival rate <30-40%. Many reasons may account for such a low survival rate. The aim of this review is to evaluate whether pharmacogenetic factors can explain treatment failure in neuroblastoma. METHODS: A literature search based on PubMed's database Medical Subject Headings (MeSH) was performed to retrieve all pertinent publications on current treatment options and new classes of drugs under investigation. One hundred and fifty-eight articles wer reviewed, and relevant data were extracted and summarised. RESULTS AND CONCLUSIONS: Few of the large number of polymorphisms identified thus far showed an effect on pharmacokinetics that could be considered clinically relevant. Despite their clinical relevance, none of the single nucleotide polymorphisms (SNPs) investigated can explain treatment failure. These findings seem to reflect the clinical context in which anti-tumour drugs are used, i.e. in combination with multimodal therapy. In addition, many pharmacogenetic studies did not assess (differences in) drug exposure, which could contribute to explaining pharmacogenetic associations. Furthermore, it remains unclear whether the significant activity of new drugs on different neuroblastoma cell lines translates into clinical efficacy, irrespective of resistance or myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN) amplification. Elucidation of the clinical role of pharmacogenetic factors in the treatment of neuroblastoma demands an integrated pharmacokinetic-pharmacodynamic approach to the analysis of treatment response data.
Assuntos
Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Farmacogenética/métodos , Criança , Humanos , Neuroblastoma/metabolismo , Polimorfismo de Nucleotídeo Único , Falha de TratamentoRESUMO
Streptococcus intermedius occasionally causes brain abscesses that can be life-threatening, requiring prompt antibiotic and neurosurgical treatment. The source is often dental, and it may spread to the eye or the brain parenchyma. We report the case of a 34-year-old man with signs of apical periodontitis, endophthalmitis, and multiple brain abscesses caused by Streptococcus intermedius. Initial treatment with meropenem and vancomycin was unsuccessful due to subtherapeutic concentrations, despite recommended dosages. Adequate concentrations could be reached only after increasing the dose of meropenem to 16 g/day and vancomycin to 1.5 g × 4. The patient exhibited high creatinine clearance consistent with augmented renal clearance, although iohexol and cystatin C clearances were normal. Plasma free vancomycin clearance followed that of creatinine. A one-day dose of trimethoprim-sulfamethoxazole led to an increase in serum creatinine and a decrease in both creatinine and urea clearances. These results indicate that increased tubular secretion of the drugs was the cause of suboptimal antibiotic treatment. The patient eventually recovered, but his left eye needed enucleation. Our case illustrates that augmented renal clearance can jeopardize the treatment of serious bacterial infections and that high doses of antibiotics are needed to achieve therapeutic concentrations in such cases. The mechanisms for regulation of kidney tubular transporters of creatinine, urea, vancomycin, and meropenem in critically ill patients are discussed.
RESUMO
Troponin T (TnT) is a useful biomarker for studying drug-induced toxicity effects on cardiac cells. We describe how a surface plasmon resonance (SPR) biosensor was applied to monitor the release of TnT from active HL-1 cardiomyocytes in vitro when exposed to cardiotoxic substances. Two monoclonal human TnT antibodies were compared in the SPR immunosensor to analyse the TnT release. The detection limit of TnT was determined to be 30 ng/ml in a direct assay set-up and to be 10 ng/ml in a sandwich assay format. Exposure of the cardiomyocytes to doxorubicin, troglitazone, quinidine and cobalt chloride for periods of 6 and 24 h gave significant SPR responses, whereas substances with low toxicity showed insignificant effects (ascorbic acid, methotrexate). The SPR results were verified with a validated immunochemiluminescence method which showed a correlation of r (2) = 0.790.
