A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48 -60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48 -60). These included such peptides as the D-amino acid-and arginine-substituted Tat-(48 -60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4. These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences. Moreover, these peptides were able to be internalized even at 4°C. These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis. Using (Arg) n (n ‫؍‬ 4 -16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n ϳ 8) for the efficient translocation.Recently, methods have been developed for the delivery of exogenous proteins into living cells with the help of membranepermeable carrier peptides such as HIV-1 1 Tat-(48 -60) and Antennapedia-(43-58) (1-11). By genetically or chemically hybridizing these carrier peptides, the efficient intracellular delivery of various oligopeptides and proteins was achieved. One of the most amazing examples is the Tat-␤-galactosidase fusion protein (4), which has a molecular mass as high as 120 kDa.Intraperitoneal injection of the protein resulted in delivery of the protein with ␤-galactosidase activity to various tissues in mice, including the brain. The peptide-mediated approaches would allow the incorporation of peptides containing unnatural amino acids or nonpeptide molecules such as fluorescence probes. These methods would become powerful tools not only for therapeutic purposes as an alternative to gene delivery, but also for the understanding of the mechanisms behind fundamental cellular events, such as signal transduction and gene transcription.Besides the potential of Tat-(48 -60) as a protein carrier, the internalization mechanism of the peptide attracted our interest. For example, Tat-(48 -60) (GRKKRRQRRRPPQ) is a highly basic and hydrophilic peptide, which contains 6 arginine and 2 lysine residues in its 13 amino acid residues. However, the peptide was reported to be translocated through the cell membranes in 5 min at a concentration of 0.1 M (2). Internalization of the peptide was not inhibited even at 4°C. The peptide is less toxic to cells than other basic membrane-interacting agents. The above features suggested that the internalization mechanism of Tat-...

Basic peptides derived from the HIV-1 1 Tat protein (Tat-(48 -60)) and Drosophila Antennapedia protein (Antp-(43-58)) have been reported to have the ability to translocate through the cell membranes and to carry exogenous molecules into the cytoplasm and nucleus (1-13). A 119-kDa protein, ␤-galactosidase, genetically fused with the former peptide segment, was successfully carried into various tissues in mice including the brain via intraperitoneal injection (6). The 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside (X-gal) staining of the tissues indicated that the fusion protein was delivered in its active form. Oligo-DNAs and metal chelates were also brought into cells using the Tat-derived peptide (4, 7). Such a method to deliver bioactive molecules into cells using membrane-permeable peptides has a great potential for therapeutic fields.We have recently demonstrated that not only Tat-(48 -60) and Antp-(43-58) but also various arginine-rich RNA-or DNAbinding peptides such as HIV-1 Rev-(34 -50) and flock house virus (FHV) coat-(35-49) were membrane-permeable and have the ability to bring exogenous protein into cells (14). Even octaarginine (Arg 8 ) gave similar results based on the fluorescence microscopic observation of the fluorescein-labeled peptides (14, 15). These peptides seem to have other similarities in translocation, namely facile internalization within 5 min, little uptake inhibition at 4°C, and localization in the nucleus and cytosol. The above results suggested the possible existence of a ubiquitous mechanism for the internalization of the argininerich peptides. Since there were no sequence similarities among these peptides except that they had several arginine residues, arginine seemed to be the key amino acid for membrane permeability.Despite the great potential of the arginine-rich peptides as carriers of proteins, nucleic acids, and other bioactive compounds, little is known about the mechanism of their internalization. Involvement of the cell surface heparan sulfate (HS) and low density lipoprotein receptor-related protein (LRP) was suggested in the translocation of the full-length Tat protein (16,17). The addition of HS and the inhibitor of LRP to the culture medium produced a significant decrease in the cellular uptake of the protein. However, the uptake of the full-length Tat protein suffered a certain decrease at 4°C (16), and some energydependent endocytosis pathway seemed to play a significant role in the internalization of the Tat protein. These results suggested that the mechanisms of internalization of the Tat-(48 -60) peptide and the full-length Tat protein may not be completely parallel. Actually, the importance of the "core" domain (Tat-(37-48)) of the Tat protein has been claimed for the LRP-dependent internalization pathway (16).We have pointed out that many arginine-rich peptides showed very similar characteristics in translocation with HIV-1 Tat-(48 -60) (14). In addition to the translocation mechanisms of these arginine-rich peptides, it is unclear whether these peptides share...

