Cytotoxic peptides with insulin‐releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae)

Peptidomic analysis of norepinephrine‐stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host‐defense peptides differing by a single amino acid residue belonging to the brevinin‐1 family (brevinin‐1ITa and ‐1ITb), a peptide belonging to the temporin family (temporin‐ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine‐sulphoxide forms of brevinin‐1ITa and ‐1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin‐1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non‐small cell lung adenocarcinoma A549 cells (LC₅₀ = 18 μM), breast adenocarcinoma MDA‐MB‐231 cells (LC₅₀ = 8 μM) and colorectal adenocarcinoma HT‐29 cells (LC₅₀ = 18 μM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC₅₀ = 7 μM). Temporin‐ITa (VFLGAIAQALTSLLGKL.NH₂) was between three and fivefold less potent against these cells. Brevinin‐1ITa inhibited growth of both Gram‐positive Staphylococcus epidermidis and Gram‐negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin‐ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN‐BD11 clonal β‐cells at concentrations ≥1 nM, but brevinin‐1ITa was cytotoxic to the cells at concentrations ≥3 μM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Immunomodulatory,insulinotropic, and cytotoxic activities of phylloseptins and plasticin‐TR from the Trinidanian leaf frog Phyllomedusa trinitatis

The aim of the study was to determine the in vitro immunomodulatory, cytotoxic, and insulin‐releasing activities of seven phylloseptin‐TR peptides and plasticin‐TR, first isolated from the frog Phyllomedusa trinitatis. The most cationic peptides, phylloseptin‐1.1TR and phylloseptin‐3.1TR, showed greatest cytotoxic potency against A549, MDA‐MB231, and HT‐29 human tumor‐derived cells and against mouse erythrocytes. Phylloseptin‐4TR was the most hydrophobic and the most effective peptide at inhibiting production of the proinflammatory cytokines TNF‐α and IL‐1β by mouse peritoneal cells but was without effect on production of the antiinflammatory cytokine IL‐10. Phylloseptin‐2.1TR and phylloseptin‐3.3TR were the most effective at stimulating the production of IL‐10. The noncytotoxic peptide, plasticin‐TR, inhibited production of TNF‐α and IL‐1β but was without effect on IL‐10 production. The results of CD spectroscopy suggest that the different properties of plasticin‐TR compared with the immunostimulatory activities of the previously characterized plasticin‐L1 from Leptodactylus laticeps may arise from greater ability of plasticin‐TR to oligomerize and adopt a stable helical conformation in a membrane‐mimetic environment. All peptides stimulated release of insulin from BRIN‐BD11 rat clonal β cells with phylloseptin‐3.2TR being the most potent and effective and phylloseptin‐2.1TR the least effective suggesting that insulinotropic potency correlates inversely with helicity. The study has provided insight into structure‐activity relationships among the phylloseptins. The combination of immunomodulatory and insulinotropic activities together with low cytotoxicity suggests that phylloseptin‐3.3TR and plasticin‐TR may represent templates for the development of agents for use in antiinflammatory and type 2 diabetes therapies.     

蛙皮抗菌肽促胰岛素分泌功能研究进展

随着对蛙皮抗菌肽功能研究的不断深入,陆续发现部分肽具有促胰岛素分泌活性,该活性对于2型糖尿病治疗具有较好的应用前景。蛙皮抗菌肽即可以通过克服注射胰岛素产生的低血糖反应,又能改善2型糖尿病胰岛素抵抗的问题,这使其有希望成为安全、高效治疗2型糖尿病药物的新药物。本文综述了具有促胰岛素分泌功能的蛙皮抗菌肽的序列特征和工作机制的研究进展,为进一步开展相关研究提供参考。     

Antimicrobial activity,bactericidal mechanism and LPS‐neutralizing activity of the cell‐penetrating peptide pVEC and its analogs

pVEC is a cell‐penetrating peptide derived from the murine vascular endothelial‐cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)‐neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4–16 μM) against Gram‐positive and Gram‐negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 μM. These peptides induced a near‐complete membrane depolarization (more than 80%) at 4 μM against Staphylococcus aureus and a significant dye leakage (35–70%) from bacterial membrane‐mimicking liposome at a concentration as low as 1 μM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog‐derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor‐α release in LPS‐stimulated mouse macrophage RAW264.7 cells at 10 to 50 μM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS‐neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses

