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ABSTRACT: The mistletoe lectins are major active components in the extract of European mistletoes that have been widely used in adjuvant chemotherapy of cancer. This study was performed to investigate the mechanism of anticancer and antimetastatic activity of the purified Korean mistletoe lectin (Viscum album L. coloratum agglutinin, VCA). C57BL6 mice inoculated with B16-BL6 melanoma cells and treated with VCA were assessed for survival and metastasis. The induction of apoptosis of B16-BL6 cells by VCA was investigated by morphological changes, DNA fragmentation characteristics, and cell cycle analysis. The antiangiogenic activity of VCA was also measured by the CAM (choriallantoic membrane) assay. Length of survival of mice was increased and lung metastasis was inhibited by VCA. Treatment of cells with VCA resulted in growth suppression, nuclear morphological changes, DNA fragmentation, and an increased fraction of cells in sub-G1 consistent with apoptosis. Antiangiogenesis of VCA was assessed by CAM assay, where vessel growth induced by fat emulsion was decreased. These results suggest that VCA inhibits tumor growth and metastasis by increasing apoptosis and inhibiting angiogenesis.
Cancer Biotherapy and Radiopharmaceuticals 11/2001; 16(5):439-47. · 1.79 Impact Factor
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ABSTRACT: Several diastereomers and an enantiomer of KKVVFKVKFKK, an antimicrobial peptide that acts on the lipid membrane of pathogens were synthesized to investigate the effect of D-amino acid substitution on stability, secondary structure, and activity. The stability of the peptide in serum was improved greatly by the D-amino acid substitutions. D-Amino acid substitutions at the N- and/or C-terminal of the peptide, which had little effect on the alpha-helical structure, and all D-amino acid substitutions that formed a left-handed alpha-helix maintained antimicrobial activity, whereas D-amino acid substitutions in the middle of the amino acid sequence disrupted the alpha-helical structure, resulting in the complete loss of activity. This result confirmed that the peptide did not interact with chiral receptors, enzymes, or any chiral component of the membrane. D-Amino acid substitutions at the termini reduced the inhibition of the activity by heat-inactivated serum, which indicated that local change of chirality or change of secondary structure induced by D-amino acid substitutions might affect the interactions between the peptide and certain components in the serum. The present study suggests that partial D-amino acid substitution is a useful technique to improve the in vivo activity of antimicrobial peptides.
Biochemical Pharmacology 01/2000; 58(11):1775-80. · 4.70 Impact Factor
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ABSTRACT: By the introduction of various amide surrogates, novel pseudopeptides corresponding to a membrane active depsipeptide were synthesized and their native characteristics compared with that of the peptide. The pseudopeptides had more resistance to serum proteases than the peptide and similar antimicrobial activities to that of the peptide without hemolytic activity. The pseudopeptides like the peptide were active against current drug resistant fungi and pathogenic fungi isolated from patients, and also had a strong synergism with current antifungal drugs against Candida albicans. The leakage assay suggested that the pseudopeptides also acted on the lipid membrane of pathogenic cells. These results indicated that the novel pseudopeptides had advantages over the peptide as a candidate for a novel antifungal drug and backbone modifications can be a tool in the development of a novel antifungal agent from membrane-active peptides isolated from natural sources or chemically synthesized.
Bioorganic & Medicinal Chemistry 12/1999; 7(11):2509-15. · 2.92 Impact Factor
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ABSTRACT: To obtain active and metabolically stable analogues, peptide backbone modifications have been incorporated into many biologically active peptides. In this study, we designed and synthesized pseudopeptides corresponding to the antimicrobial peptide that acted on the lipid membrane of the pathogen. Most pseudopeptides exhibited a longer half-life than the peptide in the presence of serum as well as a considerable activity against test bacteria and fungi. Circular dichroism spectra and retention times of the pseudopeptides helped us to elucidate the effect of the incorporation of backbone modifications on the structural parameters necessary for the activity, indicating that alpha-helical structure was the most important factor for the activity and hydrophobicity had a considerable effect on the activity. Backbone modifications employed in this study can be a useful tool for structure-activity relationship studies and the development of therapeutic agents from membrane-active antimicrobial peptides.