Membrane-permeable arginine-rich peptides, such as HIV-1 Tat-(48-60), HIV-1 Rev-(34-50), and flock house virus (FHV) coat-(35-49), have been shown to possess the ability to transfect COS-7 cells with luciferase-coding plasmid as efficiently as polyarginine (MW 5000-15 000) and polylysine (MW 9800). Not only these virus-derived cationic peptides but also oligoarginines of 4-16 residues were found to be able to transfect cells. In the case of the Tat, FHV, and octaarginine peptides, N-terminal stearylation of the peptides increases the transfection efficiency by approximately 100 times to reach the same order of magnitude as that of LipofectAMINE, one of the most efficient commercially available transfection agents. Also, a certain correlation was observed between the transfection efficiency of stearyl-(Arg)n peptides (stearyl-Rn: n = 4, 8, 12, 16) and the membrane permeability of the corresponding (Arg)n peptides (Rn).

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A basic peptide derived from HIV-1 Tat has been reported to have the ability to translocate through cell membranes and to bring exogenous proteins into cells. We have demonstrated that these features could be observed among many arginine-rich peptides, and the presence of a ubiquitous internalization mechanism for arginine-rich oligopeptides has been suggested. In this report, we report that these features are also applicable to the peptides having branched-chain structures. Peptides that have arginine residues on four branched chains (R(n))(4) [n (number of arginine residues)= 0-6] were prepared. Fluorescence microscopic observation revealed that the (R(2))(4) peptide exhibited the most efficient translocation. The dependence on the number of arginine residues of the translocation efficiency and cellular localization was also observed for the branched-chain peptides as was seen in the linear peptides. Quite interestingly, efficient translocation was also recognized in the (RG(3)R)(4) peptide, where three glycine residues intervened between two arginine residues on each chain of (R(2))(4). The results strongly suggested that a linear structure was not indispensable for the translocation of arginine-rich peptides and that there could be considerable flexibility in the location of the arginine residue in the molecules.

Background: IL-6 signaling is a key component of inflammatory diseases.Results: Modified DNA aptamers that inhibit IL-6 signaling were discovered and optimized.Conclusion: Modified aptamers are stable in serum and block the interaction of IL-6 with its receptor IL-6Rα.Significance: Modified aptamers are a new class of antagonist with properties potentially suitable for clinical treatment of inflammation.

The ability to flexibly adjust movement timing is important for everyday life. Although the basal ganglia and cerebellum have been implicated in monitoring of supra- and sub-second intervals, respectively, the underlying neuronal mechanism remains unclear. Here, we show that in monkeys trained to generate a self-initiated saccade at instructed timing following a visual cue, neurons in the caudate nucleus kept track of passage of time throughout the delay period, while those in the cerebellar dentate nucleus were recruited only during the last part of the delay period. Conversely, neuronal correlates of trial-by-trial variation of self-timing emerged earlier in the cerebellum than the striatum. Local inactivation of respective recording sites confirmed the difference in their relative contributions to supra- and sub-second intervals. These results suggest that the basal ganglia may measure elapsed time relative to the intended interval, while the cerebellum might be responsible for the fine adjustment of self-timing.

Peptide-mediated protein delivery into living cells has been attracting our attention. Among the peptides that have been reported to have carrier activity, the one from the human immunodeficient virus (HIV)-1 Tat has been most often used for the introduction of exogenous macromolecules into cells. We have shown that not only the Tat peptide, but also various arginine-rich peptides showed very similar characteristics in translocation, and the possible presence of ubiquitous internalization mechanisms among the arginine-rich peptides has also been suggested. These arginine-rich peptides includes ones derived from HIV-1 Rev and flock house virus coat proteins. The linear- and branched-chain peptides containing approximately 8 residues of arginine also show a similar ability. In this review, we present the structural variety of membrane permeable peptides and provide a survey of the findings on the translocation of these peptides through the cell membranes.