Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma.

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68+ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8+ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4+ and CD8+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8+ T cells in HCC.

Antimicrobial and Immunomodulatory Properties and Applications of Marine-Derived Proteins and Peptides.

Marine organisms provide an abundant source of potential medicines. Many of the marine-derived biomaterials have been shown to act as different mechanisms in immune responses, and in each case they can significantly control the immune system to produce effective reactions. Marine-derived proteins, peptides, and protein hydrolysates exhibit various physiologic functions, such as antimicrobial, anticancer, antioxidant, antihypertensive, and anti-inflammatory activities. Recently, the immunomodulatory properties of several antimicrobial peptides have been demonstrated. Some of these peptides directly kill bacteria and exhibit a variety of immunomodulatory activities that improve the host innate immune response and effectively eliminate infection. The properties of immunomodulatory proteins and peptides correlate with their amino acid composition, sequence, and length. Proteins and peptides with immunomodulatory properties have been tested in vitro and in vivo, and some of them have undergone different clinical and preclinical trials. This review provides a comprehensive overview of marine immunomodulatory proteins, peptides, and protein hydrolysates as well as their production, mechanisms of action, and applications in human therapy.

Pse-T2, an Antimicrobial Peptide with High-Level, Broad-Spectrum Antimicrobial Potency and Skin Biocompatibility against Multidrug-Resistant Pseudomonas aeruginosa Infection.

Pseudin-2, isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity but also cytotoxicity. In an effort to develop clinically applicable antimicrobial peptides (AMPs), we designed pseudin-2 analogs with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. In addition, truncated analogs of pseudin-2 and Lys-substituted peptides were synthesized to produce linear 18-residue amphipathic α-helices, which were further investigated for their mechanism and functions. These truncated analogs exhibited higher antimicrobial activity and lower cytotoxicity than pseudin-2. In particular, Pse-T2 showed marked pore formation, permeabilization of the outer/inner bacterial membranes, and DNA binding. Fluorescence spectroscopy and scanning electron microscopy showed that Pse-T2 kills bacterial cells by disrupting membrane integrity. In vivo, wounds infected with multidrug-resistant (MDR) Pseudomonas aeruginosa healed significantly faster when treated with Pse-T2 than did untreated wounds or wounds treated with ciprofloxacin. Moreover, Pse-T2 facilitated infected-wound closure by reducing inflammation through suppression of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α). These data suggest that the small antimicrobial peptide Pse-T2 could be useful for future development of therapeutic agents effective against MDR bacterial strains.

Single-Molecule, Real-Time Dissecting of Peptide Nucleic Acid-DNA Duplexes with a Protein Nanopore Tweezer.

Peptide nucleic acids (PNAs) are artificial, oligonucleotides analogues, where the sugar-phosphate backbone has been substituted with a peptide-like N-(2-aminoethyl)glycine backbone. Because of their inherent benefits, such as increased stability and enhanced binding affinity toward DNA or RNA substrates, PNAs are intensively studied and considered beneficial for the fields of materials and nanotechnology science. Herein, we designed cationic polypeptide-functionalized, 10-mer PNAs, and demonstrated the feasible detection of hybridization with short, complementary DNA substrates, following analytes interaction with the vestibule entry of an α-hemolysin (α-HL) nanopore. The opposite charged state at the polypeptide-functionalized PNA-DNA duplex extremities, facilitated unzipping of a captured duplex at the lumen entry of a voltage-biased nanopore, followed by monomers threading. These processes were resolvable and identifiable in real-time, from the temporal profile of the ionic current through a nanopore accompanying conformational changes of a single PNA-DNA duplex inside the α-HL nanopore. By employing a kinetic description within the discrete Markov chains theory, we proposed a minimalist kinetic model to successfully describe the electric force-induced strand separation in the duplex. The distinct interactions of the duplex at either end of the nanopore present powerful opportunities for introducing new generations of force-spectroscopy nanopore-based platforms, enabling from the same experiment duplex detection and assessment of interstrand base pairing energy.

Antibacterial activity and mechanism of action of analogues derived from the antimicrobial peptide mBjAMP1 isolated from Branchiostoma japonicum.