European Journal of Allergy and Clinical Immunology 09/1999; 54(2):129-36. · 1.30 Impact Factor
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ABSTRACT: In the present study, we investigated the antifungal activity and cytotoxicity of a novel membrane-active peptide, KKVVFKVKFKK (MP). MP inhibited the growth of various pathogenic fungi isolated from patients and of fluconazole-resistant fungi at concentrations of 2 to 32 microg/ml. MP had potent fungicidal activity; the minimal fungicidal concentrations of the peptide were no more than fourfold greater than the MICs. Time course experiments of MP-induced killing of Candida albicans ATCC 36232 showed that the rate of killing was rapid and depended on the concentration of MP. MP had a strong synergism with other antifungal drugs; the fractional inhibitory concentration index values of MP with amphotericin B and fluconazole for C. albicans were 0.16 and 0.02, respectively. The 50% inhibitory concentrations of MP for NIH 3T3 and Jurkat cells were approximately 100 times higher than the MIC for C. albicans ATCC 36232, indicating that MP had high selectivity between the fungal and mammalian cells. These results suggest that MP has great advantages in the development of antifungal agents.
Antimicrobial Agents and Chemotherapy 08/1999; 43(7):1704-7. · 4.84 Impact Factor
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ABSTRACT: Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an alpha-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an alpha-helical structure correlated with the antimicrobial activity and a beta-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of L-amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the beta-sheet structure retained antifungal activity in the same condition.
European Journal of Allergy and Clinical Immunology 01/1999; 53(1):41-6. · 1.30 Impact Factor
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ABSTRACT: Tenecin 1, an inducible antibacterial protein secreted in the larvae of Tenebrio molitor, has a long N-terminal loop and common structural feature of insect defensin family corresponding to cysteine stabilized alpha/beta motif. To study the function of the N-terminal loop and disulfide bridges, N-terminal loop deleted tenecin 1, reduced tenecin 1 and tenecin 1 were chemically synthesized and their activities were measured. N-terminal loop deleted tenecin and reduced tenecin 1 did not show antibacterial activity. Circular dichroism (CD) spectroscopy data revealed that the alpha-helical content of tenecin 1 and the other proteins increased in the presence of 50% (v/v) trifluoroethanol (TFE) and the alpha-helical content of tenecin 1 was much higher than that of the other proteins in buffer with or without 50% (v/v) TFE. These results suggest that disulfide bridges are necessary for the activity structure and the N-terminal loop plays an important role in the increase of alpha-helix in the membrane mimetic environment and the activity.
FEBS Letters 12/1998; 439(1-2):41-5. · 3.54 Impact Factor
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ABSTRACT: Novel combinatorial libraries consisting of simplified amino acid sequences were designed to screen for peptides active against the Candida albicans membrane. A novel decapeptide, KKVVFKVKFK, that had a unique primary amino acid sequence was identified in this work. This peptide irreversibly inhibited the growth of C. albicans and showed a broad range of antibacterial activity but no hemolytic activity. Circular dichroism spectra revealed that the predominant secondary structure of this peptide strongly depended on the membrane-mimetic environments; the peptide preferred to form an amphipathic alpha-helical structure in the presence of 50% trifluoroethanol, while it preferred to adopt a distorted alpha-helical structure in the presence of sodium dodecyl sulfate micelles. Experiments in which dye was released from vesicles indicated that this novel antimicrobial peptide killed microorganisms through the action on the membrane as its primary target. Replacement of amino acids in this active decapeptide on the basis of information from the libraries could provide unique information about factors affecting its antimicrobial activity such as its secondary structure, net positive charge, and hydrophobicity.
