Piscidin 4, an antimicrobial peptide recently isolated from mast cells of hybrid striped bass (Morone chrysops female × Morone saxatilis male), is unusual in that it is twice as long (44 amino acids) as the typical members of the piscidin family. We previously showed that native piscidin 4 had a modified amino acid at position 20, but synthetic piscidin 4 (having an unmodified Trp at position 20) had similar potent activity against a number of both human and fish bacterial pathogens. In this study, the structure and membrane topology of synthetic piscidin 4 were examined using liposomes as model bilayers. Circular dichroism analyses revealed that it had a disordered structure in aqueous solution and folded to form a relatively weak α-helical structure in both membrane-mimetic trifluoroethanol solutions and liposome suspensions. Fluorescence data (piscidin 4 embedded in liposomes) and leakage experiments indicated that piscidin 4 interacted strongly with the hydrophobic part of the liposome. Binding of piscidin 4 to liposomes induced significant blue shifts of the emission spectra of the single Trp residue (Trp20). Quenching of Trp20 by water-soluble quencher (either acrylamide or I-) indicated that the fluorescence of Trp20 decreased more in the presence of liposomes than in buffer solution, thus revealing that Trp20 is less accessible to the quenchers in the presence of liposomes. The relative leakage abilities of piscidin 4 (1 μM) with liposomes were in the following order: DPPC (100%)≥EYPC (94%)>DPPC/DPPG (65%)>EYPC/EYPG (0%). This high activity against DPPC and EYPC liposomes was contrary to our data suggesting that piscidin 4 has a much weaker tendency to form an α-helix than other piscidins, such as piscidin 1. However, the structural similarity of protozoan membranes to EYPC liposomes might explain our discovery of the potent activity of piscidin 4 against the important skin/gill parasite ich (Ichthyophthirius multifiliis), but its negligible hemolytic activity against vertebrate membranes (hybrid striped bass or human erythrocytes). It also suggests that other conformation(s) in addition to the α-helix of this peptide may be responsible for its selective activity. This differential toxicity also suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass and may be capable of functioning extracellularly.
The jellyfish Nemopilema nomurai was hydrolyzed with papain and a novel dipeptide purified via ultrafiltration, gel filtration chromatography with Sephadex LH-20, and reverse phase chromatography using C 18 and C 12 columns. The IR, 1H NMR, 13C NMR, and MS spectrometer analyses showed that the dipeptide comprised tyrosine-isoleucine (Tyr-Ile). The IC 50 and K i values were 6.56 ± 1.12 and 3.10 ± 0.28 μM, respectively, indicating competitive inhibition of angiotensin-Ι-converting enzyme (ACE). As a novel ACE-inhibitory active peptide, Tyr-Ile may have potential for use in antihypertensive therapy.
The in vitro antioxidant activities of five melania snails in the genus Semisulcospira (S. coreana, S. forticosta, S. libertina, S. tegulata and S. gottschei) were tested in detail. The total phenolic contents of the snails ranged from 32.3±1.0 to 87.9±6.9 mg gallic acid equivalent (GAE)/g dry sample. The EC 50 values for the 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities were 2.245±0.179 mg/mL for S. coreana and 9.319±1.276 mg/mL for S. gottschei and differed significantly (P<0.05) among the tested species. The superoxide dismutase (SOD)-like activity was highest for S. gottschei at 67.2% and lowest for S. forticosta at 4.7%. However, no significant differences among the species were recognized for the peroxynitrite anion scavenging activity. Comparing the correlation coefficients between the total phenolic contents and the DPPH radical and peroxynitrite anion scavenging activities, there was a low level relationship between each activity.
Apidaecin Ib had strong antimicrobial activity against several tested Gram-negative bacteria including Escherichia coli, Enterobacter cloacae, and Shigella flexneri (MECs; 0.3-1.5 µg/mL), but showed no activity against all the tested Gram-positive bacteria including Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and one yeast, Candida albicans (MECs; >125 µg/mL). Interestingly, this peptide showed potent antibacterial activity only against Edwardsiella species (MECs; 0.6-3.6 µg/mL) among the tested fish pathogenic bacteria through a bacteriostatic process and showed no significant hemolytic activity. Apidaecin Ib took an unordered structure in all environments and also had very weak membrane perturbation activity even at 25 µM. Anti-Edwardsiella activity of apidaecin Ib is stronger than those of other antimicrobial polypeptides or antibiotics, but its activity is salt-sensitive. These results suggest that apidaecin Ib has Edwardsiella speciesspecific antibacterial activity and could be applied as new preventive or control additives for Edwardsiella species infection in freshwater fish aquaculture.
The Nemopilema nomurai hydrolysate was produced by the reaction of papain, and an angiotensin-Ι converting enzyme (ACE)-inhibitory peptide was purified by using the molecular cutoffs membrane filter, the gel filtration chromatography with Sephadex LH-20 and the reverse phase chromatographic method using C 18 and C 12 columns. Purification yield of the active peptide was estimated to be 0.2 ± 0.1%, starting from the lyophilized jellyfish. The infrared (IR), proton nuclear magnetic resonance spectroscopy (1H NMR), carbon nuclear magnetic resonance (13C NMR) and mass spectrometry (MS) spectrometer analyses elucidated that the structure of the purified peptide is tyrosine-isoleucine (Tyr-Ile). The inhibitory concentration at 50% (IC 50) and K i values were calculated to be 2.0 ± 0.3 μg/ml and 3.3 ± 0.3 μM, respectively, which acts as a competitive inhibitor to ACE.
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