The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to 3-galactosidase, horseradish peroxidase, RNase A, and domain Ill ofPseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-f-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages.Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.The potential for intracellular therapeutic use of proteins, peptides, and oligonucleotides has been limited by the impermeable nature of the cell membrane to these compounds. A wide variety of methods have been proposed for the delivery of proteins and other macromolecules into living cells for either experimental or therapeutic uses, including microinjection, scrape loading, electroporation, liposomes (1-7), bacterial toxins (8)(9)(10), and receptor-mediated endocytosis (11-16). Most of these methods are either inefficient or time-consuming, cause appreciable cell death, or result in uptake into intracellular vesicles without efficient cytoplasmic delivery. Several approaches (15-18) rely on binding of macromolecules to the cell surface, followed by internalization via the endocytic route. However, since proteins that have entered this pathway remain enclosed within lipid vesicles, they do not have access to the cell cytoplasm, most typically the target environment. It seems reasonable to assume that the escape from endocytic vesicles is the ratelimiting step in achieving true cellular delivery, yet many assays fail to measure this.Recently the Tat protein from human immunodeficiency virus 1 (HIV-1) was shown to enter cells when added exogenously (19,20). Tat protein can simply be added to medium at concentrations as low as 1 nM, and biological responses can be detected. Since the assay for this process was the transactivation of a transfected reporter gene, activity reflects those molecules that had been targeted to the nucleus, presumably after cytoplasmic delivery. The mechanism by which Tat traverses a membrane and the precise intracellular location of this event remain unclear. However, Tat binds efficiently to cells, with >107 sites per cell and then is internalized by an adsorptive endocytosis process (20). In characterizing the uptake process using iodinated Tat, Mann and Frankel (20) observed that only 3% of the Tat becameThe publication costs of this article were...
Towards the end of 2006, routine Electronic Distance Measurement (EDM) prism monitoring highlighted rapid acceleration of one prism. At this stage, there were no visible signs of failure close to the prism and all other monitoring points in the vicinity could no longer be read. The prism itself was situated on a berm that was no longer accessible. A week later the prism was still accelerating with the only evidence of wall movements being two sets of cracks, one 30 m below the prism near the pit floor and the other about 50 m above the prism, suggesting the possibility of a large scale wall failure. A GroundProbe Slope Stability Radar was available for a few days and was deployed to scan a large section of wall. After a day of radar monitoring it was apparent that movement was occurring over an area approximately 265 m wide and nearly 100 m high. Movement rates were up to 30 mm per day and accelerating rapidly. The eastern half of the pit below the affected area was closed off and failure commenced two days later. The radar remained at the site for the initial part of the failure allowing correlation of movement rates and magnitudes with those recorded by prism monitoring.It appears that the failure mechanism is a combination of sliding on a fault plane, sliding on the contact between the Footwall Zone and the Mount McRae Shale and rock mass failure through the Dales Gorge Member to produce significant floor heave at the base of the failure.
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