RESUMO
Two fluorescent adenosine analogs, 4-amino-6-methyl-8-(2-deoxy-beta-d-ribofuranosyl)-7(8H)-pteridone (6MAP) and 4-amino-2,6-dimethyl-8-(2'-deoxy-beta-d-ribofuranosyl)-7(8H)-pteridone (DMAP), have been synthesized as phosphoramidites. These probes are site-selectively incorporated into oligonucleotides using automated DNA synthesis. Relative quantum yields are 0.39 for 6MAP and 0.48 for DMAP as monomers and range from >0.01 to 0.11 in oligonucleotides. Excitation maxima are 310 (6MAP) and 330 nm (DMAP) and the emission maximum for each is 430 nm. Fluorescence decay curves of each are monoexponential exhibiting lifetimes of 3.8 and 4.8 ns for 6MAP and DMAP, respectively. When these probes are incorporated into oligonucleotides they display quenching of fluorescence intensity, increases in the complexity of decay curves, and decreases in mean lifetimes. Because these changes are apparently mediated by interactions with neighboring bases, spectral changes that occur as probe-containing oligonucleotides meet and react with other molecules provide a means of monitoring these interactions in real time. These probes are minimally disruptive to DNA structure as evidenced by melting temperatures of probe-containing oligonucleotides that are very similar to those of controls. Digestion of probe-containing oligonucleotides with P1 nuclease confirms probe stability as fluorescence levels are restored to those expected for each monomer. These adenosine analog probes are capable of providing information on DNA structure as it responds to binding or catalysis through interaction with other molecules.
Assuntos
Adenosina/síntese química , Pteridinas/síntese química , Adenosina/análogos & derivados , Adenosina/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Estrutura Molecular , Compostos Organofosforados/química , Pteridinas/química , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Espectrometria de FluorescênciaRESUMO
Self-assembly of HIV-1 integrase (IN) in solution has been studied previously by time-resolved fluorescence, using tryptophan anisotropy decay. This approach provides information on the size of macromolecules via the determination of rotational correlation times (theta). We have shown that, at submicromolar concentration, IN is characterized by a long rotational correlation time (theta(20 degrees C) = 90-100 ns) corresponding to a high-order oligomeric form, likely a tetramer. In the present work, we investigated the self-assembly properties of the DNA-bound IN by using three independent fluorophores. Under enzymatic assay conditions (10(-7) M IN, 2 x 10(-8) M DNA), using either fluorescein-labeled or fluorescent guanosine analog-containing oligonucleotides that mimic a viral end long terminal repeat sequence, we found that the DNA-IN complex was characterized by shorter theta(20 degrees C) values of 15.5-19.5 and 23-27 ns, calculated from experiments performed at 25 degrees C and 37 degrees C, respectively. These results were confirmed by monitoring the Trp anisotropy decay as a function of the DNA substrate concentration: the theta of IN shifted from 90-100 ns to lower values (<30 ns) upon increasing the DNA concentration. Again, the normalized theta(20 degrees C) values were significantly higher when monitored at 37 degrees C as compared with 25 degrees C. These results indicate that upon binding the viral DNA end, the multimeric enzyme undergoes a dissociation, most likely into a homogeneous monomeric form at 25 degrees C and into a monomer-dimer equilibrium at 37 degrees C.
