{"doi":"10.1074/jbc.m208762200","title":"Translocation of Analogues of the Antimicrobial Peptides Magainin and Buforin across Human Cell Membranes","abstract":null,"journal":"Journal of Biological Chemistry","year":2003,"id":588408,"datarank":10.15515405013675,"base_score":5.351858133476067,"endowment":5.351858133476067,"self_citation_contribution":0.8027787200214102,"citation_network_contribution":9.35237533011534,"self_endowment_contribution":0.8027787200214102,"citer_contribution":9.35237533011534,"corpus_percentile":null,"corpus_rank":null,"citation_count":210,"citer_count":200,"citers_with_citation_signal":184,"citers_with_endowment":184,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1505325,"name":"Akinori Chikushi","orcid":null,"position":1,"is_corresponding":false},{"id":1505326,"name":"Kyung-Kwon Lee","orcid":null,"position":2,"is_corresponding":false},{"id":1505327,"name":"Shin Yonehara","orcid":null,"position":3,"is_corresponding":false},{"id":1505328,"name":"Katsumi Matsuzaki","orcid":null,"position":4,"is_corresponding":false},{"id":1505324,"name":"Kenta Takeshima","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Translocation of Analogues of the Antimicrobial Peptides Magainin and Buforin across Human Cell Membranes","abstract":"Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47–57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 μm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide. Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47–57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 μm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide. bis(sulfosuccinimidyl)suberate Dulbecco's modified Eagle's medium fetal bovine serum fluoren-9-ylmethoxycarbonyl green fluorescent protein 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide phosphate-buffered saline Cationic antimicrobial peptides play important roles in innate immunity (1Hancock R.E.W. Lehrer R. Trends Biotechnol. 1998; 16: 82-88Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar, 2Tossi A. Sandri L. Giangaspero A. Biopolymers. 2000; 55: 4-30Crossref PubMed Scopus (1046) Google Scholar, 3Ganz T. Lehrer R.I. Lohner K. Development of Novel Antimicrobial Agents: Emerging Strategies. Horizon Scientific Press, Wymondham2001: 139-147Google Scholar, 4Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6798) Google Scholar). Broad antimicrobial spectra, highly selective toxicity, as well as difficult resistance development make these compounds promising candidates as novel antibiotics for clinical use. Intensive investigations on peptide-bacteria interactions have shown that some peptides kill bacteria mainly by permeabilizing the membranes, whereas others target intracellular compounds, such as nucleic acids, although recent studies have suggested that many peptides possess both modes of action (2Tossi A. Sandri L. Giangaspero A. Biopolymers. 2000; 55: 4-30Crossref PubMed Scopus (1046) Google Scholar, 4Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6798) Google Scholar, 5Hancock R.E.W. Rozek A. FEMS Microbiol. Lett. 2002; 206: 143-149Crossref PubMed Google Scholar). Magainin 2 (Table I) isolated from the skin of the African clawed frog Xenopus laevis is a representative membrane-acting peptide composed of 23 amino acid residues (6Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5449-5453Crossref PubMed Scopus (2031) Google Scholar, 7Matsuzaki K. Biochim. Biophys. Acta. 1998; 1376: 391-400Crossref PubMed Scopus (538) Google Scholar, 8Matsuzaki K. Biochim. Biophys. Acta. 1999; 1462: 1-10Crossref PubMed Scopus (855) Google Scholar). Addition of the peptide to bacterial cells immediately induces the permeabilization of both outer and inner membranes, leading to cell death (9Matsuzaki K. Sugishita K. Harada M. Fujii N. Miyajima K. Biochim. Biophys. Acta. 1997; 1327: 119-130Crossref PubMed Scopus (315) Google Scholar, 10Park C.B. Kim H.S. Kim S.C. Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (694) Google Scholar). A fluorescent magainin peptide mainly associates with bacterial membranes and does not enter cells (10Park C.B. Kim H.S. Kim S.C. Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (694) Google Scholar), although native magainin 2 was recently reported to enter cells (11Haukland H.H. Ulvante H. Sandvik K. Vorland L.H. FEBS Lett. 2001; 508: 389-393Crossref PubMed Scopus (123) Google Scholar). An example of the peptides targeting intracellular substances is buforin 2, which was discovered in the stomach of the Asian toad Bufo bufo gargarizans (12Park C.B. Kim M.S. Kim S.C. Biochem. Biophys. Res. Commun. 1996; 218: 408-413Crossref PubMed Scopus (244) Google Scholar). A fluorescent buforin 2 rapidly enters bacterial cells without perturbing the barrier properties of the membranes, binding to DNA and RNA (10Park C.B. Kim H.S. Kim S.C. Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (694) Google Scholar).Table IAmino acid sequences of the peptides used in this studyPeptideSequence1-aThe underlined residues show differences from the original peptide.Charge1-bApproximate charge at physiological pH (7.4).Magainin 2GIGKFLHSAKKFGKAFVGEIMNS+3MG2dGIGKFLHSAKKWGKAFVGQIMNC (Texas Red)-amide+5Buforin 2TRSSRAGLQFPVGRVHRLLRK+7BF2dTRSSRAGLQWPVGRVHRLLRKGGC (Texas Red)-amide+7Tat (47–57)Texas Red-YGRKKRRQRRR+81-a The underlined residues show differences from the original peptide.1-b Approximate charge at physiological pH (7.4). Open table in a new tab On the other hand, interactions of cationic antimicrobial peptides with human cells are not well characterized. The information on cell penetration available is limited to dermaseptins (13Ghosh J.K. Shaool D. Guillaud P. Cicéron L. Mazier D. Kustanovich I. Shai Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 14Krugliak M. Feder R. Zolotarev V.Y. Gaidukov L. Dagan A. Ginsburg H. Mor A. Antimicrob. Agents Chemother. 2000; 44: 2442-2451Crossref PubMed Scopus (105) Google Scholar, 15Feder R. Nehushtai R. Mor A. Peptides. 2001; 22: 1683-1690Crossref PubMed Scopus (46) Google Scholar), PR-39 (16Chan Y.R. Gallo R.L. J. Biol. Chem. 1998; 273: 28978-28985Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and protegrin 1 (17Drin G. Temsamani J. Biochim. Biophys. Acta. 2002; 1559: 160-170Crossref PubMed Scopus (38) Google Scholar), although many investigators examined hemolytic activity (9Matsuzaki K. Sugishita K. Harada M. Fujii N. Miyajima K. Biochim. Biophys. Acta. 1997; 1327: 119-130Crossref PubMed Scopus (315) Google Scholar, 18Kobayashi S. Takeshima K. Park C.B. Kim S.C. Matsuzaki K. Biochemistry. 2000; 39: 8648-8654Crossref PubMed Scopus (183) Google Scholar) and cytotoxicity (19Cruciani R.A. Barker J.L. Zasloff M. Chen H.-C. Colamonici O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3792-3796Crossref PubMed Scopus (361) Google Scholar, 20Ohsaki Y. Gazdar A.F., H.-C., C. Johnson B.E. Cancer Res. 1992; 52: 3534-3538PubMed Google Scholar, 21Baker M.A. Maloy W.L. Zasloff M. Jacob L.S. Cancer Res. 1993; 53: 3052-3057PubMed Google Scholar, 22Haimovich B. Tanaka J.C. Biochim. Biophys. Acta. 1995; 1240: 149-158Crossref PubMed Scopus (35) Google Scholar). In contrast, short Arg-rich cationic peptides have recently been shown to enter mammalian cells by an energy (ATP)-independent, non-endocytotic pathway and have been utilized as vectors for delivering membrane-impermeable drugs, oligonucleotides, and proteins into cells (23Lindgren M. Hällbrink M. Prochiantz A. Langel Ü. Trends Pharmacol. Sci. 2000; 21: 99-103Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). Model membrane studies revealed that the membrane-acting magainin peptide also translocates across lipid bilayers (24Matsuzaki K. Murase O. Fujii N. Miyajima K. Biochemistry. 1995; 34: 6521-6526Crossref PubMed Scopus (313) Google Scholar, 25Matsuzaki K. Murase O. Miyajima K. Biochemistry. 