Assuntos
Técnicas Biossensoriais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Miocárdio/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Troponina T/metabolismo , Anticorpos Monoclonais/imunologia , Linhagem Celular , Humanos , Luminescência , Troponina T/imunologiaRESUMO
Several transcripts have been claimed to be clinically valuable for detecting minimal disease in neuroblastoma, but they have not been prospectively compared in a standardized manner. Tyrosine hydroxylase (TH), dopa decarboxylase (DDC) and GD2 synthase (GD2S) mRNAs were analyzed in 554 blood (PB) and bone marrow (BM) samples from 58 children with neuroblastoma. Samples from 44 children with other diseases served as controls. High transcript concentrations of TH, GD2S or DDC in PB or BM at diagnosis were associated with poor prognosis. TH in BM above median indicated worse outcome for a homogenous cohort with high-risk neuroblastoma (survival probability 91% for TH below median versus 33% for TH above median, p = 0.009). The number of children with localized neuroblastoma with increased results in PB did not differ between the three transcripts. In these children, all without morphologically detectable neuroblastoma in BM, the number of patients with elevated GD2S in BM at diagnosis was significantly higher than for the other transcripts (10/16 elevated, p = 0.012). GD2S was elevated in PB from 10/28 controls without neuroblastoma compared to 1/28 for TH and DDC (p < 0.001). In BM from these children GD2S was significantly elevated. We conclude that high expression of TH and DDC both in PB and BM corresponds to metastatic neuroblastoma at diagnosis, residual disease, and poor outcome. Children with high-risk neuroblastoma and low levels of TH in BM at diagnosis may be cured by current therapy. GD2S is less specific than TH and DDC mRNA for neuroblastoma detection in PB and BM.
Assuntos
Biomarcadores Tumorais/genética , Medula Óssea/enzimologia , Dopa Descarboxilase/genética , N-Acetilgalactosaminiltransferases/genética , Neuroblastoma/enzimologia , Tirosina 3-Mono-Oxigenase/genética , Adolescente , Estudos de Casos e Controles , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Estadiamento de Neoplasias , Neuroblastoma/genética , Neuroblastoma/patologia , Valor Preditivo dos Testes , Prognóstico , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medição de Risco , Fatores de Risco , Sensibilidade e Especificidade , Suécia , Resultado do TratamentoRESUMO
Prostaglandin E(2) (PGE(2)) has been shown to play important roles in several aspects of tumor development and progression. PGE(2) is synthesized from arachidonic acid by cyclooxygenases (COX) and prostaglandin E synthases (PGES) and mediates its biological activity through binding to the four prostanoid receptors EP(1) through EP(4). In this study, we show for the first time that medulloblastoma (MB), the most common malignant childhood brain tumor, expresses high levels of COX-2, microsomal prostaglandin E synthase-1, and EP(1) through EP(4) and secretes PGE(2). PGE(2) and the EP(2) receptor agonist butaprost stimulated MB cell proliferation. Treatment of MB cells with COX inhibitors suppressed PGE(2) production and induced caspase-dependent apoptosis. Similarly, specific COX-2 silencing by small interfering RNA inhibited MB cell growth. EP(1) and EP(3) receptor antagonists ONO-8713 and ONO-AE3-240, but not the EP(4) antagonists ONO-AE3-208 and AH 23848, inhibited tumor cell proliferation, indicating the significance of EP(1) and EP(3) but not EP(4) for MB growth. Administration of COX inhibitors at clinically achievable nontoxic concentrations significantly inhibited growth of established human MB xenografts. Apoptosis was increased, proliferation was reduced, and angiogenesis was inhibited in MBs treated with COX inhibitors. This study suggests that PGE(2) is important for MB growth and that therapies targeting the prostanoid metabolic pathway are potentially beneficial and should be tested in clinical settings for treatment of children with MB.
