Identification and characterization of two novel cathelicidins from the frog Odorrana livida.

Antimicrobial peptides (AMPs) are a group of gene-encoded small peptides that play pivotal roles in the host immune system of multicellular organisms. Cathelicidins are an important family of AMPs that exclusively exist in vertebrates. Many cathelicidins have been identified from mammals, birds, reptiles and fish. To date, however, cathelicidins from amphibians are poorly understood. In the present study, two novel cathelicidins (OL-CATH1 and 2) were identified and studied from the odorous frog Odorrana livida. Firstly, the cDNAs encoding the OL-CATHs (780 and 735 bp in length, respectively) were successfully cloned from a lung cDNA library constructed for the frog. Multi-sequence alignment was carried out to analyze differences between the precursors of the OL-CATHs and other representative cathelicidins. Mature peptide sequences of OL-CATH1 and 2 were predicted (33 amino acid residues) and their secondary structures were determined (OL-CATH1 showed a random-coil conformation and OL-CATH2 demonstrated a-helical conformation). Furthermore, OL-CATH1 and 2 were chemically synthesized and their in vitro functions were determined. Antimicrobial and bacterial killing kinetic analyses indicated that OL-CATH2 demonstrated relatively moderate and rapid antimicrobial potency and exhibited strong anti-inflammatory activity. At very low concentrations (10 µg/mL), OL-CATH2 significantly inhibited the lipopolysaccharide (LPS)-induced transcription and production of pro-inflammatory cytokines TNF-a, IL-1b and IL-6 in mouse peritoneal macrophages. In contrast, OL-CATH1 did not exhibit any detectable antimicrobial or anti-inflammatory activities. Overall, identification of these OL-CATHs from O. livida enriches our understanding of the functions of cathelicidins in the amphibian immune system. The potent antimicrobial and anti-inflammatory activities of OL-CATH2 highlight its potential as a novel candidate in anti-infective drug development.

Effects of C-terminal amidation and heptapeptide ring on the biological activities and advanced structure of amurin-9KY, a novel antimicrobial peptide identified from the brown frog, Rana kunyuensis.

Rana kunyuensis is a species of brown frog that lives exclusively on Kunyu Mountain, Yantai, China. In the current study, a 279-bp cDNA sequence encoding a novel antimicrobial peptide (AMP), designated as amurin-9KY, was cloned from synthesized double-strand skin cDNA of R. kunyuensis. The amurin-9KY precursor was composed of 62 amino acid (aa) residues, whereas the mature peptide was composed of 14 aa and contained two cysteines forming a C-terminal heptapeptide ring (Rana box domain) and an amidated C-terminus. These structural characters represent a novel amphibian AMP family. Although amurin-9KY exhibited high similarity to the already identified amurin-9AM from R. amurensis, little is known about the structures and activities of amurin-9 family AMPs so far. Therefore, amurin-9KY and its three derivatives (amurin-9KY1-3) were designed and synthesized. The structures and activities were examined to evaluate the influence of C-terminal amidation and the heptapeptide ring on the activities and structure of amurin-9KY. Results indicated that C-terminal amidation was essential for antimicrobial activity, whereas both C-terminal amidation and the heptapeptide ring played roles in the low hemolytic activity. Circular dichroism (CD) spectra showed that the four peptides adopted an a-helical conformation in THF/H2O (v/v 1:1) solution, but a random coil in aqueous solution. Elimination of the C-terminal heptapeptide ring generated two free cysteine residues with unpaired thiol groups, which greatly increased the concentration-dependent anti-oxidant activity. Scanning electron microscopy (SEM) was also performed to determine the possible bactericidal mechanisms.

[Proteomics analysis of Paragonimus skrjabini].

Objective: To perform a proteomics analysis for metacercariae, juvenile and adult worms of Paragonimus skrjabini. Methods: Crabs were collected in P. skrjabini endemic areas of Kaiyang and Bijie in Guizhou Province. Metacercariae were isolated, placed in PBS, and were used to infect three SD rats(10 metacercariae/rat) by intragastric administration and infect three male dogs(20 metacercariae/dog) through feeding. The rats were sacrificed at 1 month after infection to obtain juvenile worms. The dogs were sacrificed at 3 months after infection to obtain adult worms. The metacercariae, juvenile and adult worms were lysed, and total protein was extracted by ultrasonication. The total protein content was determined by Bradford method and separated by SDS-PAGE and two dimensional gel electrophoresis. Images of two dimensional gel electrophoresis were analyzed using the PDquest 8.0 software. The dots with difference were digested and analyzed with mass spectrometry. Finally, online searches in NCBI and local databases were performed. Results: Results of SDS-PAGE showed that the total protein of metacercariae, juvenile and adult worms was concentrated within Mr of 25 000-116 000. Fifty-one protein dots with difference were found by two dimensional gel electrophoresis, comprising of 20 dots for metacercariae, 25 for juvenile worms and 6 for adult worms. Thirty-six peptide sequences of metacercariae and juvenile worms were analyzed. They were basically determined to be Achrornobacter lyticus protease Ⅰ(a lysine-specific serine protease), ascorbate reductase protein, glutathione s-transferase DHAR2 analogue, heat shock proteins Hsp82 and Hsp96-ß, actin, cystatin, etc., by online searches, and cysteine, actin and heat shock protein by local searches in the diginea database(downloaded to a local computer from NCBI). Mass spectrometry was not performed for adult worms, as the variation of grayscale value between their spots was far less than those for metacercariae and juvenile worms. Conclusion: One difference is that the metacercariae of P. skrjabini have actin, while the juvenile worms have detoxification proteins and stress proteins. But they both have hydrolases and cysteine enzymes.