Antimicrobial Agents and Chemotherapy 11/1998; 42(10):2534-41. · 4.84 Impact Factor
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ABSTRACT: Brevinin 1E, consisting of 24 amino acid residues, from Rana esculenta has potent antimicrobial and hemolytic activity. From a structural point of view, this peptide has a N-terminal hydrophobic region, a proline hinge region in the middle and a C-terminal loop region delineated by an intra-disulfide bridge, which is a common structural feature of antimicrobial peptides from Rana species. To investigate the structural features for antimicrobial and hemolytic activity, truncated and linearized brevinin 1E amides were synthesized and characterized. A deletion of three amino acids from the N-terminal region did not greatly affect antimicrobial activity but dramatically reduced hemolytic activity. The contribution of the intra-disulfide bridge to antimicrobial and hemolytic activity was somewhat different between brevinin 1E amide and truncated fragments. In brevinin 1E amide, the elimination of the intra-disulfide bridge did not greatly affect antimicrobial and hemolytic activity whereas the elimination of the intra-disulfide bridge in the truncated fragments did not decrease antimicrobial activity but did decrease hemolytic activity. Circular dichroism spectra and the retention time on the C18 reverse phase column revealed that the intra-disulfide bridge (i, i+6) formed an amphipathic loop which increased hydrophobicity and helped to induce the alpha-helical structure in the membrane-mimetic environment. Even though the intra-disulfide bridge and the N-terminal region were responsible for the alpha-helical structure and hydrophobicity, these two structural features were not essential for antimicrobial activity. The hemolytic activity of brevinin 1E amide and its analogs also correlated well with the retention time rather than the alpha-helicity.
Biochimica et Biophysica Acta 10/1998; 1387(1-2):239-48. · 4.66 Impact Factor
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ABSTRACT: An active fragment was identified from tenecin 1, an antibacterial protein belonging to the insect defensin family, by synthesizing the peptides corresponding to the three regions of tenecin 1. Only the fragment corresponding to the C-terminal beta-sheet domain showed activity against fungi as well as Gram-positive and Gram-negative bacteria, whereas tenecin 1, the native protein, showed activity only against Gram-positive bacteria. CD spectra indicated that each fragment in a membrane-mimetic environment might adopt a secondary structure corresponding to its region in the protein. The leakage of dye from liposomes induced by this fragment suggested that this fragment acts on the membrane of pathogens as a primary mode of action. A comparison between the structure and the activity of each fragment indicated that a net positive charge was a prerequisite factor for activity. To the best of our knowledge this is the first report in which the fragment corresponding to the beta-sheet region in antibacterial proteins, which consists of alpha-helical and beta-sheet regions, has been identified as a primary active fragment.
Biochemical Journal 09/1998; 334 ( Pt 1):99-105. · 4.90 Impact Factor
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ABSTRACT: A function of the intra-disulfide bridge located at the C-terminal of Rana peptides has not been extensively studied. To investigate the function of the disulfide bridge related to the activity and the structure, we chose Gaegurin-6, isolated from Rana rugosa as a model peptide and synthesized linear analogs. The reduction of the disulfide bridge resulted in the complete loss of antimicrobial activity while replacements of cysteines by serines retained antimicrobial activity. Circular dichroism spectra from a titration of the peptides in sodium dodecyl sulfate indicated that the disulfide bridge of Gaegurin-6 might stabilize the induction of an alpha helical structure in lipid membranes and the alpha helical forming propensity of the peptides correlated with antimicrobial activity.
Peptides 02/1998; 19(10):1653-8. · 2.43 Impact Factor
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ABSTRACT: The structure of an active analog of the antibacterial peptide gaegurin was investigated by CD and NMR spectroscopy. The NOE connectivities showed that 21 out of 24 residues formed an a-helix despite the presence of a central proline. CD and NMR analysis indicates that the helix is in fast equilibrium with random coil. From chemical shift analysis of the amide protons, the distances of hydrogen bonding in the helix were calculated, and manifested obvious periodicity which implied a kink in the middle of the helix. 1D amide proton exchange experiments provided further evidence of an exceptionally stable kink. It is inferred that this kink is important not only to the function of the peptide but also to the early stage of the folding as a nucleation site.
FEBS Letters 10/1996; 392(3):309-12. · 3.54 Impact Factor