Assuntos
Neoplasias Cerebelares/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Meduloblastoma/metabolismo , Transdução de Sinais/fisiologia , Adolescente , Adulto , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Criança , Pré-Escolar , Ciclo-Oxigenase 2/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The clinical utility of detecting minimal residual disease (MRD) in children with neuroblastoma (NB) by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) is not clear. This in part reflects the lack of uniform methodology for analysis and reporting. Reference laboratories across Europe have therefore established standard operating procedures (SOPs) for the detection of NB cells by QRT-PCR. Haemopoietic samples are collected into PAXgene blood RNA tubes, which stabilise mRNA for 48 h at room temperature and more than 6 months at -80 degrees C. Tyrosine hydroxylase (TH) was selected as the target for NB cell detection, expression is normalised to beta2-microglobulin and reported using the DeltaDeltaCt method. The sensitivity of QRT-PCR increased from 58% to 90% following the development of SOPs. A robust, transferable, objective method for the detection of NB cells by QRT-PCR has been defined to improve the power and consistency of studies on MRD in children with NB.
Assuntos
Neoplasia Residual/diagnóstico , Neuroblastoma/diagnóstico , Linhagem Celular Tumoral , Criança , Pré-Escolar , Humanos , Lactente , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Controle de Qualidade , RNA Neoplásico/análise , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Manejo de Espécimes/normasRESUMO
Malignant melanomas are more often seen in subjects with light colored skin who tan poorly than in persons who tan more rapidly. This has been attributed to the structure of their pigment, pheomelanin, which differs markedly from the eumelanin of persons with darker skin. To study the hydrolysis products of pheomelanin pigments a new method was developed for analysis of 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP). Pheomelanin samples were hydrolyzed and extracted with solid-phase extraction columns using strong cation-exchange (SCX) cartridges. Separation of 4-AHP and 3-AHP was achieved on a ZIC-HILIC column (150 mm x 2.1mm I.D.) with a mobile phase consisting of acetonitrile: 0.1 M ammonium acetate buffer, pH 4.5 (82:18, v/v). Detection was performed with an electrochemical detector at +400 mV. Run time was 30 min. The limits of detection were 73 pg and 51 pg for 4-AHP and 3-AHP respectively, using 2 microl injections. Good linearity was found within the range 0.05-5.0 microg/ml. Absolute recovery was 70% and relative recovery was 100%. The AHPs were stable for 1 year in the hydrolyzed samples, for 4 days in the eluates from solid-phase sorbents stored in the refrigerator, and for 2 days diluted with mobile phase and stored in the autosampler at 10 degrees C. The within-day imprecision was <5% and the between-day imprecision was <7% for the two analytes. The method, applied to the analysis of pheomelanin in urine from human melanoma patients, allows the analysis of 30 samples in one set and is suitable for routine work with human hair and melanoma cells. By using the ZIC-HILIC stationary phase, ion-pairing reagents could be avoided, which makes the method suitable to further analysis of degradation products from pheomelanins using mass spectrometric detection.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Melaninas/química , Tirosina/análise , Estrutura Molecular , Reprodutibilidade dos Testes , Temperatura , Tirosina/análogos & derivados , Tirosina/químicaRESUMO
A quantitative and precise measure of treatment response is warranted in neuroblastoma patients. We compared three quantitative methods often used for detection of minimal residual disease in such patients. Specificity, sensitivity and concordance of immunocytochemistry, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry were compared using experimental cell suspensions (n = 8) and clinical samples (n = 126). Neuroblastoma cells were identified by immunocytochemistry and flow cytometry using anti-GD2 (14.G2a) and anti-NCAM (5.1H11) antibodies, whereas tyrosine hydroxylase mRNA was the molecular target for quantitative RT-PCR. The sensitivity using flow cytometry was 1-2 logs less than using immunocytochemistry or quantitative RT-PCR. All control samples (n = 35) tested negative by immunocytochemistry, whereas 2/34 (6%) and 1/14 (7%) were false positive by quantitative RT-PCR and flow cytometry respectively. Concordant results were obtained in 85% of patient samples (n = 116) analyzed in parallel by quantitative RT-PCR and immunocytochemistry, whereas 71% of samples analyzed by flow cytometry and immunocytochemistry were concordant (n = 35). The correlation between tumor cell levels analyzed by quantitative RT-PCR and immunocytochemistry was high (r = 0.78, p < 0.001). Quantitative RT-PCR and immunocytochemistry both reliably detected very low levels of neuroblastoma cells in clinical samples. The agreement and correlation between these methods were high. In comparison, flow cytometry was less sensitive.
