RESUMO
Cancer stem cells (CSCs) have been successfully isolated from solid tumors and are believed to be initiating cells of primary, metastatic and recurrent tumors. Imaging and therapeutic reagents targeted to CSCs have potential to detect subclinical tumors and completely eradicate the disease. Previously, we have demonstrated that Mab CC188 binds to colon cancer CD133- and CD133+ (CSCs) cells. In this study, we examined the reactivity of Mab CC188 to ovarian cancer cells including CD133+ cells and primary tumor tissues using immunofluorescence staining methods and tissue microarray technique. We also explored the feasibility of using NIR dye-labeled Mab CC188 probe to image ovarian tumors in vivo. Mab CC188 stains both CD133- and CD133+ cells of ovarian cancer. Tissue microarray analysis reveals that 75% (92/123) of ovarian cancer cases are positively stained with Mab CC188. Weak positive (±), positive (+), strong positive (++) and very strong positive (+++) stains are 14.8, 3.7, 11 and 24.4%, respectively. In contrast, Mab CC188 staining is low in normal cells and tissues. In vivo study show that significant amounts of the probe accumulates in the excretion organs in the early period postinjection. At 24 hr, the imaging probes have largely accumulates in the tumor, while the intensity of the imaging probe decreases in the liver. The tumor uptake was still evident at 120-hr postinjection. Our work suggests that Mab CC188-based imaging and therapeutic reagents are capable of detecting early stage ovarian tumors and effectively treating the tumor.
Assuntos
Anticorpos Monoclonais , Antígenos CD/análise , Glicoproteínas/análise , Células-Tronco Neoplásicas/química , Neoplasias Ovarianas/diagnóstico , Peptídeos/análise , Antígeno AC133 , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Estadiamento de Neoplasias , Neoplasias Ovarianas/patologia , Análise Serial de TecidosRESUMO
Recently we described a new method for in situ localization of specific DNA sequences, based on lac operator/repressor recognition (Robinett, C.C., A. Straight, G. Li, C. Willhelm, G. Sudlow, A. Murray, and A.S. Belmont. 1996. J. Cell Biol. 135:1685-1700). We have applied this methodology to visualize the cell cycle dynamics of an approximately 90 Mbp, late-replicating, heterochromatic homogeneously staining region (HSR) in CHO cells, combining immunostaining with direct in vivo observations. Between anaphase and early G1, the HSR extends approximately twofold to a linear, approximately 0.3-mum-diam chromatid, and then recondenses to a compact mass adjacent to the nuclear envelope. No further changes in HSR conformation or position are seen through mid-S phase. However, HSR DNA replication is preceded by a decondensation and movement of the HSR into the nuclear interior 4-6 h into S phase. During DNA replication the HSR resolves into linear chromatids and then recondenses into a compact mass; this is followed by a third extension of the HSR during G2/ prophase. Surprisingly, compaction of the HSR is extremely high at all stages of interphase. Preliminary ultrastructural analysis of the HSR suggests at least three levels of large-scale chromatin organization above the 30-nm fiber.
Assuntos
Replicação do DNA , Heterocromatina , Interfase , Animais , Células CHO , Núcleo Celular/metabolismo , Cricetinae , Fase G1 , Imuno-Histoquímica , Mitose , Fase S , Coloração e Rotulagem/métodos , Telófase , Fatores de TempoRESUMO
Analysis of the relationship between transcriptional activators and chromatin organization has focused largely on lower levels of chromatin structure. Here we describe striking remodeling of large-scale chromatin structure induced by a strong transcriptional activator. A VP16-lac repressor fusion protein targeted the VP16 acidic activation domain to chromosome regions containing lac operator repeats. Targeting was accompanied by increased transcription, localized histone hyperacetylation, and recruitment of at least three different histone acetyltransferases. Observed effects on large-scale chromatin structure included unfolding of a 90-Mbp heterochromatic chromosome arm into an extended 25-40-micrometers chromonema fiber, remodeling of this fiber into a novel subnuclear domain, and propagation of large-scale chromatin unfolding over hundreds of kilobase pairs. These changes in large-scale chromatin structure occurred even with inhibition of ongoing transcription by alpha-amanitin. Our results suggest a functional link between recruitment of the transcriptional machinery and changes in large-scale chromatin structure. Based on the observed long-range propagation of changes in large-scale chromatin structure, we suggest a possible rationale for the observed clustering of housekeeping genes within Mbp-sized chromosome bands.