Assuntos
DNA Viral/metabolismo , Integrase de HIV/química , Integrase de HIV/metabolismo , Sequência de Bases , DNA Viral/genética , Polarização de Fluorescência , HIV-1/genética , HIV-1/metabolismo , Humanos , Técnicas In Vitro , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Subunidades Proteicas , Triptofano/químicaRESUMO
HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Conformação de Ácido Nucleico , Sítios de Ligação , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Dimerização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Polarização de Fluorescência , Corantes Fluorescentes/química , Substâncias Macromoleculares , Oligonucleotídeos/química , Ligação Proteica , Espectrometria de Fluorescência , Ultracentrifugação , Xantopterina/análogos & derivados , Xantopterina/químicaRESUMO
Pteridine nucleoside analog probes are highly fluorescent and offer different approaches to monitor subtle DNA interactions with other molecules. Similarities in structure and size to native nucleosides make it possible to incorporate these probes into oligonucleotides through the standard deoxyribose linkage. These probes are formulated as phosphoramidites and incorporated into oligonucleotides using automated DNA synthesis. Their position within the oligonucleotide renders them exquisitely sensitive to changes in structure as the oligonucleotide meets and reacts with other molecules. Changes are measured through fluorescence intensity, anisotropy, lifetimes, spectral shifts, and energy transfer. The fluorescence properties of pteridine nucleoside analogs as monomers and incorporated into single and double stranded oligonucleotides are reviewed. The two guanosine analogs, 3MI and 6MI, and two adenosine analogs, 6MAP and DMAP, are reviewed in detail along with applications utilizing them.
Assuntos
DNA/metabolismo , Corantes Fluorescentes/farmacologia , Pteridinas/química , Espectrometria de Fluorescência/métodos , Adenina/química , Anisotropia , DNA/química , Guanosina/química , Modelos Químicos , Oligonucleotídeos/química , Temperatura , Fatores de TempoRESUMO
O6-alkylguanine-DNA alkyltransferase (AGT) is a DNA-repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6-position of guanine. We developed a real-time AGT assay that utilizes a fluorescent guanosine analog (3-methylisoxantopterin, 3-MI). 3-MI fluorescence is quenched in DNA and fluorescence intensity increases substantially with digestion of the oligonucleotide and release of 3-MI. The substrate is a doubled-stranded oligonucleotide with 3'-overhangs on each end and a PvuII recognition site. PvuII is inhibited by O6-methylguanine, positioned within the restriction site. 3-MI is incorporated in the opposite strand just outside of the PvuII restriction site. AGT repairs O6-methylguanine; PvuII cleaves at its restriction site, yielding a blunt-ended double strand, which is then digested by exonuclease III. This releases 3-MI from the oligonucleotide, resulting in an increase in fluorescence intensity. All reaction components (100-microL volume) are monitored in a single microcuvette. Rate of increase in fluorescence intensity is related to the amount of AGT in the reaction mixture. We measured AGT levels in extracts from a leukemia cell line, from leukemic lymphoblasts from patients, and from peripheral blood mononuclear cells from normal controls. This method may prove useful for mechanistic studies of AGT.
Assuntos
O(6)-Metilguanina-DNA Metiltransferase/análise , Sequência de Bases , Humanos , Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Proteínas Recombinantes/análise , Sensibilidade e Especificidade , Espectrometria de FluorescênciaRESUMO
The fluorescence properties of 3-methyl-isoxanthopterin (3-MI) incorporated into different oligonucleotides have been determined. This highly fluorescent guanosine analog has its absorption and fluorescence spectra well resolved from those of the normal nucleotides and the aromatic amino acids. The small shifts observed in absorption and fluorescence emission spectra upon incorporation of 3-MI into these oligonucleotides are consistent with a general solvent effect and do not suggest any contribution from the position of the probe from the 5' end, the sequence of nucleotides immediately 5' or 3' to the probe, or the single- or double-stranded nature of the oligomer. However, steady-state and time-resolved fluorescence studies indicate that the presence of a purine immediately 5' or 3' to the probe results in some dynamic but mostly static quenching in the single-stranded oligomer. Furthermore, a 3' purine is more effective than a 5' purine, and an adenine appears to be more effective than a guanine for these static quenching interactions. Formation of the double-stranded oligomer leads to an additional loss of quantum yield, which can also be ascribed primarily to static quenching. These results show that this new class of spectrally enhanced fluorescent purine analogs will be able to provide useful information concerning the perturbation of nucleic acid structures.