Objectives: The worldwide increase in antibiotic-resistant bacteria is a growing threat to public health. Antimicrobial peptides (AMPs) are potentially effective alternatives to conventional antibiotics. We therefore tested analogues of the AMP mBjAMP1 from Branchiostoma japonicum, which we produced by adding and/or replacing amino acids to increase antimicrobial activity against Gram-negative bacteria. Methods: We compared the antimicrobial activities of mBjAMP1 analogues against Gram-negative bacteria reference strains and 52 strains of Klebsiella pneumoniae isolated from patients. Antibiofilm activity and cytotoxicity were evaluated, and the mechanisms of action were then studied. Results: Analogue peptides exhibited greater antimicrobial and antibiofilm activities than mBjAMP1. In particular, the analogue IARR-Anal10 displayed not only the greatest antimicrobial and antibiofilm activities, but also no toxicity against human red blood cells or other mammalian cells. IARR-Anal10 had little or no effect on bacterial outer membrane permeability, membrane polarization or membrane integrity. Instead, it appears IARR-Anal10 binds bacterial DNA, as evidenced in DNA gel retardation assays. Thus, IARR-Anal10 likely kills bacteria through an intracellular mechanism. We also confirmed that IARR-Anal10 suppresses the virulence of K. pneumoniae to a degree similar to tigecycline, used to treat carbapenem-resistant Enterobacteriaceae infections. Notably, IARR-Anal10 did not induce development of resistance by K. pneumoniae, though both meropenem and tigecycline did so within a short time. Conclusions: These results suggest that IARR-Anal10 is a promising agent for treating infections caused by bacteria resistant to tigecycline and meropenem.

Therapeutic Properties and Biological Benefits of Marine-Derived Anticancer Peptides.

Various organisms exist in the oceanic environment. These marine organisms provide an abundant source of potential medicines. Many marine peptides possess anticancer properties, some of which have been evaluated for treatment of human cancer in clinical trials. Marine anticancer peptides kill cancer cells through different mechanisms, such as apoptosis, disruption of the tubulin-microtubule balance, and inhibition of angiogenesis. Traditional chemotherapeutic agents have side effects and depress immune responses. Thus, the research and development of novel anticancer peptides with low toxicity to normal human cells and mechanisms of action capable of avoiding multi-drug resistance may provide a new method for anticancer treatment. This review provides useful information on the potential of marine anticancer peptides for human therapy.

Antimicrobial Peptide CMA3 Derived from the CA-MA Hybrid Peptide: Antibacterial and Anti-inflammatory Activities with Low Cytotoxicity and Mechanism of Action in Escherichia coli.

CA-MA is a hybrid antimicrobial peptide (AMP) derived from two naturally occurring AMPs, cecropin A and magainin 2. CA-MA shows strong antimicrobial activity against Gram-negative and Gram-positive bacteria but also exhibits cytotoxicity toward mammalian cells. Our objective was to identify CA-MA analogues with reduced cytotoxicity by systematic replacement of amino acids with positively charged R groups (His and Lys), aliphatic R groups (Leu), or polar R groups (Glu). Among the CA-MA analogues studied (CMA1 to -6), CMA3 showed the strongest antimicrobial activity, including against drug-resistant Escherichia coli and Pseudomonas aeruginosa strains isolated from hospital patients. CMA3 appeared to act by inducing pore formation (toroidal model) in the bacterial membrane. In cytotoxicity assays, CMA3 showed little cytotoxicity toward human red blood cells (hRBCs) or HaCaT cells. Additionally, no fluorescence was released from small or giant unilamellar vesicles exposed to 60 µM CMA3 for 80 s, whereas fluorescence was released within 35 s upon exposure to CA-MA. CMA3 also exerted strong lipopolysaccharide (LPS)-neutralizing activity in RAW 264.7 cells, and BALB/c mice exposed to LPS after infection by Escherichia coli showed improved survival after administration of one 0.5-mg/kg of body weight or 1-mg/kg dose of CMA3. Finally, in a mouse model of septic shock, CMA3 reduced the levels of proinflammatory factors, including both nitric oxide and white blood cells, and correspondingly reduced lung tissue damage. This study suggests that CMA3 is an antimicrobial/antiendotoxin peptide that could serve as the basis for the development of anti-inflammatory and/or antimicrobial agents with low cytotoxicity.

Marine peptides and their anti-infective activities.