Assuntos
Citometria de Fluxo/métodos , Imuno-Histoquímica/métodos , Neoplasia Residual/diagnóstico , Neuroblastoma/diagnóstico , Neuroblastoma/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Anticorpos/química , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Pré-Escolar , Reações Falso-Positivas , Gangliosídeos/imunologia , Humanos , Lactente , Recém-Nascido , Moléculas de Adesão de Célula Nervosa/imunologia , Neuroblastoma/metabolismo , RNA Mensageiro/metabolismo , Sensibilidade e Especificidade , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Molecular tools for fluorescent imaging of cells and their components are vital for understanding the function and activity of cells. Here, we report an imidazole functionalized pentameric oligothiophene, p-HTIm, that can be utilized for fluorescent imaging of cells. p-HTIm fluorescence in normal cells appeared in a peripheral punctate pattern partially co-localized with lysosomes, whereas a one-sided perinuclear Golgi associated localization of the dye was observed in malignant cells. The uptake of p-HTIm was temperature dependent and the intracellular target was reached within 1 h after staining. The ability of p-HTIm to stain cells was reduced when the imidazole side chain was chemically altered, verifying that specific imidazole side-chain functionalities are necessary for achieving the observed cellular staining. Our findings confirm that properly functionalized oligothiophenes can be utilized as fluorescent tools for vital staining of cells and that the selectivity toward distinct intracellular targets are highly dependent on the side-chain functionalities along the conjugated thiophene backbone.
RESUMO
BACKGROUND: Carbohydrate deficient transferrin (CDT) is used for detection of alcohol abuse and follow-up. High performance liquid chromatography (HPLC) of transferrin glycoforms is highly specific for identification of alcohol abuse, but unresolved disialo- and trisialotransferrin glycoforms sometimes makes interpretation difficult. The cause of this phenomenon is unknown, cannot be explained by genetic variants of transferrin, but seems to be associated with liver disease. METHODS: Nineteen serum samples showing di-tri bridging when analyzed by HPLC were collected. Transferrin was purified by affinity chromatography, and N-linked oligosaccharides were released enzymatically. The N-glycans were further analyzed by high performance anion-exchange chromatography with pulsed amperometric detection and MALDI-TOF mass spectrometry. RESULTS: The HPLC-analysis showed three different types of glycoform patterns. The N-glycans from fifteen samples showed patterns with increased number of triantennary structures containing one or two fucose residues. One sample contained an increased amount of triantennary glycans without fucose. Three samples showed a glycosylation pattern similar to normal transferrin. CONCLUSIONS: The di-tri bridging phenomenon was associated with alterations in transferrin glycosylation in the majority of cases. Transferrin contained a higher extent of triantennary and often fucosylated N-linked oligosaccharides. These results may be important in future diagnostic approaches to liver diseases.
