Researchers pave the way to protein therapy in humans

a compound whose uptake by cells could be monitored. The compound was a dye called fluorescein, which turns green when exposed to fluorescent lighting. It normally doesn't enter cells because of its size-- 2,000 daltons compared with the 500 dalton limit placed on most drugs.

The researchers injected mice with this combined PTD-fluorescein protein and isolated cells from the animals' blood and spleen. All the cells fluoresced green when checked 20 minutes after the injection. Cells in muscle and brain tissue also had soaked up the combined protein. "It was very encouraging to discover that we could get a mouse with an entirely green brain," Dowdy says, noting that the blood-brain barrier, a layer of cells lining the brain's blood vessels, normally prevents most proteins from entering.

Dowdy and colleagues then linked a bacterial enzyme to the PTD and fluorescein. Their fluorescent analysis revealed that the 120,000 dalton enzyme, beta-galactosidase, entered all the cell types tested.

Beta-galactosidase was chosen because its activity could reveal whether an enzyme could continue to function after it had been transported into cells by the PTD. Cells take up proteins better if they are at least partially unfoled, as was true for the enzyme.

Dowdy's team tested whether beta-galactosidase trapped inside cells of injected mice converted the enzyme's clear chemical target into a blue dye. A vibrant blue image of the kidney of the first mouse tissue they evaluated is pinned on a bulletin board above Dowdy's desk. "Once we got this first result, we realized that the protein would be biologically active in the rest of the body," Dowdy says.

The liver, lung, and other tissues of the injected mice also turned blue when exposed to the enzyme's target. The animals' entire brains also stained blue within four hours of injection, indicating that a lot of the bacterial enzyme had refolded there by then. Importantly, the

Contact: Barbra Rodriguez
Washington University School of Medicine

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