Assuntos
Desoxiguanosina/análogos & derivados , Corantes Fluorescentes/química , Oligonucleotídeos/química , Xantopterina/análogos & derivados , Sequência de Bases , Fenômenos Biofísicos , Biofísica , Desoxiguanosina/química , Polarização de Fluorescência , Polidesoxirribonucleotídeos/química , Teoria Quântica , Espectrometria de Fluorescência , Espectrofotometria , Xantopterina/químicaRESUMO
Eighteen fluorescent pteridine-based nucleoside analogs have been prepared that are suitable for synthesis as phosphormidites and site-specific incorporation into oligonucleotides. Their quantum yields ranged from < or = 0.03 to 0.88. The maximum excitation and emission wavelenghts of seven selected probes with quantum yields > 0.15 ranged from 334 to 358 and 400 to 444 nm, respectively. Fluorescence decay curves of the seven probes were biexponential, and the mean intensity-weighted lifetimes ranged from 0.87 to 6.54 ns. Incorporation of probes 4 and 17 (3-methylisoxanthopterin and 6-methylisoxanthopterin) into oligonucleotides significantly quenched their fluorescence signal, and the degree of quench correlated with the number and proximity of purines in the oligonucleotide. Incorporation also resulted in a shift in absorbance-, emission-, and decay-associated spectra for 6-methylisoxanthopterin. An increase in the complexity of the decay curve and a decrease in the mean lifetime occurred for both probes. Formation of double-stranded oligonucleotides did not substantially increase the degree of quenching but generally increased the complexity of decay curves and decreased the mean lifetimes. Melting temperature, Tm, depression equivalent to that of a single base pair mismatch was observed in 3-methylisoxanthopterin-containing double-stranded oligonucleotides, while the Tm of 6-methylisoxanthopterin-containing double-stranded oligonucleotides were unperturbed, e.g., equivalent to unlabeled double-stranded oligonucleotides. This new class of fluorophore yields promising probes for the study of protein/DNA interactions.
Assuntos
Nucleosídeos/química , Oligonucleotídeos/química , Pteridinas/química , Corantes Fluorescentes , Espectrometria de FluorescênciaRESUMO
We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Desoxiguanosina/análogos & derivados , Corantes Fluorescentes , Guanosina/análogos & derivados , HIV-1/enzimologia , Xantopterina/análogos & derivados , Sequência de Bases , Desoxiguanosina/síntese química , Desoxiguanosina/metabolismo , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/metabolismo , Genoma Viral , Guanosina/metabolismo , Humanos , Integrases , Dados de Sequência Molecular , Sensibilidade e Especificidade , Xantopterina/síntese química , Xantopterina/metabolismoRESUMO
The pharmacokinetics of 2',3'-dideoxyadenosine (ddA), didanosine, 2',3'-dideoxyguanosine (ddG), and 6-halogenated prodrugs of ddG, 6-chloro-ddG and 6-iodo-ddG, in plasma and cerebrospinal fluid (CSF) were studied in a non-human primate model. ddA was rapidly and completely deaminated to didanosine, such that didanosine concentration profiles in plasma and CSF were identical following administration of ddA and didanosine. The mean clearance of didanosine was 0.50 liters/h/kg, the terminal half-life was 1.8 h, and the CSF-to-plasma ratio was 4.8%. The disposition of ddG was similar, with a clearance of 0.70 liters/h/kg and a half-life of 1.7 h. The adenosine deaminase-mediated conversion of the 6-halogenated-ddG prodrugs to ddG was rapid but incomplete (48% for 6-chloro-ddG and 29% for 6-iodo-ddG). The CSF-to-plasma ratios of ddG with equimolar doses of ddG, 6-chloro-ddG, and 6-iodo-ddG were 8.5, 24, and 17%, respectively, but the actual ddG exposures in CSF (area under the CSF concentration-time curve) were comparable for ddG (12.1 microM.h) and the 6-halogenated-ddG prodrugs (18.8 microM.h for 6-chloro-ddG, 9.3 microM.h for 6-iodo-ddG).6-Chloro-ddG was not detectable in plasma or CSF, and the CSF-to-plasma ratio of 6-iodo-ddG was 9.4%, so the higher CSF-to-plasma ratios of ddG with the administration of the 6-halogenated-ddG prodrugs does not appear to be the result of enhanced penetration of the prodrug and subsequent dehalogenation to ddG. The penetration of ddG into CSF exceeds that of didanosine and is enhanced by administration of the 6-halogenated prodrugs, although the mechanism of this enhanced penetration is unclear.
