An immature retroviral RNA genome resembles a kinetically trapped intermediate state.

Unlabelled: Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions.

Femtomole SHAPE reveals regulatory structures in the authentic XMRV RNA genome.

Higher-order structure influences critical functions in nearly all noncoding and coding RNAs. Most single-nucleotide resolution RNA structure determination technologies cannot be used to analyze RNA from scarce biological samples, like viral genomes. To make quantitative RNA structure analysis applicable to a much wider array of RNA structure-function problems, we developed and applied high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to structural analysis of authentic genomic RNA of the xenotropic murine leukemia virus-related virus (XMRV).

Separations in poly(dimethylsiloxane) microchips coated with supported bilayer membranes.

Hybrid microchannels composed of poly(dimethylsiloxane) and glass were coated with supported bilayer membranes (SBMs) by the process of vesicle fusion. The electroosmotic mobility (mu(eo)) of zwitterionic, positively charged, and negatively charged phospholipid membranes was measured over a 4 h time to evaluate the stability of the coatings in an electric field. Coated microchips with a simple cross design were used to separate the fluorescent dyes fluorescein and Oregon Green.

Chemical analysis of single cells.

Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques.

Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis.

Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis.

Determination of sphingosine kinase activity for cellular signaling studies.

Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate.

Demonstration and use of nanoliter sampling of in vivo rat vitreous and vitreoretinal interface.

Purpose: An understanding of the chemical microenvironments at different locations on the retina can provide unique insights into retinal neurochemistry and pathology. The anatomical shape and the small volumes available from a spatially restricted volume greatly complicate these types of measurements. The aim of this study was to demonstrate an in vivo sampling system to probe different regions of the rat retina.

Determination of amino acids in rat vitreous perfusates by capillary electrophoresis.

In vivo determinations of amino acids are important for improving our understanding of physiological states of biological tissue function and dysfunction. However, the chemically complex matrix of different biological fluids complicates the assay of this important class of molecules. We introduce a method for characterizing the amino acid composition of submicroliter volumes of vitreous humor perfusates.

Determination of nitrate and nitrite in rat brain perfusates by capillary electrophoresis.

A fast and simple method for the direct, simultaneous detection of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in rat striatum has been developed using a capillary electrophoresis separation of low-flow push-pull perfusion samples. The method was optimized primarily for nitrite because nitrite is more important physiologically and is found at lower levels than nitrate. We obtained a complete separation of NO(2) (-) and NO(3) (-) in rat striatum within 1.

Solid-phase immunoassay detection of peptides from complex matrices without a separation.

A simple and sensitive solid-phase fluorescence immunoassay method was developed to detect peptides without separating them from a biological matrix. A near infrared fluorescence detection system was constructed for scanning analyte spots blotted onto protein binding membranes. Hydrophobic membranes were used with a modified vacuum spot blotting system to concentrate the peptide solution into a small area and the overall assay time was thus reduced by eliminating blocking steps.

Demonstration of low flow push-pull perfusion.

Methods to follow in vivo chemical composition provide information regarding the processes of intercellular communication. There is a need for methods that provide chemical information from small volumes of the central nervous system (CNS) without sacrificing neurochemical recovery. One method that offers potential for providing such information is push-pull perfusion.