Assuntos
Fucose/química , Transferrina/análogos & derivados , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Feminino , Glicosilação , Humanos , Masculino , Pessoa de Meia-Idade , Oligossacarídeos/sangue , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Transferrina/análise , Transferrina/química , Transferrina/isolamento & purificação , Adulto JovemRESUMO
PURPOSE: To characterize resistance mechanisms to the nucleoside analog 9-ß-D-arabinofuranosylguanine (AraG) in the T-cell acute lymphoblastic leukemia cell line MOLT-4 and its AraG-resistant variant. METHODS: A gene expression microarray analysis was performed, as well as gene expression and enzyme activity measurements of key enzymes in the activation of AraG. Cytotoxicity of AraG and cross-resistance to other compounds were evaluated using a standard cytotoxicity assay. RESULTS: Gene expression microarray analysis revealed that fetal hemoglobin genes and the multidrug resistance ABCB1 gene, encoding the drug efflux pump P-gp, were the most highly upregulated genes in the resistant cells, while genes traditionally associated with nucleoside analog resistance were not. Fetal hemoglobin and ABCB1 induction can be due to global DNA hypomethylation. This phenomenon was studied using AraG during a period of 4 weeks in MOLT-4 cells and the lung adenocarcinoma cell line A549, leading to up-regulation of hemoglobin gamma and ABCB1 as well as DNA hypomethylation. Inhibiting P-gp in the AraG-resistant MOLT-4 cells led to decreased proliferation, reduced hemoglobin expression, and highly induced ABCB1 expression. CONCLUSIONS: We show that AraG can cause hypomethylation of DNA and induce the expression of the fetal hemoglobin gamma gene and the ABCB1 gene. We speculate that the induction of ABCB1/P-gp may occur in order to help with excretion of hemoglobin degradation products that would otherwise be toxic to the cells, and we present data supporting our theory that P-gp may be linked to the induction of hemoglobin.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Arabinonucleosídeos/farmacologia , Hemoglobina Fetal/análise , Subfamília B de Transportador de Cassetes de Ligação de ATP , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/efeitos dos fármacos , Humanos , Análise em Microsséries , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio , TiazóisRESUMO
BACKGROUND: Currently used as structural markers for pheomelanin identification and quantitation, benzothiazole compounds derived from isomers of cysteinyldopa have been indicated by recent in vitro studies as new potential pheomelanogenesis intermediates. The presence of benzothiazole compounds in the urine of patients with melanoma with or without diffuse melanosis was investigated. METHODS: Hydrophilic interaction liquid chromatography with zwitterionic stationary phase (ZIC-HILIC) and photo-diode array (PDA) detection was used for analysis of 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole-2-carboxylic acid (BTCA-5), and 7-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole-2-carboxylic acid (BTCA-2), derived from 5-S-cysteinyldopa (5-S-CD) and 2-S-cysteinyldopa (2-S-CD) isomers, respectively. After minimal sample preparation, isocratic chromatography allowed efficient separation of the compounds, which were safely identified by their typical absorption features. RESULTS: Three patients with diffuse melanosis, 16 patients with melanoma (stages III and IV) and three healthy subjects were investigated. The urinary BTCAs were found to be highly associated with melanosis but more loosely to excreted 5-S-CD. Analysis of the pigmented fraction of urine following alkaline hydrogen peroxide degradation and quantitation of BTCAs provided evidence for the presence of pheomelanins at high levels in patients with melanosis. CONCLUSION: Identification of free BTCA isomers in urine provides a significant contribution in the field of urinary melanogens, and has important implications for biosynthetic activity of normal and pathologic melanocytes.
Assuntos
Benzotiazóis/urina , Melaninas/urina , Melanoma/urina , Melanose/urina , Adulto , Cromatografia Líquida/métodos , Humanos , Isomerismo , Masculino , Pessoa de Meia-Idade , Espectrofotometria UltravioletaRESUMO
Human embryonic stem cell (hESC) derived cardiomyocytes are in the present study being used for testing drug-induced cardiotoxicity in a biosensor set-up. The design of an in vitro testing alternative provides a novel opportunity to surpass previous methods based on rodent cells or cell lines due to its significantly higher toxicological relevance. In this report we demonstrate how hESC-derived cardiomyocytes release detectable levels of two clinically decisive cardiac biomarkers, cardiac troponin T and fatty acid binding protein 3, when the cardiac cells are exposed to the well-known cardioactive drug compound, doxorubicin. The release is monitored by the immuno-biosensor technique surface plasmon resonance, particularly appropriate due to its capacity for parallel and high-throughput analysis in complex media.