1995; 34: 12553-12559Crossref PubMed Scopus (170) Google Scholar), although less effectively than buforin 2 (18Kobayashi S. Takeshima K. Park C.B. Kim S.C. Matsuzaki K. Biochemistry. 2000; 39: 8648-8654Crossref PubMed Scopus (183) Google Scholar). Therefore, cationic antimicrobial peptides, including those of the membrane-permeabilizing class, can penetrate human cells. However, although this issue has rarely been investigated, it is important for the development of novel peptides for therapeutic use as well as for understanding the roles of antimicrobial peptides in innate immunity. In this study, we investigated the translocation of fluorescent-labeled analogues of magainin 2 and buforin 2 across human cell membranes in comparison with the Tat-(47–57) peptide (Table I), a representative Arg-rich cell-penetrating peptide. Both tumor (human cervical carcinoma HeLa) and normal (human fibroblastic TM12, Ref. 26Iwahana H. Yanagisawa K. Ito-Kosaka A. Kuroiwa K. Tango K. Komatsu N. Katashima R. Itakura M. Tominaga S. Eur. J. Biochem. 1999; 264: 397-406Crossref PubMed Scopus (191) Google Scholar) cells were used because magainin peptides are known to exhibit selective toxicity for tumor cells (19Cruciani R.A. Barker J.L. Zasloff M. Chen H.-C. Colamonici O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3792-3796Crossref PubMed Scopus (361) Google Scholar, 20Ohsaki Y. Gazdar A.F., H.-C., C. Johnson B.E. Cancer Res. 1992; 52: 3534-3538PubMed Google Scholar, 21Baker M.A. Maloy W.L. Zasloff M. Jacob L.S. Cancer Res. 1993; 53: 3052-3057PubMed Google Scholar, 27Soballe P.W. Maloy W.L. Myrga M.L. Jacob L.S. Herlyn M. Int. J. Cancer. 1995; 60: 280-284Crossref PubMed Scopus (68) Google Scholar). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides crossed cell membranes through different mechanisms. Furthermore, the buforin peptide, covalently attached to the 28-kDa green fluorescent protein also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. The reagents for peptide synthesis were purchased from Applied Biosystems (Foster City, CA). All fluorescent probes for peptide labeling were obtained from Molecular Probes (Eugene, OR). Dulbecco's modified Eagle's medium (DMEM) was supplied by nacalai tesque (Kyoto, Japan). Bis(sulfosuccinimidyl)suberate (BS3)1 linker and MTT were purchased from Pierce and Sigma, respectively. GFP was supplied by Upstate     All other  from    were of   The peptide was  by a  fluoren-9-ylmethoxycarbonyl     on an Applied Biosystems Model  peptide  as    K. Murase O.  H.  S. Fujii N. Miyajima K. Biochemistry.    PubMed Scopus  Google Scholar).  labeling of the peptides was  as    from the  the   of the magainin peptide  the buforin   in       was  with      in    The   of the Tat peptide on the  was  with      and   with  and  and   from the  The  of the  peptides was  by        and     HeLa and TM12 cells were  in  with  fetal bovine serum  The cells were     at   The cells    were  on     and      of the  the cells were  to peptide      in  with   for   at      The peptide  were   and the cells were  three  with phosphate-buffered saline  of pH  and  with   of   in  The protein  of the cells was  by the   protein    CA). The internalized   peptides were   with a    at an   of   and an   of   The  were  as   with the  that the cells were  for  min  at    in the  of     in  with   at   The   peptide  was  at         The cells    were  on        and   with peptide  as           was  to the peptide    three  with  the cells were  with    in  for  min at  temperature,   with  and  in a           was  on a      The cells    were  in   and   in       of the  the cells were  with   of peptide  in the medium for   at     of a   MTT  in  was   and the cells were   for    the  MTT was   in   of    in    The  cell  was  by      for     for  cells.  