Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF2α—F2-iPs—in urine has reflected lipid peroxidation in humans. However, up to 64 F2-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F2-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F2-iPs, using liquid chromatography/tandem mass spectrometry (LC/MS/MS), in which sample preparation is minimized. Authentic standards of F2-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF2α-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F2-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F2-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC/MS and LC/MS/MS of iPF2α-VI in hypercholesterolemia and of 8,12-iso-iPF2α-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.

The life sciences Global Image Database (GID)

Although a vast amount of life sciences data is generated in the form of images, most scientists still store images on extremely diverse and often incompatible storage media, without any type of metadata structure, and thus with no standard facility with which to conduct searches or analyses. Here we present a solution to unlock the value of scientific images. The Global Image Database (GID) is a web-based (http://www.g wer.ch/qv/gid/gid.htm) structured central repository for scientific annotated images. The GID was designed to manage images from a wide spectrum of imaging domains ranging from microscopy to automated screening. The annotations in the GID define the source experiment of the images by describing who the authors of the experiment are, when the images were created, the biological origin of the experimental sample and how the sample was processed for visualization. A collection of experimental imaging protocols provides details of the sample preparation, and labeling, or visualization procedures. In addition, the entries in the GID reference these imaging protocols with the probe sequences or antibody names used in labeling experiments. The GID annotations are searchable by field or globally. The query results are first shown as image thumbnail previews, enabling quick browsing prior to original-sized annotated image retrieval. The development of the GID continues, aiming at facilitating the management and exchange of image data in the scientific community, and at creating new query tools for mining image data.

A novel assay for drug-DNA binding mode, affinity, and exclusion number: scanning force microscopy.

Determining the mode-of-binding of a DNA ligand is not always straightforward. Here, we establish a scanning force microscopic assay for mode-of-binding that is (i) direct: lengths of individual DNA-ligand complexes are directly measured; (ii) rapid: there are no requirements for staining or elaborate sample preparation; and (iii) unambiguous: an observed increase in DNA length upon addition of a ligand is definitive evidence for an intercalative mode-of-binding. Mode-of-binding, binding affinity, and site-exclusion number are readily determined from scanning force microscopy measurements of the changes in length of individual drug-DNA complexes as a function of drug concentration. With this assay, we resolve the ambiguity surrounding the mode of binding of 2,5-bis(4-amidinophenyl) furan (APF) to DNA and show that it binds to DNA by nonintercalative modes. APF is a member of an important class of aromatic dicationic drugs that show significant activity in the treatment of Pneumocystis carinii pneumonia, an opportunistic infection that is the leading cause of death in AIDS patients.