Marine bioresources are a valuable source of bioactive compounds with industrial and nutraceutical potential. Numerous clinical trials evaluating novel chemotherapeutic agents derived from marine sources have revealed novel mechanisms of action. Recently, marine-derived bioactive peptides have attracted attention owing to their numerous beneficial effects. Moreover, several studies have reported that marine peptides exhibit various anti-infective activities, such as antimicrobial, antifungal, antimalarial, antiprotozoal, anti-tuberculosis, and antiviral activities. In the last several decades, studies of marine plants, animals, and microbes have revealed tremendous number of structurally diverse and bioactive secondary metabolites. However, the treatments available for many infectious diseases caused by bacteria, fungi, and viruses are limited. Thus, the identification of novel antimicrobial peptides should be continued, and all possible strategies should be explored. In this review, we will present the structures and anti-infective activity of peptides isolated from marine sources (sponges, algae, bacteria, fungi and fish) from 2006 to the present.

The effects of marine carbohydrates and glycosylated compounds on human health.

Marine organisms have been recognized as a valuable source of bioactive compounds with industrial and nutraceutical potential. Recently, marine-derived carbohydrates, including polysaccharides and low molecular weight glycosylated oligosaccharides, have attracted much attention because of their numerous health benefits. Moreover, several studies have reported that marine carbohydrates exhibit various biological activities, including antioxidant, anti-infection, anticoagulant, anti-inflammatory, and anti-diabetic effects. The present review discusses the potential industrial applications of bioactive marine carbohydrates for health maintenance and disease prevention. Furthermore, the use of marine carbohydrates in food, cosmetics, agriculture, and environmental protection is discussed.

Synergistic effects and antibiofilm properties of chimeric peptides against multidrug-resistant Acinetobacter baumannii strains.

The increasing prevalence of drug-resistant pathogens highlights the need to identify novel antibiotics. Here we investigated the efficacies of four new antimicrobial peptides (AMPs) for potential drug development. The antibacterial activities, synergistic effects, and antibiofilm properties of the four chimeric AMPs were tested against Acinetobacter baumannii, an emerging Gram-negative, nosocomial, drug-resistant pathogen. Nineteen A. baumannii strains resistant to ampicillin, cefotaxime, ciprofloxacin, tobramycin, and erythromycin were isolated at a hospital from patients with cholelithiasis. All four peptides exhibited significant antibacterial effects (MIC=3.12 to 12.5 µM) against all 19 strains, whereas five commercial antibiotics showed little or no activity against the same pathogens. An exception was polymyxin, which was effective against all of the strains tested. Each of the peptides showed synergy against one or more strains when administered in combination with cefotaxime, ciprofloxacin, or erythromycin. The peptides also exhibited an ability to prevent biofilm formation, which was not seen with cefotaxime, ciprofloxacin, or erythromycin, though polymyxin also inhibited biofilm formation. Indeed, when administered in combination with ciprofloxacin, the AMP HPMA exerted a potent synergistic effect against A. baumannii biofilm formation. Collectively, our findings indicate that the AMPs tested have no cytotoxicity but possess potent antibacterial and antibiofilm activities and may act synergistically with commercial antibiotics.

The role of biophysical parameters in the antilipopolysaccharide activities of antimicrobial peptides from marine fish.

Numerous antimicrobial peptides (AMPs) from marine fish have been identified, isolated and characterized. These peptides act as host defense molecules that exert antimicrobial effects by targeting the lipopolysaccharide (LPS) of Gram-negative bacteria. The LPS-AMP interactions are driven by the biophysical properties of AMPs. In this review, therefore, we will focus on the physiochemical properties of AMPs; that is, the contributions made by their sequences, net charge, hydrophobicity and amphipathicity to their mechanism of action. Moreover, the interactions between LPS and fish AMPs and the structure of fish AMPs with LPS bound will also be discussed. A better understanding of the biophysical properties will be useful in the design of AMPs effective against septic shock and multidrug-resistant bacterial strains, including those that commonly produce wound infections.

Colonization and infection of the skin by S. aureus: immune system evasion and the response to cationic antimicrobial peptides.

Staphylococcus aureus (S. aureus) is a widespread cutaneous pathogen responsible for the great majority of bacterial skin infections in humans. The incidence of skin infections by S. aureus reflects in part the competition between host cutaneous immune defenses and S. aureus virulence factors. As part of the innate immune system in the skin, cationic antimicrobial peptides (CAMPs) such as the ß-defensins and cathelicidin contribute to host cutaneous defense, which prevents harmful microorganisms, like S. aureus, from crossing epithelial barriers. Conversely, S. aureus utilizes evasive mechanisms against host defenses to promote its colonization and infection of the skin. In this review, we focus on host-pathogen interactions during colonization and infection of the skin by S. aureus and methicillin-resistant Staphylococcus aureus (MRSA). We will discuss the peptides (defensins, cathelicidins, RNase7, dermcidin) and other mediators (toll-like receptor, IL-1 and IL-17) that comprise the host defense against S. aureus skin infection, as well as the various mechanisms by which S. aureus evades host defenses. It is anticipated that greater understanding of these mechanisms will enable development of more sustainable antimicrobial compounds and new therapeutic approaches to the treatment of S. aureus skin infection and colonization.

