It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 3 Ala (
The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. Fluorescence activated cell sorting (FACS) analysis and confocal microscopy characterization showed that TAT-SA is internalized by Jurkat T-cells and NIH 3T3 cells alone and when complexed to phycoerythrin, whereas the native streptavidin is not. Additionally, biotinylated alkaline phosphatase is successfully internalized and retains its activity when complexed to TAT-SA and incubated with Jurkat T-cells. Confocal microscopy suggested, however, that internalized TAT-SA and TAT-SA complexes were largely compartmentalized in vesicular compartments, rather than freely diffusing in the cytoplasmic compartment. To effect cytoplasmic delivery, the endosomal releasing polymer, poly(propylacrylic acid) (PPAA), was biotinylated and complexed to TAT-SA. Endosomal release and cytoplasmic delivery of fluorescently labeled TAT-SA complexes with PPAA was shown by the diffuse distribution of fluorescent protein in the cytoplasm. Taken together, these results demonstrate that TAT-SA can be used to direct intracellular delivery of large biotinylated cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired.
We tested the effect of anti-sense RNA on the replication of avian retroviruses in cultured cells. The replication of a recombinant retrovirus carrying a neomycin resistance gene (neor) in the anti-sense orientation was blocked when the cells expressed high steady-state levels of RNA molecules with neor sequences in the sense orientation, i.e., complementary to the viral sequence. Viral DNA bearing neor sequences was not detected specifically in host cells where this anti-sense RNA inhibition of viral replication occurred. These observations suggest that anti-sense RNA inhibition may be a useful strategy for the inhibition of retroviral infections.The anti-sense RNA-mediated inhibition of gene expression (5) has potential as a tool for genetic analysis (1,8,10,11) and has also been suggested as a novel approach to inhibiting viral infections (5; E. C. M. Mariman, Letter, Nature (London) 318:414, 1985; R. Tellier and J. M. Weber, Letter, Nature (London) 318:414, 1985 were killed completely in 5 to 7 days by the same concentration of G418.The neor sequences of N-10 and aN-10 virion RNAs were detected selectively with 32P-labeled single-stranded neor RNA probes complementary to sense and anti-sense neor sequences, respectively, prepared by using an SP6-T7 plasmid system (pGEM4; Promega Biotec, Madison, Wis.). Both sense and anti-sense neor inserts were equally stable in viral RNA upon chronic passage in both chicken embryo fibroblasts and quail cells. Figure 2, panel I, shows a dot blot analysis of neor sequences in N-10 and aN-10 viral stocks grown on quail cells. The level of neor sequences relative to total viral RNA was comparable in both stocks. RNA blot hybridization analysis with a viral sequence probe of N-10 and aN-10 viral stocks from chronic passages also showed the accumulation of virions which had deleted neor inserts. These deleted genomes never amounted to more than 50 to 65% of the total viral stock (Fig. 2, panel III), in comparison with the 10-fold or greater excess of analogous transformation-defective deletions common in wild-type stocks of chronically passaged RSV (7).The host cell system we used to test possible mutual anti-sense effects on both viral replication and target host cell gene expression was derived from a chemically transformed Japanese quail cell line, QT35 (9), which had been made G418 resistant by transfection with a defective retroviral vector carrying the neor gene. QT35 cells and neor derivatives were propagated under the same conditions as those described earlier for chicken embryo fibroblasts, except that the medium was supplemented with 1% dimethyl sulfoxide. Figure 3 shows a slot blot analysis of virion RNA produced by cultures of three clones of G418-resistant QT35 derivatives infected with either N-10 or aN-10. The three clones were derived from three independent transfections of QT35 with constructs that express neor from an avian retroviral promoter. The steady-state levels of neor transcripts in each of the clones was determined by neor hybridization by slot blot an...
Traditional cancer registries have often been siloed efforts, established by single groups with limited objectives. There is the potential for registry data to support a broad range of research, audit and education initiatives. Here, we describe the establishment of a series of comprehensive cancer registries across the spectrum of common solid cancers. The experience and learnings of each registry team as they develop, implement and then use collected data for a range of purposes, that informs the conduct and output of other registries in a virtuous cycle. Each registry is multi-site, multi-disciplinary and aims to collect data of maximal interest and value to a broad range of enquiry, which would be accessible to any researcher with a high-quality proposal. Lessons learnt include the need for careful and continuous curation of data fields, with regular database updates, and the need for a continued focus on data quality. The registry data as a standalone resource has supported numerous projects, but linkage with external datasets with patients in common has enhanced the audit and research potential. Multiple projects have linked registry data with matched tissue specimens to support prognostic and predictive biomarker studies, both validation and discovery. Registry-based biomarker trials have been successfully supported, generating novel and practice-changing data. Registry-based clinical trials, particularly randomised studies exploring the optimal use of available therapy options are now complementing the research conducted in traditional clinical trials. More recent projects supported by the registries include health economic studies, personalised patient education material, and increased consumer engagement, including consumer entered data.
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