RESUMO
A fiber-optic Raman probe fitted with a microscope objective was used to obtain on-line normal Raman spectra of adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate and uridine 5'-monophosphate separated by capillary isotachophoresis. With multimode optical fiber, the system interrograted a 40-micron length of capillary. Fiber-optic coupling facilitated use of an unmodified spectrograph and conventional capillary mounting systems. Raman spectra were excited with a 2W 532 nm NdYVO4, laser as the excitation source, with collection of 1 spectrum per second. Even at 2.10(-5) M initial concentration, Raman spectra were obtained at a good signal-to-noise ratio.
Assuntos
Ribonucleotídeos/análise , Eletroforese , Tecnologia de Fibra Óptica , Indicadores e Reagentes , Inosina Trifosfato/análise , Fibras Ópticas , Análise Espectral RamanRESUMO
Nucleic acid sequence-based amplification (NASBA) according to the standard protocol failed to amplify cRNA of viroids, probably because of their GC-rich and intramolecular base-paired structure. However, NASBA in the presence of inosine 5'-triphosphate successfully amplified the cRNAs to viroids in total nucleic acid extracts from citrus plants. As sequence specificity of the cRNA to viroids was confirmed by northern analysis, the amplification and fidelity of cRNAs are sufficient for the sensitive and specific detection of viroids.
Assuntos
Citosina , Guanina , Inosina Trifosfato/análise , Técnicas de Amplificação de Ácido Nucleico , Vírus de Plantas/genética , RNA Viral/química , Viroides/genética , Composição de Bases , Sequência de Bases , Primers do DNA , Inosina Trifosfato/metabolismo , Vírus de Plantas/metabolismo , RNA Viral/metabolismo , Sensibilidade e Especificidade , Viroides/metabolismoRESUMO
A rapid and sensitive high-performance liquid chromatographic method for the determination of multiple inositol phosphates and inositol trisphosphate isomers was developed. The separation of inositol phosphates was optimized by controlling the ionic strength with stepped gradient programs and the pH of mobile phase. Six inositol phosphates were determined within 22 min or the six compounds plus an inositol trisphosphate isomer within 24 min using a single anion-exchange column containing the quaternary ammonium functional group. This technique was successfully applied to the determination of inositol phosphatide turnover by AlF4-stimulation in a small amount (5.10(5)-1.10(6) cells) of cultured retinal capillary pericytes. Because of its efficiency, accuracy and applicability to the separation of inositol phosphates from biological samples, this method may be useful in signal transduction studies in cellular systems.
Assuntos
Inosina Trifosfato/análise , Fosfatos de Inositol/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Isomerismo , Músculo Liso Vascular/química , Músculo Liso Vascular/citologiaRESUMO
ATP is known to be easily determined fluorometrically after it is utilized to produce the corresponding amount of NADPH by combined reactions of hexokinase and glucose-6-phosphate dehydrogenase. We studied further whether nucleoside triphosphates other than ATP can be also determined in a similar manner if they were incubated for a longer period with an increased amount of hexokinase. It was shown that CTP, GTP, ITP, and UTP can be utilized to produce the corresponding amount of NADPH after an incubation of at least 60 min and that 0 to 50 nmols of these nucleotides were able to be determined fluorometrically.