Long-term outcomes of single-port laparoscopic hepatectomy for hepatocellular carcinoma: a retrospective comparative analysis.

Purpose: Numerous efforts have been made to achieve minimally invasive surgery, such as single-port laparoscopic surgery. However, few studies have provided long-term follow-up information, and the number of patients with hepatocellular carcinoma (HCC) in previous studies has been small. The purpose in this study is to compare the long-term oncological outcomes of HCC patients who underwent single-port laparoscopic hepatectomy (SPLH) with those of patients who underwent multiport laparoscopic hepatectomy (MPLH). Methods: We retrospectively reviewed the medical records of 135 patients with HCC who underwent laparoscopic liver between January 2008 and December 2018. Of the 135 patients, 53 underwent MPLH, and 82 underwent SPLH. Results: From January 2008 to December 2018, 135 patients underwent laparoscopic hepatectomy for HCC. Among them, 82 patients underwent SPLH, and 53 patients underwent MPLH. Neither long-term overall survival (P = 0.849) nor recurrence-free survival (P = 0.057) differed significantly between the 2 groups, even though the recurrence rate was higher in the SPLH group. In the univariable analysis of risk factors for recurrence, multiple tumors, SPLH method, and portal vein invasion were statistically significant (P < 0.05). Multivariable analysis showed that the SPLH method and portal vein invasion were independent adverse prognostic factors for recurrence-free survival. Conclusion: In terms of both short-term and long-term outcomes, the SPLH method seems to be a feasible approach for HCC in select patients. Because the potential risk of margin recurrence might produce poor oncological outcomes, strict patient selection is essential to ensure that an adequate safety margin can be secured.

Liver Transplantation at a Small Volume Center-The Role of Network System.

BACKGROUND: Liver transplantation (LT) is typically performed at specialized, high-volume centers. However, some smaller centers also offer liver transplantation services, but their outcomes and safety have been a subject of debate. To overcome these difficulties, we tried to build a Catholic Medical Center (CMC) network to share our experiences and overcome the lack of volume. In this study, we reviewed the overall outcome of patients undergoing LT at a small-volume procedure center, with a focus on patient and graft survival rates. METHODS: Between July 2014 and September 2021, 60 adults underwent LT at Bucheon Saint Mary's Hospital. The overall outcomes were analyzed in terms of perioperative outcomes, complications, and overall survival rate. In addition, the patients were divided into a benign end-stage liver disease (ESLD) group (n = 44) and a hepatocellular carcinoma (HCC) group (n = 16). The baseline characteristics, perioperative outcomes, complications, and overall survival rate were analyzed between the 2 groups. RESULTS: Of a total of 60 LT, living donor liver transplantation (LDLT) was 26, and deceased donor liver transplantation was 34. LDLT was 14 (31.8%) in the ESLD group and 12 (75.0%) in the HCC group. The overall 1-year, 3-year, and 5-year survival rates were 86.7%, 79.7%, and 77.7%, respectively. The survival difference was not statistically significant (P = .214) between the 2 groups. CONCLUSION: We suggest that with appropriate patient selection and adequate resources, LT can be safely performed at smaller centers with the assistance of the CMC network, thus expanding access to this life-saving procedure.

Pioneering PGC-1α-boosted secretome: a novel approach to combating liver fibrosis.