Innate immune mechanisms of defense are especially important to ectothermic vertebrates in which adaptive immune responses may be slow to develop. One innate defense in amphibian skin is the release of abundant quantities of antimicrobial peptides. Chytridiomycosis is an emerging infectious disease of amphibians caused by the skin fungus, Batrachochytrium dendrobatidis . Susceptibility to chytridiomycosis varies among species, and mechanisms of disease resistance are not well understood. Previously, we have shown that Australian and Panamanian amphibian species that possess skin peptides that effectively inhibit the growth of B. dendrobatidis in vitro tend to survive better in the wild or are predicted to survive the first encounter with this lethal pathogen. For most species, it has been difficult to experimentally infect individuals with B. dendrobatidis and directly evaluate both survival and antimicrobial peptide defenses. Here, we demonstrate differences in susceptibility to chytridiomycosis among four Australian species ( Litoria caerulea, Litoria chloris, Mixophyes fasciolatus and Limnodynastes tasmaniensis ) after experimental infection with B. dendrobatidis , and show that the survival rate increases with the in vitro effectiveness of the skin peptides. We also observed that circulating granulocyte, but not lymphocyte, counts differed between infected and uninfected Lit. chloris . This suggests that innate granulocyte defenses may be activated by pathogen exposure. Taken together, our data suggest that multiple innate defense mechanisms are involved in resistance to chytridiomycosis, and the efficacy of these defenses varies by amphibian species.     

Antimicrobial and anti-inflammatory activities of chemokine CXCL14-derived antimicrobial peptide and its analogs

CXCL14 is a CXC chemokine family that exhibits antimicrobial activity and contains an amphipathic cationic α-helical region in the C-terminus, a characteristic structure of antimicrobial peptides (AMPs). In this study, we designed three analogs of CXCL14_59–75 (named CXCL14-C17) corresponding to the C-terminal α-helix of CXCL14, which displayed potential antimicrobial activity against a wide variety of gram-negative and gram-positive bacteria with minimum inhibitory concentrations of 4?16?μM without mammalian cell toxicity. Furthermore, two CXCL14-C17 analogs (CXCL14-C17-a1 and CXCL14-C17-a3) with improved cell selectivity were engineered by introducing Lys, Arg, or Trp in CXCL14-C17. Additionally, CXCL14-C17 analogs showed much greater synergistic effect (FICI: 0.3125–0.375) with chloramphenicol and ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa (MDRPA) than LL-37 did (FICI: 0.75–1.125). CXCL14-C17 analogs were more active against antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), MDRPA, and vancomycin-resistant Enterococcus faecium (VREF) than LL-37 and melittin. In particular, CXCL14-C17-a2 and CXCL14-C17-a3 completely inhibited the biofilm formation at sub-MIC and all of the peptides were able to eliminate pre-formed biofilm as well. Membrane depolarization, flow cytometry, sytox green uptake, ONPG hydrolysis and confocal microscopy revealed the possible target of the native peptide (CXCL14-C17) to likely be intracellular, and the amphipathic designed analogs targeted the bacterial membrane. CXCL14-C17 also showed DNA binding characteristic activity similar to buforin-2. Interestingly, CXCL14-C17-a2 and CXCL14-C17-a3 effectively inhibited the production and expression of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 from lipopolysaccharide (LPS)-stimulated RAW264.7 cells, suggesting that these peptides could be promising anti-inflammatory and antimicrobial agents.     

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3-18) and its analog [Phe(8)]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.     

Development of a novel antimicrobial peptide, AG-30, with angiogenic properties

The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)-30, that possesses both antimicrobial and pro-inflammatory activities. AG-30 has an α-helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose-dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG-30 in vivo , we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody). These data suggest that the novel peptide, AG-30, may have therapeutic potential for ischaemic diseases.     

