Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ? 98 %). All samples were analyzed following injection (100 ?l) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 ?m, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients

Photo-Active Biological Molecular Materials: From Photoinduced Dynamics to Transient Electronic Spectroscopies

We present an overview of a methodology for the simulation of the photo-response of biological (macro)molecules, designed around a Quantum Mechanics / Molecular Mechanics (QM/MM) subtractive scheme. The resulting simulation workflow, that goes from the characterization of the photo-active system to the modeling of (transient) electronic spectroscopies is implemented in the software COBRAMM, but is completely general and can be used in the framework of any specific QM/MM implementation. COBRAMM is a smart interface to existing state-of-the-art theoretical chemistry codes, combining different levels of description and different algorithms to realize tailored problem-driven computations. The power of this approach is illustrated by reviewing the studies of two fundamental problems involving biological light-sensitive molecules. First, we will consider the photodynamics of the retinal molecule, the pigment of rhodopsin, a visual receptor protein contained in the rod cells of the retina. Retinal, with its light-induced isomerization, triggers a cascade of events leading to the production of the nerve impulse. Then, we will review some studies focusing on the interaction of DNA systems with ultraviolet (UV) light, a problem that has become one of the benchmark for the development of nonlinear spectroscopy, because of the ultrashort excited state lifetimes that arise from very efficient radiationless excited state decay and consent self-protection of DNA against UV damage