Assuntos
Cromatina/química , Cromatina/metabolismo , Proteínas de Escherichia coli , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Proteínas de Saccharomyces cerevisiae , Acetilação , Acetiltransferases/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CHO , Cromatina/genética , Cricetinae , Genoma , Proteína Vmw65 do Vírus do Herpes Simples/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Histona Acetiltransferases , Histonas/metabolismo , Repressores Lac , Conformação Molecular , Regiões Operadoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo , Transcrição Gênica/genética , Ativação Transcricional , TransfecçãoRESUMO
We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.
Assuntos
Cromatina/ultraestrutura , Óperon Lac/fisiologia , Anáfase/fisiologia , Animais , Sequência de Bases , Células CHO/química , Células CHO/fisiologia , Células CHO/ultraestrutura , Cromátides/fisiologia , Cromátides/ultraestrutura , Cromatina/química , Cromatina/genética , Cromossomos/fisiologia , Cromossomos/ultraestrutura , Cricetinae , DNA/análise , Amplificação de Genes , Dosagem de Genes , Proteínas de Fluorescência Verde , Hibridização In Situ , Proteínas Luminescentes/análise , Microscopia Eletrônica , Mitose/fisiologia , Dados de Sequência Molecular , Proteínas Recombinantes/análise , Sequências Repetitivas de Ácido Nucleico , Coloração e Rotulagem , Fatores de Tempo , Leveduras/genéticaRESUMO
To determine whether nitro group reduction occurs in mammalian tissues, metronidazole (0.021, 0.064 and 10 mg/kg), misonidazole (0.015 mg/kg) and nitrofurazone (0.13 mg/kg), respectively, were administered to germfree rats. A reduced metabolite [1-(2-aminoimidazol-1-yl)-3-methoxypropan-2-ol] and two of its hydrolysis products, urea and (2-hydroxy-3-methoxypropyl)-guanidine, were found in the urines of germfree rats that received misonidazole. When nitrofurazone was administered, a reduced metabolite, 4-cyano-2-oxobutyraldehyde semicarbazone, was detected in the urines. However, acetamide and N-(2-hydroxyethyl)oxamic acid, fragmentation products from the reduction of metronidazole, were not found in significant concentrations in the urine when germfree rats received metronidazole. Apparently metronidazole is reduced so much more slowly than misonidazole and nitrofurazone in the tissues of germfree rats that its reductive metabolites are not detectable. This observation may be explained by the one-electron reduction potentials (E1 7) of these drugs, that of metronidazole (E1 7 = -486 mV) being lower than those of either misonidazole (E1 7 = -389 mV) or nitrofurazone (E1 7 = -257 mV). Under these circumstances, metronidazole reduction is not detected, either because its radical anion forms more slowly than that of the other nitroheterocyclic compounds or because its radical anion interacts more rapidly with oxygen to restore the parent compound.
Assuntos
Metronidazol/metabolismo , Misonidazol/metabolismo , Nitrofurazona/metabolismo , Nitroimidazóis/metabolismo , Acetamidas/urina , Animais , Etanolamina , Etanolaminas/urina , Vida Livre de Germes , Masculino , Metronidazol/urina , Misonidazol/urina , Nitrofurazona/urina , Ácido Oxâmico/análogos & derivados , Ácido Oxâmico/urina , Oxirredução , Ratos , Ratos EndogâmicosRESUMO
The fatty acid contents of thirteen commercial preparations of human albumin were found to be in the range 0.03 to 9 mol of fatty acid/mol albumin. Marked differences were found between the preparations in the binding of the fluorescent probes, 5-dimethylamino-naphthalene-1-sulfonamide (DNSA) and dansylsarcosine. The displacement of these probes by ibuprofen and phenylbutazone also showed marked differences between preparations. The differences between the commercial albumin samples correlated well with their fatty acid contents and were abolished by treatment with charcoal. They were similar to the changes observed when oleic acid was added to fatty acid free albumin. The source and fatty acid content of commercial albumin preparations should be considered when comparing studies of the binding of drugs to human albumin.