A peptide of the phylloseptin family from the skin of the frog Hylomantis lemur (Phyllomedusinae) with potent in vitro and in vivo insulin-releasing activity

A peptide with the ability to release insulin from the rat BRIN-BD11 clonal β cell line was isolated from norepinephrine-stimulated skin secretions of the Lemur leaf frog Hylomantis lemur Boulenger,1882. Determination of the primary structure (FLSLIPHVISALSSL.NH₂) demonstrated that the peptide belongs to the phylloseptin family whose members have previously been identified in other Phyllomedusinae species. A synthetic replicate of the peptide, termed phylloseptin-L2, produced a significant stimulation of insulin release (134% of basal rate, P < 0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (301% of basal rate, P < 0.001) at a concentration of 3 μM. Phylloseptin-L2 did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 μM, indicating that the integrity of the plasma membrane had been preserved. The stimulatory action was maintained in the absence of extracellular Ca²⁺ and in the presence of verapamil (50 μM) and diazoxide (300 μM) suggesting that mechanism of action of the peptide did not primarily involve influx of Ca²⁺ or closure of ATP-sensitive K⁺ channels. Administration of phylloseptin-L2 (50 nmol/kg body weight) into mice significantly (P < 0.05) increased total release of insulin and improved glucose tolerance during the 60 min period following an intraperitoneal injection of glucose (18 mmol/kg body weight). It is concluded that the peptide shows potential for development into a therapeutically valuable agent for the treatment of Type 2 diabetes.     

Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activities

Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment-friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components of the extract and whether they may be produced in recombinant form are unknown. Here we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Staphylococcus, Streptococcus, and Legionella species. Thus, this polypeptide displays the unprecedented feature of combining water purification and disinfectant properties. Identification of an active principle derived from the seed extracts points to a range of potential for drinking water treatment or skin and mucosal disinfection in clinical settings.     

In vitro cytotoxic,antibacterial, antifungal and urease inhibitory activities of some N4- substituted isatin-3-thiosemicarbazones

A series of 15 previously reported N⁴-substituted isatin-3-thiosemicarbazones 3a-o has been screened for cytotoxic, antibacterial, antifungal and urease inhibitory activities. Compounds 3b, 3e and 3n proved to be active in cytotoxicity assay; 3e exhibited a high degree of cytotoxic activity (LD₅₀ = 1.10 × 10^{? 5} M). Compound 3h exhibited significant antibacterial activity against B. subtilis, whereas compounds 3a, 3k and 3l displayed significant antifungal activity against one or more fungal strains i.e. T. longifusus, A. flavus and M. canis. In human urease enzyme inhibition assay, compounds 3g, 3k and 3m proved to be the most potent inhibitors, exhibiting relatively pronounced inhibition of the enzyme. These compounds, being non-toxic, could be potential candidates for orally effective therapeutic agents to treat certain clinical conditions induced by bacterial ureases like H. pylori urease. This study presents the first example of inhibition of urease by isatin-thiosemicarbazones and as such provides a solid basis for further research on such compounds to develop more potent inhibitors.     

Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna,Katsuwonus pelamis

A 3.4 kDa of antimicrobial peptide was purified from an acidified skin extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea–polyacrylamide gel electrophoresis and C₁₈ reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high sequence homology with the YFGAP (Yellowfin tuna Glyceraldehyde-3-phosphate dehydrogenase-related Antimicrobial Peptide); thus, this peptide was identified as the skipjack tuna GAPDH-related antimicrobial peptide (SJGAP). SJGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2–17.0 μg/mL), Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1–12.0 μg/mL), and against Candida albicans (MEC, 16.0 μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide is heat-stable but salt-sensitive. According to the secondary structural prediction and the homology modeling, this peptide consists of three secondary structural motifs, including one α-helix and two parallel β-strands, and forms an amphipathic structure. This peptide showed neither membrane permeabilization ability nor killing ability, but did display a small degree of leakage ability. These results suggest that SJGAP acts through a bacteriostatic process rather than bactericidal one. SJGAP is another GAPDH-related antimicrobial peptide isolated from skipjack tuna and likely plays an important role for GAPDH in the innate immune defense of tuna fish.     

Synthesis and cytotoxic and analgesic activities of some 1, 5-diaryl-3-ethoxycarbonylpyrrole derivatives

Some 1,5-diaryl-3-ethoxycarbonyl-2-methylpyrrole derivatives were obtained by reacting 1-aryl-3-ethoxycarbonylpent-1,4-diones and a suitable aniline derivative or sulfanilamide under Paal-Knorr pyrrole synthesis conditions. The cytotoxicity of the compounds was tested and all compounds, except for compound 2 h, showed a time-dependent increase in cytotoxic activity. Analgesic activities of the compounds were determined by using the tail-flick and tail-immersion methods; some of the compounds showed potent analgesic activity.     