and    at  and   The   of the buforin peptide  in      was  with     in    were  by  The  peptide   and GFP   were  in       the protein of the amino  linker    in      at a concentration of 10  was  to the      the  was  with  1  The  were  by  and  the  reagents in the  were  from the   by        The   of attached buforin peptide   GFP was  as  by   The magainin 2    was  as  The translocation  upon the  of a pore (24Matsuzaki K. Murase O. Fujii N. Miyajima K. Biochemistry. 1995; 34: 6521-6526Crossref PubMed Scopus (313) Google Scholar). The translocation  is known to be  by  the    which  the intermediate pore  K.  A. Murase O. Sugishita K. Fujii N. Miyajima K. Biochemistry. 1997;   PubMed Scopus  Google Scholar). Therefore, the   of magainin 2 was  with  and the   was     the charge by    which is  and has a   than other  used  fluorescent  and is  to   was  to the    at the   The   was for   of the  peptide by   at   The   to the buforin 2   was for the   The    was  to     the   in   In this study,   was covalently attached to the peptide for   The Tat-(47–57) peptide  the   for the cellular uptake of the human   Tat protein  of the         K.    L.   Proc. Natl. Acad.  U. S. A. 2000;   PubMed Scopus  Google Scholar). The   of the peptide was also  with this  The labeling     on    also  a   with the  at the   The concentration and time dependence of peptide uptake were   to those of   not  The fluorescent peptides   were  with HeLa  TM12 cells for  min at     and the intracellular  of the peptides were examined by        translocated across the cell membranes of both cells   A and  and was     the cells  for the  in which the peptide was less    be    by  with the     1    were also obtained at    1  In  to the magainin  the   of   1  and Tat-(47–57)  1  were similar to those in the  The  with    that   entered the   1  The uptake of these peptides was also not       The  of   the cell   with the other two    that  peptide  were not    we  the   of HeLa cells  with  as in  1 A. The   was  from  to  in the    of   at an  of     are shown in      was   in the   of the  In  to     on the cellular uptake of the peptides are  for the  of translocation mechanisms.    has  been utilized for this       K.    L.   Proc. Natl. Acad.  U. S. A. 2000;   PubMed Scopus  Google Scholar). An     was used in this  The  of peptide   was   after cell   the  time, the  of cellular proteins was  as a  of the  of cells. The  of peptide uptake   of cellular protein  a   at   is  as a  of peptide concentration for HeLa and TM12 cells in   A  respectively.  these   the  of peptide  to the cell  it is  from  1 and 2 that  peptide  were  The uptake of  into both cells was  cooperative with  to peptide concentration    The      of     and     for HeLa and TM12 cells,   not  In contrast, the uptake of  was  less concentration-dependent    The Tat-(47–57) peptide was  into the cells   to the peptide concentration of at        are known to be  at    by ATP      J.  Biol.    PubMed Scopus  Google Scholar,  Y.  M.  I.  J.  T.  A.  J.  1998;   Google Scholar,  S.       1997;   PubMed Scopus  Google Scholar). Open    A show the peptide uptake by HeLa cells at    to that at    The uptake of  was  by  whereas those of  and Tat-(47–57) were  to   in   A show the  of   which  ATP synthesis by    on peptide  The  of  was    by the    that of  was not  by the  In contrast, the     the concentration dependence of Tat uptake     a  was not  for   not    A and  show the time dependence of peptide uptake by HeLa and TM12 cells, respectively.    the uptake  was  A   was  for the   A      which was  at  min. The time course of   was  different  The uptake was  and  the  at 10  and    In  to these antimicrobial peptides, the Tat-(47–57) peptide was    at   to 120 min  The  of the peptides without   labeling were  by the  MTT  which    of cells and is  used as a  of cell  The   is  as a  of the peptide concentration in   In the  of  the cell  was    10 and   for HeLa   and TM12 cells   respectively. In contrast,  was   at   to   for both cells   Tat-(47–57)   the cell  was  as a  of peptide concentration  the  of  to  The  of any significant cytotoxicity  that the buforin peptide is a promising  as a vector for delivering  into cells.   