Assuntos
Didesoxinucleosídeos/farmacocinética , Animais , Antivirais/sangue , Antivirais/líquido cefalorraquidiano , Sistema Nervoso Central/metabolismo , Didanosina/sangue , Didanosina/líquido cefalorraquidiano , Didanosina/farmacocinética , Didesoxiadenosina/sangue , Didesoxiadenosina/líquido cefalorraquidiano , Didesoxiadenosina/farmacocinética , Didesoxinucleosídeos/sangue , Didesoxinucleosídeos/líquido cefalorraquidiano , Macaca mulatta , Masculino , Taxa de Depuração Metabólica , Modelos BiológicosRESUMO
The thiopurines have a wide array of effects on purine metabolism, but the primary mechanism of cytotoxicity for both 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) appears to be incorporation of drug into DNA following conversion to the thioguanylate form. In murine leukemic cell lines exposed to a range of thiopurine concentrations in vitro, cell survival curves have displayed a phenomenon termed paradoxical cytotoxicity, defined as a decrease in cytotoxicity with increasing drug concentration. The paradoxical cytotoxicity of thiopurines has usually been attributed to concentration-dependent perturbations in the cell cycle. The present study assessed whether the paradoxical cytotoxicity of 6-MP occurred in cultured human leukemic cells, and investigated the biochemical and cell-cycle alterations occurring in these lines at thiopurine concentrations associated with the reverse of cytotoxicity. Paradoxical cytotoxicity was observed in the two human leukemic cell lines examined, but only when 6-MP concentrations exceeded 100 microM. The extent of incorporation of 6-MP metabolites into DNA as thiol-versus non-thiol-containing metabolites was analyzed by performing parallel experiments with 14C- and 35S-radiolabeled drug. With 5 microM 6-MP, approximately 50% of drug was incorporated into DNA as a thionucleotide; however, with increasing drug concentrations, the degree of thionucleotide incorporation remained unchanged or decreased, and the amount incorporated as the desulfurated metabolite (presumably adenylate or guanylate) increased. With 500 microM 6-MP, less than 10% of the drug was incorporated as the thionucleotide. Perturbations in cell cycle reflected the relative amounts of thiol- and non-thiol-containing nucleotide formed at various concentrations of 6-MP. These results suggest that thiopurines may be vulnerable to a unique mechanism of detoxification, in which a human cell can metabolize a cytotoxic drug to a comparatively potent "self-rescue" agent.
Assuntos
DNA/metabolismo , Mercaptopurina/farmacologia , Radioisótopos de Carbono , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Hipoxantina , Hipoxantina Fosforribosiltransferase/metabolismo , Hipoxantinas/farmacologia , Inativação Metabólica , Mercaptopurina/química , Mercaptopurina/metabolismo , Radioisótopos de Enxofre , Tioguanina/farmacologia , Tionucleotídeos/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The pharmacokinetics of intravenous and oral 2',3'-dideoxyinosine (ddI) and the relationships between pharmacokinetic parameters and measures of response were studied in 48 human immunodeficiency virus-infected children. Disappearance of ddI from plasma after the intravenous dose was rapid and biexponential, with half-lives of 12 min and 1.0 h and a total clearance of 510 +/- 180 ml/min/m2. After oral administration, ddI absorption was limited and variable (mean bioavailability, 19% +/- 17%). A plasma ddI concentration-response relationship was observed for both decline in viral p24 antigen levels and improvement in intelligence quotient score. A limited sampling model was developed that accurately predicts the area under the ddI plasma concentration-time curve from one to three plasma samples. Although this pharmacokinetic study was done in children, the results also have relevance to adults and suggest that individualization of dose and schedule through therapeutic drug monitoring may be necessary to achieve optimal response.
