A rapid diffusion immunoassay in a T-sensor

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 microl of reagents and sample, and is applicable to a wide range of analytes.     

Testosterone delivery using glutamide-based complex high axial ratio microstructures

Complex high axial ratio microstructures (CHARMs) were evaluated for delivery of testosterone in vivo. Methods to incorporate testosterone included noncovalent mixing and covalent attachment of testosterone to the lipid to form a prodrug monomer. When prepared by covalent attachment, testosterone-loaded CHARMs were resistant to in vitro spontaneous hydrolysis; when injected into rats, testosterone was released with biphasic kinetics consisting of a burst followed by a much slower phase. Some CHARM material associated with testosterone persisted at the site of injection for at least 9 days.     

Statistical mechanical analysis of Raman spectroscopic order parameter changes in pressure-induced lipid bilayer phase transitions.

The statistical mechanical cluster theory of Fisher as applied by Kanehisa and Tsong to phospholipid bilayers is modified to describe the effects of hydrostatic pressure on the state of an aqueous dispersion of the phospholipid dipalmitoyl phosphatidylcholine. A high pressure Raman scattering cell has been built to obtain the Raman spectra of aqueous dispersions of phospholipids as a function of the applied hydrostatic pressure from 0 to 100 atmospheres. Predicted thermal and pressure-induced phase transitions are compared with an experimentally obtained Raman order parameter derived from the ratio of two bands in the C-H stretching region of the Raman spectrum of the sample. The parameters of the theory are adjusted to obtain a satisfactory fit of the Raman order parameter versus temperature. The theory is then found to give an excellent prediction of the observed pressure dependence of the Raman order parameter with no changes in the adjustable parameters. The implications of the success of the theoretical fit is discussed. Particularly of interest is the rather high value of the critical temperature, Tc, for lipid bilayers which is predicted by the model.     

Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein.

We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein.     

Extracts of black and brown rice powders improve hepatic lipid accumulation via the activation of PPARα in obese and diabetic model mice

Rice powder extract (RPE) from black and brown rice (Oryza sativa L. indica) improves hepatic lipid accumulation in obese and diabetic model mice via peroxisomal fatty acid oxidation. RPE showed PPARα agonistic activity which did not differ between black and brown RPE despite a higher anthocyanin content in black RPE.