Purpose: Liver fibrosis is a critical health issue with limited treatment options. This study investigates the potential of PGC-Sec, a secretome derived from peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-overexpressing adipose-derived stem cells (ASCs), as a novel therapeutic strategy for liver fibrosis. Methods: Upon achieving a cellular confluence of 70%-80%, ASCs were transfected with pcDNA-PGC-1α. PGC-Sec, obtained through concentration of conditioned media using ultrafiltration units with a 3-kDa cutoff, was assessed through in vitro assays and in vitro mouse models. Results: In vitro, PGC-Sec significantly reduced LX2 human hepatic stellate cell proliferation and mitigated mitochondrial oxidative stress compared to the control-secretome. In an in vivo mouse model, PGC-Sec treatment led to notable reductions in hepatic enzyme activity, serum proinflammatory cytokine concentrations, and fibrosis-related marker expression. Histological analysis demonstrated improved liver histology and reduced fibrosis severity in PGC-Sec-treated mice. Immunohistochemical staining confirmed enhanced expression of PGC-1α, optic atrophy 1 (a mitochondrial function marker), and peroxisome proliferator-activated receptor alpha (an antifibrogenic marker) in the PGC-Sec-treated group, along with reduced collagen type 1A expression (a profibrogenic marker). Conclusion: These findings highlight the therapeutic potential of PGC-Sec in combating liver fibrosis by enhancing mitochondrial biogenesis and function, and promoting antifibrotic processes. PGC-Sec holds promise as a novel treatment strategy for liver fibrosis.

Proposal of a Network System to Solve the Problem of Small Volume in Liver Transplantation; Catholic Medical Center Network.

INTRODUCTION: Liver transplantation (LT) is a complex and demanding procedure associated with significant perioperative challenges and risks. Concerns have arisen regarding LT outcomes in low-volume centers. We implemented an integrated training and surgical team network to address these concerns within the Catholic Medical Center (CMC) network. This study presents a comprehensive review of our 9-year LT experience within the CMC medical network. METHOD: A retrospective study of LT procedures conducted between January 2013 and August 2021 in 6 CMC-affiliated hospitals was performed. One center was categorized as a high-volume center, conducting over 60 cases annually, and the remaining 5 were considered small-volume centers. The primary endpoints assessed were 1-year and 5-year survival rates. RESULTS: A total of 793 LTs were performed during the study period. The high-volume center performed 411 living donor LT (LDLT) cases and 127 deceased donor LT (DDLT) cases. Also, 146 LDLT cases and 109 DDLT cases were performed in 5 small-volume centers. One-year and 5-year patient survival for LDLT recipients was 88.3% and 78.8% in the high-volume center and 85.6% and 80.6% in the low-volume center. Five-year survival was not significantly different in small-volume centers (P = .903). For DDLT recipients, 1-year and 5-year patient survival was 80.3% and 70.6% in the high-volume center and 76.1% and 67.6% in the low-volume center. In DDLT cases, 5-year survival was not significantly different in small-volume centers (P = .445). CONCLUSION: In conclusion, comparable outcomes for liver transplantation can be obtained in a small-volume center with a high level of integrated training systems and networks.

Effect of repetitive lysine-tryptophan motifs on the bactericidal activity of antimicrobial peptides.

Previous studies identified lysine- and tryptophan-rich sequences within various cationic antimicrobial peptides. In the present study, we synthesized a series of peptides composed of lysine (K)-tryptophan (W) repeats (KW)( n ) (where n equals 2, 3, 4 or 5) with amidation of the C-terminal to increase cationicity. We found that increases in chain length up to (KW)(4) enhanced the peptides' antibacterial activity; (KW)(5) exhibited somewhat less bactericidal activity than (KW)(4). Cytotoxicity, measured as lysis of human red blood cells, also increased with increasing chain length. With (KW)(5), reduced antibacterial activity and increased cytotoxicity correlated with greater hydrophobicity and self-aggregation in the aqueous environment. The peptides acted by inducing rapid collapse of the bacterial transmembrane potential and induction of membrane permeability. The mode of interaction of the peptides and the phosphate groups of lipopolysaccharide was dependent upon the peptides' ability to permeate the membrane. Longer peptides [(KW)(4) and (KW)(5)] but not shorter peptides [(KW)(2) and (KW)(3)] strongly bound and partially inserted into negatively charged, anionic lipid bilayers. These longer peptides also induced membrane permeabilization and aggregation of lipid vesicles. The peptides had a disordered structure in aqueous solution, and only (KW)(4) and (KW)(5) displayed a folded conformation on lipid membranes. Moreover, (KW)(4) destroyed and agglutinated bacterial cells, demonstrating its potential as an antimicrobial agent. Collectively, the results show (KW)(4) to be the most efficacious peptide in the (KW)( n ) series, exhibiting strong antibacterial activity with little cytotoxicity.

Anti-microbial, anti-biofilm activities and cell selectivity of the NRC-16 peptide derived from witch flounder, Glyptocephalus cynoglossus.