Assuntos
Didanosina/farmacocinética , Infecções por HIV/metabolismo , Absorção , Administração Oral , Adolescente , Criança , Pré-Escolar , Didanosina/administração & dosagem , Didanosina/uso terapêutico , Avaliação de Medicamentos , Feminino , Seguimentos , Infecções por HIV/tratamento farmacológico , Meia-Vida , Humanos , Lactente , Infusões Intravenosas , MasculinoRESUMO
The last few years have seen a tremendous growth in videocassette usage, not only for entertainment, but also as a viable teaching method. Most homes in American now have at least one VCR. At Ozarka Vo Tech in Melbourne, AR, educators recognize the educational value of videotapes and have combined efforts to produce several quality computer-generated videotapes. With funding from projects, P189-01 A Competency-Based, Computer-Assisted, And Computer-Managed Model LPN Program, and the PI 2B061 Comprehensive Learning Lab, educators have been able to produce teacher-made videotapes to augment the different educational programs at our school. This article describes the method used, equipment needed, and general hints for improving the quality of teacher-made videotapes.
Assuntos
Enfermagem Prática/educação , Instruções Programadas como Assunto/normas , Ensino/métodos , Gravação de Videoteipe/normas , Humanos , Técnicas de Planejamento , Gravação de Videoteipe/instrumentação , Gravação de Videoteipe/métodosAssuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Cromatografia Líquida de Alta Pressão/métodos , Zalcitabina/sangue , Síndrome da Imunodeficiência Adquirida/sangue , Adolescente , Cromatografia Líquida de Alta Pressão/estatística & dados numéricos , Humanos , Masculino , Análise de Regressão , Zalcitabina/farmacocinética , Zalcitabina/uso terapêuticoRESUMO
Steady-state chemostat cultures of Azotobacter vinelandii strain CA11, carrying a deletion of genes encoding the structural polypeptides of nitrogenase nifHDK, were established in a simple defined medium chemically purified to minimize contamination by Mo. The medium contained no utilizable N source. Growth was dependent on N2 (1.1 X 10(8) viable cells X ml-1 at D = 0.176 h-1), and was inhibited by Mo (20 nM). DNA hybridization showed the deletion to be stable during prolonged (55 days) growth in the chemostat (132 doublings). Since batch cultures, using unsupplemented 'spent' chemostat medium, showed good growth (1.9 X 10(8) cells X ml-1), no requirement for subnanomolar concentrations of Mo was found. The biomass yield, as the dilution rate (D) was varied, showed that the N content of the culture, protein and dry wt. increased as D was decreased, indicating that neither N2 nor O2 were limiting growth. The limiting nutrient was not identified. Substantial amounts of H2 were evolved by the chemostat cultures, probably as the result of inhibition of O2-dependent hydrogenase activity by nitrilotriacetic acid present in the medium. Over a range of D values approx. 50% of the electron flux through the alternative system was allocated to H+ reduction. C2H2 was a poor substrate, being reduced at 0.14-0.1 times the rate of N2 fixation, calculated from the N content of the cells. SO4(2-)-limited steady-state continuous cultures of strain UW136 (wild-type for nifHDK) had a 2-fold greater biomass in the presence of MoO4(2-) (1 microM). The significance of this finding for 'Mo-limited' continuous cultures [Eady & Robson (1984) Biochem. J. 224, 853-862] is discussed.