Assuntos
Ácidos Graxos/análise , Albumina Sérica , Corantes Fluorescentes , Humanos , Ibuprofeno , Fenilbutazona , Ligação Proteica , Sarcosina , Albumina Sérica/análise , Espectrometria de FluorescênciaRESUMO
Near-infrared (NIR) water-dispersible fluorescent tags are of big importance for biomedical imaging. Bright, stable, biocompatible NIR fluorescent nanoparticles have great translation potential to improve diagnosis of early stages of different diseases. Here we report on the synthesis of exceptionally bright ("ultrabright") fluorescent meso(nano)porous silica nanoparticles of 28 ± 3 nm in diameter. The NIR fluorescent dye LS277 is encapsulated inside these silica nanoparticles. The wavelengths of the maximum excitation/fluorescence of the particles are 804/815 nm. The absorptivity coefficient of the particles is 2.1 × 108 M-1 cm-1 at 805 nm and the quantum yield of the dye increased by a factor of 5 after encapsulating to 1.5%. The fluorescent brightness of these particles is more than 2000× higher than the fluorescence of one molecule of LS277 in water. When exited in NIR spectral region (>700 nm), these particles are up to 4× brighter than QD800 commercial quantum dots emitting at 800 nm. We demonstrate that the synthesized NIR mesoporous silica nanoparticles easily internalize 4T1luc breast tumor cells, and remain bright for more than 9 weeks whereas the dye is completely bleached by that time.
Assuntos
Acetilcolina/análogos & derivados , Colina/análogos & derivados , Órgão Elétrico/metabolismo , Peixes/metabolismo , Neurotransmissores , Acetilação , Acetilcolina/metabolismo , Animais , Colina/metabolismo , Órgão Elétrico/fisiologia , Frações Subcelulares/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Sinaptossomos/metabolismoAssuntos
Bactérias Anaeróbias/metabolismo , Nitroimidazóis/metabolismo , Animais , Bactérias Anaeróbias/genética , Biotransformação , Ceco/microbiologia , Reparo do DNA , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Vida Livre de Germes , Humanos , Metronidazol/metabolismo , Mutação/efeitos dos fármacos , Nitroimidazóis/uso terapêutico , Nitroimidazóis/toxicidade , Oxirredução , Relação Estrutura-Atividade , Xantina Oxidase/metabolismoAssuntos
Citosina/análise , Cromatografia em Papel , Citosina/sangue , Citosina/urina , Flúor , Fluorometria , Humanos , Indicadores e ReagentesRESUMO
1. The binding of racemic mixtures of warfarin and warfarin-alcohol to human serum albumin (HSA) is accompanied by an increase in the fluorescence quantum yield of these compounds. This property has been used to measure the characteristics of the binding of warfarin and warfarin-alcohol to HSA at 22 degrees C and 37 degrees C. Within the limits of the technique, no significant differences between the number of binding sites and strength of binding at the tight site at either temperature were observed. 2. The fluorescence of warfarin and warfarin-alcohol was used to label their binding site on HSA and to study the effects of other drugs on their binding. The results indicate that these two molecules are bound to the same site on HSA. 3. The validity of using changes in the fluorescence of warfarin as a measure of its displacement from HSA was investigated. Good correlations were observed between drug-induced decreases in the fluorescence of bound warfarin and displacement as measured by equilibrium dialysis. The displacement of warfarfin, as detected by fluorescence, correlates well with the increase in free warfarin resulting from addition of therapeutic drug concentrations to undiluted human serum. 4. The most potent displacing agents, by all the methods used, were iophenoxic acid, phenylbutazone and oxyphenylbutazone. The first of these is no longer used clinically, but the latter two are and have been reported to cause hypoprothrominaemia by displacing warfarin from HSA. The present study indicates that changes in the fluorescence of warfarin bound to HSA can be used to measure displacement of bound warfarin and to screen drugs that may cause clinically significant interactions by this mechanism.