Previous studies had identified novel antimicrobial peptides derived from witch flounder. In this work, we extended the search for the activity of peptide that showed antibacterial activity on clinically isolated bacterial cells and bacterial biofilm. Pseudomonas aeruginosa was obtained from otitis media and cholelithiasis patients, while Staphylococcus aureus was isolated from otitis media patients. We found that synthetic peptide NRC-16 displays antimicrobial activity and is not sensitive to salt during its bactericidal activity. Interestingly, this peptide also led to significant inhibition of biofilm formation at a concentration of 4-16 µM. NRC-16 peptide is able to block biofilm formation at concentrations just above its minimum inhibitory concentration while conventional antibiotics did not inhibit the biofilm formation except ciprofloxacin and piperacillin. It did not cause significant lysis of human RBC, and is not cytotoxic to HaCaT cells and RAW264.7 cells, thereby indicating its selective antimicrobial activity. In addition, the peptide's binding and permeation activities were assessed by tryptophan fluorescence, calcein leakage and circular dichroism using model mammalian membranes composed of phosphatidylcholine (PC), PC/cholesterol (CH) and PC/sphingomyelin (SM). These experiments confirmed that NRC-16 does not interact with any of the liposomes but the control peptide melittin did. Taken together, we found that NRC-16 has potent antimicrobial and antibiofilm activities with less cytotoxicity, and thus can be considered for treatment of microbial infection in the future.

Effect of repetitive lysine-tryptophan motifs on the eukaryotic membrane.

In a previous study, we synthesized a series of peptides containing simple sequence repeats, (KW)(n)-NH(2) (n = 2,3,4 and 5) and determined their antimicrobial and hemolytic activities, as well as their mechanism of antimicrobial action. However, (KW)(5) showed undesirable cytotoxicity against RBC cells. In order to identify the mechanisms behind the hemolytic and cytotoxic activities of (KW)(5), we measured the ability of these peptides to induce aggregation of liposomes. In addition, their binding and permeation activities were assessed by Trp fluorescence, calcein leakage and circular dichrorism using artificial phospholipids that mimic eukaryotic liposomes, including phosphatidylcholine (PC), PC/sphingomyelin (SM) (2:1, w/w) and PC/cholesterol (CH) (2:1, w/w). Experiments confirmed that only (KW)(5) induced aggregation of all liposomes; it formed much larger aggregates with PC:CH (2:1, w/w) than with PC or PC:SM (2:1, w/w). Longer peptide (KW)(5), but not (KW)(3) or (KW)(4), strongly bound and partially inserted into PC:CH compared to PC or PC:SM (2:1, w/w). Calcein release experiments showed that (KW)(5) induced calcein leakage from the eukaryotic membrane. Greater calcein leakage was induced by (KW)(5) from PC:CH than from PC:SM (2:1, w/w) or PC, whereas (KW)(4) did not induce calcein leakage from any of the liposomes. Circular dichroism measurements indicated that (KW)(5) showed higher conformational transition compared to (KW)(4) due to peptide-liposome interactions. Taken together, our results suggest that (KW)(5) reasonably mediates the aggregation and permeabilization of eukaryotic membranes, which could in turn explain why (KW)(5) displays efficient hemolytic activity.

PG-2, a Potent AMP against Pathogenic Microbial Strains, from Potato (Solanum tuberosum L cv. Gogu Valley) Tubers Not Cytotoxic against Human Cells.

In an earlier study, we isolated potamin-1 (PT-1), a 5.6-kDa trypsin-chymotrypsin protease inhibitor, from the tubers of a potato strain (Solanum tuberosum L cv. Gogu Valley). We established that PT-1 strongly inhibits pathogenic microbial strains, but not human bacterial strains, and that its sequence shows 62% homology with a serine protease inhibitor. In the present study, we isolated an antifungal and antibacterial peptide with no cytotoxicity from tubers of the same potato strain. The peptide (peptide-G2, PG-2) was isolated using salt-extraction, ultrafiltration and reverse-phase high performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) showed the protein to have a molecular mass of 3228.5 Da, while automated Edman degradation showed the N-terminal sequence of PG-2 to be LVKDNPLDISPKQVQALCTDLVIRCMCCC-. PG-2 exhibited antimicrobial activity against Candida albicans, a human pathogenic yeast strain, and Clavibacter michiganensis subsp. michiganensis, a plant late blight strain. PG-2 also showed antibacterial activity against Staphylococcus aureus, but did not lyse human red blood cells and was thermostable. Overall, these results suggest PG-2 may be a good candidate to serve as a natural antimicrobial agent, agricultural pesticide and/or food additive.