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Tat-derived peptides have attracted much interest as molecular carriers for intracellular delivery as they incorporate specific attributes required for efficient cargo delivery to sub-cellular domains. Little is known, however, about intracellular trafficking and interactions of Tat peptide-tagged cargoes, although some in vitro studies have suggested the relevance of active processes in Tat peptide-driven nuclear translocation. These issues are addressed by comparing Tat peptide-induced transport properties with well-established passive diffusion and active import benchmarks in living cells. Specifically, we examine several constructs of increasing molecular weight (MW) both below and above the threshold for passive diffusion through the nuclear pore. The resulting sub-cellular localization is analyzed by confocal imaging, and construct intracellular dynamics is investigated by fluorescence recovery after photobleaching (FRAP) real-time imaging. Our experiments yield the characteristic transport parameters of Tat peptide intra-cytoplasm dynamics and nucleus/cytoplasm shuttling. These results allow us to elucidate the mechanism of Tat peptide-driven nuclear permeation, demonstrating that it crosses the nuclear envelope (NE) by passive diffusion. Finally, we discuss the limitations of this route in terms of acceptable cargo size.

Reactive oxygen species generating activity of tartrate-resistant acid phosphatase (TRACP) has been suggested to have several functions in TRACP expressing bone resorbing osteoclasts, macrophages, and dendritic cells. This work aimed to study the TRACP knock down phenotype in osteoclasts by using antisense DNA and RNA interference methods. Unexpectedly, both TRACP specific DNA oligonucleotides and siRNA molecules extensively increased the TRACP expression in human osteoclasts and monocytes. Toll-like receptor 9 (TLR9) is an immunity sensor for CpG motifs in DNA. We cultured bone marrow-derived osteoclast precursor cells from wild-type and TLR9-/- mice with CpG and non-CpG DNA oligonucleotides, and observed that the increased TRACP expression was sequence and TLR9 independent. In contrast, cells with increased TRACP activity showed decreased activity of tartrate-sensitive acid phosphatases. Conclusion: DNA oligonucleotides and RNA molecules extensively increase TRACP expression in monocyte-macrophage lineage. These results suggest a potential role of TRACP in pathogen recognition and in innate immunity. © 2007 Elsevier Inc. All rights reserved.

With the ultimate purpose of testing the hypothesis that, as shown in yeast mutants, any malfunction of DNA topoisomerase II might result in aberrant mitosis due to defective chromosome segregation, we have chosen three chemicals of different nature, recently reported to catalytically inhibit the enzyme. The endpoint selected to assess any negative effect on the ability of topoisomerase II to properly carry out decatenation of fully replicated chromosomes in the G2/M phase of the cell cycle was the presence of metaphases showing diplochromosomes as a result of endoreduplication, i.e. two successive rounds of DNA replication without intervening mitosis. The anti-topoisomerase drugs selected were the anthracycline antibiotic and antineoplastic agent aclarubicin, the respiratory venom sodium azide, and 9-aminoacridine, a chemical compound with planar topology capable of intercalation between DNA bases. Our results show that the three chemicals tested are able to induce endoreduplication to different degrees. These observations seem to lend support to the proposal that topoisomerase II plays a central role in chromosome segregation in mammalian cells.

Zanamivir (4-guanidino-Neu5Ac2en [4-GU-DANA]) inhibits not only the neuraminidase activity but also the receptor interaction of the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN), blocking receptor binding and subsequent fusion promotion. All activities of the HPIV3 variant ZM1 HN (T193I/I567V) are less sensitive to 4-GU-DANA's effects. The T193I mutation in HN confers both increased receptor binding and increased neuraminidase activity, as well as reduced sensitivities of both activities to 4-GU-DANA inhibition, consistent with a single site on the HN molecule carrying out both catalysis and binding. We now provide evidence that the HPIV3 variant's resistance to receptor-binding inhibition by 4-GU-DANA is related to a reduced affinity of the HN receptor-binding site for this compound as well as to an increase in the avidity of HN for the receptor. Newcastle disease virus (NDV) HN and HPIV3 HN respond differently to inhibition in ways that suggest a fundamental distinction between them. NDV HN-receptor binding is less sensitive than HPIV3 HN-receptor binding to 4-GU-DANA, while its neuraminidase activity is highly sensitive. Both HPIV3 and NDV HNs are sensitive to receptor-binding inhibition by the smaller molecule DANA. However, for NDV HN, some receptor binding cannot be inhibited. These data are consistent with the presence in NDV HN of a second receptor-binding site that is devoid of enzyme activity and has a negligible, if any, affinity for 4-GU-DANA. Avidity for the receptor contributes to resistance by allowing the receptor to compete effectively with inhibitors for interaction with HN, while the further determinant of resistance is the reduced binding of the inhibitor molecule to the binding pocket on HN. Based upon our data and recent three-dimensional structural information on the HPIV3 and NDV HNs, we propose mechanisms for the observed sensitivity and resistance of HN to receptor-binding inhibition and discuss the implications of these mechanisms for the distribution of HN functions.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising natural anticancer therapeutic agent because through its “death receptors”, TRAIL-R1 and TRAIL-R2, it induces apoptosis in many transformed tumor cells, but not in the majority of normal cells. Hence, agonistic compounds directed against TRAIL death receptors have the potential of being excellent cancer therapeutic agents, with minimal cytotoxicity in normal tissues. Here, we report the selection and characterization of a new single-chain fragment variable (scFv) to TRAIL-R2 receptor isolated from a human phage-display library, produced as minibody (MB), and characterized for the in vitro anti-leukemic tumoricidal activity. The anti-TRAIL-R2 MB2.23 efficiently and specifically bound to membrane-associated TRAIL-R2 on different leukemic cell lines and could act as a direct agonist in vitro, initiating apoptotic signaling as well as complement-dependent cytotoxicity and antibody-dependent cell cytotoxicity, providing a rationale for further investigations of MB2.23 in anticancer therapy.