Assuntos
Ligação Proteica , Albumina Sérica/metabolismo , Varfarina/metabolismo , Álcoois/metabolismo , Sítios de Ligação , Interações Medicamentosas , Humanos , Cinética , Métodos , Espectrometria de FluorescênciaRESUMO
Intracellular trafficking of the epidermal growth factor receptor (EGF-R) is regulated by receptor occupancy. To investigate this, we developed an assay to study endosomal sorting under steady-state conditions. Using a cell line transfected with EGF-R variants, we found that the fraction of internalized EGF.EGF-R complexes sorted to lysosomes was a function of the number of intracellular complexes and required sequences in the cytoplasmic domain of the receptor. As the number of intracellular occupied wild-type receptors increased from 3 x 10(2) to 2 x 10(5)/cell, the fraction of internalized EGF that was degraded dropped from 70 to 20%. Transforming growth factor-alpha, which dissociates from the EGF-R at endosomal pH, was degraded to a uniform extent of approximately 50% at all intracellular ligand concentrations. EGF internalized by receptors lacking a cytoplasmic domain (c'647) was degraded to an extent of only 5-10% independent of the number of intracellular complexes. Mutant receptors truncated either at residues 1022 or 973 displayed sorting patterns intermediate between wild-type and c'647 receptors. Despite large differences in their internalization rates, the fractional sorting patterns of c'1022 and c'973 receptors were indistinguishable. Receptor tyrosine kinase activity appeared to have a small effect on sorting pattern, but only in the context of full-length receptors. Our results indicate that the default pathway of internalized receptors is rapidly recycling and that lysosomal targeting of occupied EGF-R is due to endosomal retention that is both specific and saturable. In addition, internalization and endosomal retention of EGF-R appear to be mediated by distinct structural elements.
Assuntos
Endocitose , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Organelas/metabolismo , Animais , Membrana Celular/metabolismo , Receptores ErbB/biossíntese , Fibroblastos/metabolismo , Humanos , Cinética , Ligantes , Matemática , Camundongos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Deleção de Sequência , Transfecção , Fator de Crescimento Transformador alfa/metabolismoRESUMO
The effects of chronic treatment with allopurinol and clofibrate on the elimination from plasma of warfarin and dicoumarol were examined in man. In addition, the binding of warfarin to human serum proteins was measured with and without clofibrate and its metabolite chlorophenoxyisobutyric acid (CPIB), both present in therapeutic concentrations. Treatment with allopurinol and clofibrate did not alter the rate of elimination of warfarin from plasma. In addition, clofibrate and CPIB caused no significant displacement of warfarin from serum proteins. This evidence supports the conclusion that the clinically significant potentiation of warfarin activity by clofibrate in man is due to an interaction at the receptor site. In contrast, treatment with allopurinol resulted in significant prolongation of the plasma half-life of dicoumarol in one of three subjects. These data are consistent with inhibition of the metabolism of dicoumarol by allopurinol in some individuals.
Assuntos
Alopurinol/farmacologia , Clofibrato/farmacologia , Dicumarol/sangue , Varfarina/sangue , Adulto , Disponibilidade Biológica , Proteínas Sanguíneas , Butiratos/farmacologia , Meia-Vida , Humanos , Masculino , Ligação ProteicaRESUMO
The Hemoccult slide test is used frequently to test for the presence of occult blood in samples of gastric juice obtained from critically ill patients. The purpose of this study was to investigate the accuracy of this test to determine the presence of blood in human gastric juice at various pH values. Gastric aspirates were collected from 10 patients who had nasogastric tubes and were receiving nothing by mouth. These aspirates were adjusted to a range of pH values varying from 1 to 8. The aspirates were then tested by the Hemoccult slide method. The tests were repeated after (1) addition of whole blood and (2) subsequent addition of antacid. False negative results were found when the pH was below 2 and false positive results were found when the pH was between 2 to 4. When 3 drops of aspirate were added to 1 ml of a 0.2 M borate buffer at pH 8.6, and the Hemoccult slide test was performed on the resulting mixture, false negative and false positive reactions were eliminated. We conclude that the addition of the gastric juice sample to a borate buffer before performing the Hemoccult slide test makes this a reliable method of determining the presence or absence of occult blood.