Assuntos
Técnicas Biossensoriais/métodos , Células-Tronco Embrionárias/metabolismo , Cardiopatias/metabolismo , Miócitos Cardíacos/metabolismo , Testes de Toxicidade/métodos , Biomarcadores/metabolismo , Contagem de Células , Linhagem Celular , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Células-Tronco Embrionárias/química , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/metabolismo , Expressão Gênica/efeitos dos fármacos , Cardiopatias/induzido quimicamente , Cardiopatias/patologia , Humanos , Imuno-Histoquímica , Miócitos Cardíacos/química , Miócitos Cardíacos/efeitos dos fármacos , Reação em Cadeia da Polimerase , Ressonância de Plasmônio de Superfície , Testes de Toxicidade/normas , Troponina T/metabolismoAssuntos
Antiparkinsonianos/sangue , Cromatografia de Afinidade/métodos , Levodopa/sangue , Antiparkinsonianos/uso terapêutico , Cromatografia de Afinidade/economia , Ácido Edético , Formiatos , Humanos , Concentração de Íons de Hidrogênio , Levodopa/uso terapêutico , Metildopa/metabolismo , Doença de Parkinson/sangue , Doença de Parkinson/tratamento farmacológico , Padrões de ReferênciaRESUMO
Melanoma is most rapidly increasing in the white population and people with pheomelanin skin type are at high risk to develop melanoma. However, little is known about the pheomelanin structure and function, and further elucidation of this melanin is therefore an important task. A GC/MS method was developed based on hydriodic acid hydrolysis of pheomelanin in the urine. Derivatization was performed with ethyl chloroformate and ethanol:pyridine (4:1, v/v). N,O-Ethoxycarbonyl-ethyl esters were extracted with chloroform and analyzed by GC/MS. 4-Amino-3-hydroxyphenylalanine and 3-amino-4-hydroxyphenylalanine together with one benzothiazinone and two benzothiazole compounds were detected and identified in hydrolyzed samples of synthetic pheomelanin and melanin from the urine of a patient with melanoma. These findings strongly suggest that heterocyclic pheomelanin-type units are incorporated in the pigment structures.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Melaninas/química , Melanoma/diagnóstico , Humanos , Hidrólise , Masculino , Melaninas/urina , Pessoa de Meia-IdadeRESUMO
BACKGROUND: There is a certain difference in opinion regarding the optimal choice of housekeeping genes used as normalization factors in gene expression analysis. We have therefore examined the suitability of three housekeeping genes, hypoxanthine phosphoribosyl transferase, beta-glucuronidase and beta2-micro-globulin, for normalization of expression data from melanoma metastases. METHODS: The expression of the three housekeeping genes was quantified by quantitative reverse transcription PCR in snap-frozen sections from 44 melanoma metastases, of which 19 were from patients treated with cisplatinum, dacarbazine and interferon-alpha2b. RESULTS: The expression of each housekeeping gene varied considerably between the different metastases. Histopathological examination of the tissue sections revealed variation in the amount of tumor cells in the tissue, necrosis, varying degrees of lymphocyte infiltration, and lymph node remnants. Based on this examination, 16 biopsies were omitted from further analysis because they had cracked, contained empty or necrotic areas, or were dominated by lymph node tissue. Even in sections with more than 90% tumor cells, a wide variation in the expression of the three housekeeping genes was found. The amount of lymphatic infiltrate in the tumors can have an effect on the expression of housekeeping genes in the metastases, whereas treatment did not seem to influence the expression. CONCLUSIONS: We conclude that the choice of housekeeping genes can have great impact on the normalization of specific genes in melanoma metastases. Furthermore, in the analysis of mRNA expression in tumor tissue, microscopic examination is of great importance to evaluate the integrity and cellular composition of the biopsy.