Sunkyu Kim

In vitro activities of native and designed peptide antibiotics against drug sensitive and resistant tumor cell lines

In order to develop peptide agents with reduced length and enhanced tumoricidal activity, we have designed gaegurin 6 (GGN6) derivatives through deletions and/or substitutions of amino acids. The deletion of hydrophobic amino terminal region completely abolished antitumor activity whereas the deletion of carboxy terminal region had little influence on antitumor activity. Antitumor activity of the PTP peptides did not correlate with antibacterial activity. PTP7, the most potent derivative, was found to have comparable antitumor activity to GGN6 in spite of reduced number of amino acids which is about half the size of gaegurin 6; furthermore, it showed little cytotoxicity on PBMCs and RBCs. GGN6 and PTP7 also showed equivalent cytotoxicity against drug sensitive (MCF-7) and multidrug-resistant cell lines (MCF-7/DOX). Plasma membrane blebbing and DNA fragmentation of peptide-treated tumor cells indicated that the peptides could induce apoptosis in tumor cells. These results suggest that GGN6 and its derivatives can be developed as new anticancer agents and may provide a new strategy for overcoming MDR which is a major problem in cancer therapy.

Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure–activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an α‐helical structure in trifluoroethanol (TFE)/H2O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H2O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic α‐helix spanning residues 3–18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic α‐helix that potentially disrupts membrane integrity, which would result in cell death.

Correlation between the activities of α-helical antimicrobial peptides and hydrophobicities represented as RP HPLC retention times

PTP7 is a 13-amino acid residue peptide designed from gaegurin 6, an antimicrobial peptide isolated from skin secretions of Rana rugosa. In order to examine the effect of hydrophobicity on antimicrobial activity, a series of PTP7 derivatives were constructed and analyzed the activity against bacteria and artificial membrane. We found that the mean hydrophobicity by simple summation of hydrophobicity of each constituent amino acid did not necessarily describe the hydrophobic property of antimicrobial peptides. The mean hydrophobicity did not show close correlation with the observed hydrophobicity by measuring reverse phase high performance liquid chromatography (RP HPLC) retention time. The observed hydrophobicity represented as RP HPLC retention time correlated well with the activity against artificial membrane and Gram positive bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, rather than mean hydrophobicity. However, antimicrobial activity against Gram negative bacteria, such as Escherichia coli, did not show correlation with RP HPLC retention time. These data indicate that the RP HPLC retention time should be exploited rather than the mean hydrophobicity in the analysis of the relationship between hydrophobicity and antimicrobial activity.

Synergistic Inhibitory Effect of Cationic Peptides and Antimicrobial Agents on the Growth of Oral Streptococci

Although chlorhexidine is one of the most efficacious antimicrobial agents used for the prevention of dental caries, side effects limit its application. The effects of gaegurin 6 (GGN6), an animal-derived cationic peptide, and its derivatives PTP6 and PTP12 on the growth of oral streptococci were investigated to assess the potential of these agents for use in the prevention of dental caries. The minimal inhibitory concentrations of the peptides for inhibition of the growth of oral streptococci (Streptococcus mutans, S. sobrinus, S. sanguis and S. gordonii) ranged from 1.2 to 8.2 µM. The peptides also exhibited marked synergistic antibacterial effects with chlorhexidine or xylitol. The most effective combinations (fractional inhibitory concentration index of 0.5) were xylitol with GGN6 against S. gordonii 10558 and chlorhexidine with either GGN6 or PTP6 against S. sobrinus OMZ-175. These results indicate that cationic peptides alone or in combination with chlorhexidine or xylitol might prove effective for the inhibition of the growth of cariogenic oral streptococci in situ.

Neuroprotective effects of consuming bovine colostrum after focal brain ischemia/reperfusion injury in rat model

To investigate the neuroprotective effects of bovine colostrums (BC), we evaluate the ability of consuming BC after focal brain ischemia/reperfusion injury rat model to reduce serum cytokine levels and infarct volume, and improve neurological outcome. Sprague-Dawley rats were randomly divided into 4 groups; one sham operation and three experimental groups. In the experimental groups, MCA occlusion (2 h) and subsequent reperfusion (O/R) were induced with regional cerebral blood flow monitoring. One hour after MCAO/R and once daily during the experiment, the experimental group received BC while the other groups received 0.9% saline or low fat milk (LFM) orally. Seven days later, serum pro-inflammatory cytokine (IL-1β, IL-6, and TNF-α) and anti-inflammatory cytokine (IL-10) levels were assessed. Also, the infarct volume was assessed by using a computerized image analysis system. Behavioral function was also assessed using a modified neurologic severity score and corner turn test during the experiment. Rats receiving BC after focal brain I/R showed a significant reduction (-26%/-22%) in infarct volume compared to LFM/saline rats, respectively (P < 0.05). Serum IL-1β, IL-6, and TNF-α levels were decreased significantly in rats receiving BC compared to LFM/saline rats (P < 0.05). In behavioral tests, daily BC intake showed consistent and significant improvement of neurological deficits for 7 days after MCAO/R. BC ingestion after focal brain ischemia/reperfusion injury may prevent brain injury by reducing serum pro-inflammatory cytokine levels and brain infarct volume in a rat model.