this   was  with GFP   by use of the      A  that GFP  did not enter HeLa cells. In contrast, the  was internalized into the cells, as  by both GFP   and         The  was   within the cells and  the   to the vector peptide  1  The uptake     than that of      and  at       in the  of       of  with lipid bilayers are well      7Matsuzaki K. Biochim. Biophys. Acta. 1998; 1376: 391-400Crossref PubMed Scopus (538) Google  and 8Matsuzaki K. Biochim. Biophys. Acta. 1999; 1462: 1-10Crossref PubMed Scopus (855) Google Scholar). The peptides   to        K. Murase O.  H.  S. Fujii N. Miyajima K. Biochemistry.    PubMed Scopus  Google Scholar).   for mammalian cells, on the  of which   are not  have been  to be  although     peptide binding  K. Sugishita K. Fujii N. Miyajima K. Biochemistry. 1995; 34:  PubMed Scopus  Google Scholar). The  of    by both cells were larger than those of  and Tat-(47–57)     the    The  of  which has   residues      was the   the three peptides (Table   membrane   magainin   a  pore    with  lipid  in  membranes  K. Murase O. Fujii N. Miyajima K. Biochemistry. 1996;   PubMed Scopus  Google Scholar).  pore  a  of the peptide  translocates across the membrane (24Matsuzaki K. Murase O. Fujii N. Miyajima K. Biochemistry. 1995; 34: 6521-6526Crossref PubMed Scopus (313) Google Scholar).  is  by a  in peptide  membrane permeabilization  K. Murase O.  H.  S. Fujii N. Miyajima K. Biochemistry.    PubMed Scopus  Google Scholar), and cytotoxicity  B. Tanaka J.C. Biochim. Biophys. Acta. 1995; 1240: 149-158Crossref PubMed Scopus (35) Google Scholar). The   in cellular uptake of    and cytotoxicity    that the magainin  also forms a pore in cell membranes and that pore  and  cell  are   to cell    is that membrane permeabilization  to cell death by  membrane  and  of intracellular    is that the peptide       Zasloff M.  D. Biochim. Biophys. Acta.    PubMed Scopus  Google Scholar),  in  The   of    also  membrane activity of this peptide. In studies     of   in  the pore   is  and   within  min (24Matsuzaki K. Murase O. Fujii N. Miyajima K. Biochemistry. 1995; 34: 6521-6526Crossref PubMed Scopus (313) Google Scholar). In cells that are  larger and have   and  that the magainin peptide can   the      with the  uptake    The uptake of  was   at        also  translocation  the   of a     temperature,  membranes are less  the  of the    pore that  membrane   be  The  of a  cationic   composed of  amino acid residues was also   at    J.  A.  B.    M.    M.  M. Biochim. Biophys. Acta. 1998;   PubMed Scopus  Google Scholar), although this  was  to the  of  cell  mechanisms.    Y.  M.  I.  J.  T.  A.  J.  1998;   Google Scholar) reported that   of a   peptide was   at    Y.  M.  I.  J.  T.  A.  J.  1998;   Google Scholar). However, in this  the peptide uptake was  at higher peptide concentrations. The   was  for    Furthermore, the peptide internalized by      and was   from the   was  for   1 and   cytotoxicity    show that     HeLa cells   TM12 cells,  with  studies (19Cruciani R.A. Barker J.L. Zasloff M. Chen H.-C. Colamonici O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3792-3796Crossref PubMed Scopus (361) Google Scholar, 20Ohsaki Y. Gazdar A.F., H.-C., C. Johnson B.E. Cancer Res. 1992; 52: 3534-3538PubMed Google Scholar, 21Baker M.A. Maloy W.L. Zasloff M. Jacob L.S. Cancer Res. 1993; 53: 3052-3057PubMed Google Scholar, 27Soballe P.W. Maloy W.L. Myrga M.L. Jacob L.S. Herlyn M. Int. J. Cancer. 1995; 60: 280-284Crossref PubMed Scopus (68) Google Scholar).  2 effectively  bacterial cell membranes (10Park C.B. Kim H.S. Kim S.C. Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (694) Google Scholar), although the mechanism has not  been  The  that peptides also  across model lipid bilayers without perturbing lipid  (18Kobayashi S. Takeshima K. Park C.B. Kim S.C. Matsuzaki K. Biochemistry. 2000; 39: 8648-8654Crossref PubMed Scopus (183) Google Scholar)  that the membrane   by a passive   the cell  of  was   by    the     The  of peptide entered the cells was  after 10  suggesting the  of an  mechanism     have been reported for a   peptide  T.  S.  M. Tanaka S.  K.  Y. J. Biol. Chem. 2002;   Full Text Full Text PDF PubMed Scopus  Google Scholar) and a  Tat peptide      J.L.      D.  Chem. 2000;   PubMed Scopus  Google Scholar). The buforin  effectively  the   1  in  with a higher  of buforin 2 with RNA and DNA  with magainin 2 (10Park C.B. Kim H.S. Kim S.C. Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (694) Google Scholar).  2 has been  to kill bacteria by binding to nucleic acids,   protein synthesis (10Park C.B. Kim H.S. Kim S.C. Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (694) Google Scholar). However,  did not show any significant   both HeLa and TM12 cells at   to     because the  of the internalized peptide were the   the peptides investigated   and because of the  of an  mechanism   The  of cytotoxicity  that  is a promising  as a vector for intracellular delivery of membrane-impermeable   A  of Arg-rich peptides have been  for this     Ref.  M. Hällbrink M. Prochiantz A. Langel Ü. Trends Pharmacol. Sci. 2000; 21: 99-103Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar).     enter cells, although the  was   than that of the vector peptide    The observations that the cellular uptake of the  was  at    in the  of    that the  and the vector  the cell membranes by different mechanisms.  short Tat peptides have been reported to  the cell membranes without perturbing membrane  and to  the       K.    L.   Proc. Natl. Acad.  U. S. A. 2000;   PubMed Scopus  Google Scholar,  T.  S.  M. Tanaka S.  K.  Y. J. Biol. 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Chem. 2000;   PubMed Scopus  Google Scholar) by HeLa and  cells  a  at  min and    whereas that of Tat-(47–57) by HeLa cells  to  at   to 120 min     Tat-(47–57) peptide was internalized  to a peptide concentration of  to      In contrast, the uptake of  was  even  10   T.  S.  M. Tanaka S.  K.  Y. J. Biol. Chem. 2002;   Full Text Full Text PDF PubMed Scopus  Google Scholar).   was reported to have   on the uptake of   T.  S.  M. Tanaka S.  K.  Y. J. Biol. Chem. 2002;   Full Text Full Text PDF PubMed Scopus  Google Scholar). However, in   the concentration dependence of uptake was  lost by this     suggesting some  of     peptide  the  of    a   in  The  for this is not  at  ATP  can    in cell membranes, including the  of   of cell    S.       1997;   PubMed Scopus  Google Scholar), the  of   of     P.     Biochim. Biophys. Acta. 1999;   PubMed Scopus  Google Scholar), and the  of    B.  D.  J.  M.  K.   Molecular  of the        Scholar). 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Biochemistry. 2000; 39: 8648-8654Crossref PubMed Scopus (183) Google Scholar). The  is not  by   a   and the cytotoxicity is   The  peptide enters the cells by an  mechanism that many Arg-rich peptides    T.  S.  M. Tanaka S.  K.  Y. J. Biol. Chem. 2002;   Full Text Full Text PDF PubMed Scopus  Google Scholar). The  and energy dependence   the peptide sequences and the cell   The peptides show moderate  at higher     P.  B. J. Biol. Chem. 1997; 272:  Full Text Full Text PDF PubMed Scopus  Google Scholar). The membrane   be  in    Matsuzaki and A.     is not   the  peptides   D.  G. Prochiantz A. Trends  Biol. 1998;   Full Text PDF PubMed Scopus  Google Scholar)  to this   not because   were reported on the translocation across lipid bilayers    D.  M.  B. FEBS Lett. 2000;   PubMed Scopus  Google Scholar,  G.  H. Temsamani J.  R. 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