Enantiomeric 9‐mer peptide analogs of protaetiamycine with bacterial cell selectivities and anti‐inflammatory activities

Protaetiamycine is an insect defensin, derived from the larvae of the beetle Protaetia brevitarsis. In our previous work, we designed 9‐mer peptide analogs of protaetiamycine, including 9Pbw2 (RLWLAIKRR‐NH₂), 9Pbw3 (RLWLAIWRR‐NH₂), and 9Pbw4 (RLWLAWKRR‐NH₂). 9Pbw2 and 9Pbw4 showed high antimicrobial activity without cytotoxicity, while 9Pbw3 with higher hydrophobicity compared to 9Pbw2 and 9Pbw4 showed high cytotoxicity as well as high antimicrobial activity (Shin et al., J. Pept. Sci. 2009; 15: 559–568). In this study, we investigated the anti‐inflammatory activities of 9Pbw2, 9Pbw3, and 9Pbw4 by quantitation of NO production in LPS‐stimulated RAW264.7 cells. The results showed that only 9Pbw3 has strong inhibition of NO production, implying that Trp⁷ as well as optimum level of hydrophobicity may play key roles in the anti‐inflammatory activity of 9Pbw3. In order to design potent anti‐inflammatory peptide with lower cytotoxicity as well as high stability from cleavage by protease compared to 9Pbw3, we synthesized 9Pbw3‐D , the all‐D ‐amino acid analog of 9Pbw3. 9Pbw3‐D showed less cytotoxicity against RAW264.7 cells as well as considerably stronger inhibition of NO production and inflammation‐induced cytokine production in LPS‐stimulated RAW264.7 cells than 9Pbw3. 9Pbw3‐D inhibited the gene expression of inflammatory‐induced cytokine significantly more than 9Pbw3 and showed high resistance to proteolytic digestion. Binding of 9Pbw3‐D with LPS caused higher enhancement of the FITC fluorescence as a result of its stronger interaction with LPS compared to that of 9Pbw3 and this result is in good agreement with their anti‐inflammatory activities. 9Pbw3‐D with higher anti‐inflammatory activity as well as lower cytotoxicity against mammalian cell compared to 9Pbw3 can be a potent noncytotoxic antibiotic candidates. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Temporin A and its retro-analogues: synthesis, conformational analysis and antimicrobial activities.

Temporin A (TA) is a hydrophobic peptide isolated from the skin of the European red frog Rana temporaria. Strong antimicrobial activity against gram-positive cocci and Candida, as well as its small molecular weight (10-13 aa residues), makes TA an interesting antimicrobial compound. However, its synthesis is rather problematic. Here, the synthesis of two retro-analogues of TA--retro-TA and (6-1)(7-13)-TA--is reported. The synthesis of retro-TA was performed without any problems, while during the synthesis of (6-1)(7-13)-TA problems similar to those encountered during the synthesis of TA were faced. Antimicrobial assays showed minimal inhibitory concentration (MIC) values of retro-TA to be, in most cases, only one dilution higher than those of original TA, but still remained relatively low. An analysis of the circular dichroism spectra of the peptides shows that TA and (6-1)(7-13)-TA adopt an alpha-helical structure in a hydrophobic environment, while retro-TA forms mainly unordered conformation under both hydrophobic and hydrophilic conditions. One can postulate that differences in conformation of the peptide chain might be responsible for the lower antimicrobial activity of retro-TA as compared to that of the parent molecule. In any case, retro-TA can be interesting owing to its simple and nonproblematic synthesis.     

Antimicrobial and Light‐Enhanced Antimicrobial Activities,Cytotoxicity and Chemical Variability of All Tunisian Eryngium Species

This study was performed on all Eryngium species growing in Tunisia in order to evaluate their intra and interspecies variabilities and to investigate their biological activities. These species are used in traditional medicine, and literature about the phytochemical investigations of most of them is scarce. Antimicrobial and light‐enhanced activities were tested against multiresistant microorganisms and extended spectrum beta‐lactamase producing bacteria (ESBL). All studied species showed antimicrobial effect with several MIC values lower than 70 μg/ml. Tested Eryngium species have proven to be a promising source of photoactive compounds, while light‐enhanced activity offers an alternative for the inactivation of pathogenic microorganisms which is currently subjected to a great interest. This is the first report of this activity in genus Eryngium. A significant improvement of antimicrobial activity with UV irradiation was observed, mainly for E. dichotomum, E. ilicifolium and E. triquetrum. Cytotoxicity, studied for the first time for the most species, was evaluated against cancer (J774) and non‐cancer (WI38) human cell lines. Chemical composition of volatile compounds presented in the most active crude extracts (petroleum ether extracts) of the aerial parts was investigated using GC/MS analysis and was submitted to statistical analyses. It revealed their high content of bioactive phytochemicals, particularly oxygenated sesquiterpenes like spathulenol, ledol and α‐bisabolol but also hydrocarbon sesquiterpenes such as β‐bisabolene and copaene, as well as polyacetylene derivatives such as falcarinol. Statistical analyses permitted to evaluate the interrelations between all Tunisian Eryngium species.     

Antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian medicinal plant Eremophila neglecta

Aims: To determine the antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian plant Eremophila neglecta. Methods and Results: Antimicrobial activities of serrulatane compounds 8,19‐dihydroxyserrulat‐14‐ene ( 1 ) and 8‐hydroxyserrulat‐14‐en‐19‐oic acid ( 2 ) were tested against Gram‐negative and Gram‐positive bacteria including human and veterinary pathogens and some multidrug‐resistant isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the compounds were determined by broth microdilution assay. Both compounds exhibited antibacterial activity against all Gram‐positive test strains. They showed antimycobacterial activity against isolates of Mycobacterium fortuitum and Mycobacterium chelonae. Of the five Gram‐negative bacteria tested, only Moraxella catarrhalis showed susceptibility to the compounds. Cytotoxic activities were tested in the Vero cell line. Compound 1 showed more activity than 2 in both antibacterial and cytotoxicity assays with cytotoxicity at concentrations similar to the MBC. Conclusions: Serrulatane compounds showed significant activity against medically important bacteria, with 1 exhibiting stronger antibacterial activity. However, they also displayed toxicity to mammalian cells. Significance and Impact of the Study: Serrulatanes are of interest as novel antibacterial compounds for use in biomedical applications; this study reports data obtained with a range of bacterial strains and mammalian cells, essential for assessing the capabilities and limitations of potential applicability of these compounds.     

Antimicrobial activity and conformation of gaegurin-6 amide and its analogs

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 helical structure in lipid membranes and the helical forming propensity of the peptides correlated with antimicrobial activity.     

The frog skin host-defense peptide frenatin 2.1S enhances recruitment,activation and tumoricidal capacity of NK cells

Frog skin is a source of peptides with various biological properties. Frenatin 2.1S, derived from norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus, exhibits immunostimulatory effects as demonstrated by the promotion of proinflammatory phenotypes of mononuclear cells in mouse peritoneal cavity and spleen. The aim of this study was to identify the populations of host cells sensitive to the action of frenatin 2.1S in vivo and to study its effects on their functional antitumor capacity. A single injection of frenatin 2.1S (100 μg) in BALB/c mice increased the presence of peritoneal CD11c⁺ dendritic cells and CD3⁺ T cells 24 h after administration and there was a significant increase in the number of IL-17 and CXCR3 expressing inflammatory T cells. Frenatin 2.1S treatment also increased the number of TNF-α expressing F4/80⁺ proinflammatory M1 macrophages. The most striking finding of the study is the marked increase of the number of peritoneal natural killer (NK) cells following frenatin 2.1S injection. Further, frenatin 2.1S administration led to activation of NK cells as evaluated by increased expression of NKG2D, FasL, CD69 and CD107a. The increased ratio of interferon-γ vs. IL-10 producing NK cells is further indication of the proinflammatory action of frenatin 2.1S. Peptide treatment enhanced the tumoricidal action of peritoneal NK cells on 4T1 mouse mammary carcinoma cells as revealed by the real-time automated monitoring of cell status. Our data demonstrate that frenatin 2.1S promotes activation and cytotoxic capacity of NK cells and should be regarded as a candidate for antitumor immunotherapy.