1. Molecular cloning of cDNAs encoding antimicrobial peptide precursors from the skin of the Chinese brown frog, Rana chensinensis
Dejing Shang, Fenghui Yu, Junfeng Li, Junjie Zheng, Lifang Zhang, Yang Li Zoolog Sci. 2009 Mar;26(3):220-6. doi: 10.2108/zsj.26.220.
Skin plays a key role in the daily survival of amphibians. In the present study, six cDNAs encoding amphibian skin antimicrobial peptide precursors from the Chinese brown frog Rana chensinensis, were cloned and identified as preprobrevinin-1CEc, preprobrevinin-1CEb, preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, and preprochensinin-1. Preprotemporin-1CEa, CEb, and CEc are members of the temporin family, which are usually short, hydrophobic, and C-terminally alpha-amidated antimicrobial peptides. Preprobrevinin-1CEa and CEb were identified as members of the brevinin-1 family of antimicrobial peptides, because both peptides contain a "Rana box" that is responsible for forming C-terminal Cys-bridged cyclic heptapeptides. The nucleotide and deduced amino acid sequences of preprochensinin-1 were not similar to any known amphibian skin defensive peptides. Four bioactive peptides were chemically synthesized according to the deduced amino acid sequences of six prepropeptides from R. chensinensis skin, and their antimicrobial, cytotoxic, and haemolytic properties were evaluated. All of the synthesized peptides inhibited the growth of Gram-positive bacteria. Brevinin-1CEa showed a broad spectrum of antimicrobial activity. The novel amphibian skin peptide chensinin-1 was active against Bacillus cereus and Streptococcus lactis at a concentration of 11.6 microM, but did not inhibit the growth of MCF-7 and HeLa cells at 200 microM, and had no haemolytic activity at a concentration of 500 microM. Temporin-1CEa exhibited the greatest ability to inhibit the growth of MCF-7 cells. Its antimicrobial and cytotoxic activities may be due to its high degree of alpha-helical confirmation and amphipathic nature.
2. [Cloning of cDNAs encoding skin antimicrobial peptide precursors from Chinese brown frogs, Rana chensinensis and determination of antimicrobial, anticancer and hemolysis activity]
Fenghui Yu, Lifang Zhang, Junfeng Li, Xiaofan Li, Xin Fu, Dejing Shang Sheng Wu Gong Cheng Xue Bao. 2009 Jan;25(1):101-8.
Amphibian skin antimicrobial peptides exhibit a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacterium and cytotoxic activity responsible for inhibiting the growth of cancer cells. In this present study, six cDNAs encoding antimicrobial peptide precursors were cloned from the skin of Chinese brown frog, Rana chensinensis by RT-PCR and 3'-RACE procedure and identified as preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, preprobrevinin-1CEa, preprobrevinin-1CEb, and preprochensinin-1, respectively. The nucleotide sequences of cDNA encoding 59-65 amino acid composed of 289-315 bp. Preprotemporin-1CEa, preprotemporin-1CEb and preprotemporin-1CEc are members of temporin family, which usually are short, hydrophobic, and C-terminally alpha-amidated antimicrobial peptides. Preprobrevinin-1CEa and preprobrevinin-1CEb were identified as the members of the brevinin-1 family of antimicrobial peptides since both peptides contain "RANA box" that it's responsible for forming Cys-bridged cyclic heptapeptides at the C-terminal region of peptide. The nucleotide acid sequence and the deduced amino acid Sequence of preprochensinin-1 were not found to be identity with any known amphibian skin defensive peptides, so, preprochensinin-1 was identified as a novel peptide precursor. Four of bioactive peptides: temporin-1CEa, temporin-1CEb, brevinin-1CEa and chensinin-1 were synthesized to investigate their antimicrobial, anticancer and haemolysis activities. The results showed that all of the synthesized antimicrobial peptides in this study inhibited the growth of the Gram-positive bacterium, and exhibited the anticancer activity against the growth of MCF-7 cells and HeLa cells. Analysis of the R. chensinensis bioactive peptides and their gene expression will be beneficial for preservation of this species.
3. Molecular cloning and functional characterization of novel antimicrobial peptides from the skin of brown frog, Rana zhenhaiensis
Baohua Xu, Helong Che, Lumei Kang, Shuangyan Zheng, Songniu Mu, Fusheng Wan Zoolog Sci. 2012 Sep;29(9):553-8. doi: 10.2108/zsj.29.553.
Rana zhenhaiensis, a species of brown frog, is widely distributed in central and south China. In the present study, a total of 14 cDNA sequences encoding eight novel antimicrobial peptides (AMPs) were cloned from the synthesized cDNAs of R. zhenhaiensis skin. The eight novel AMPs belong to four families: brevinin-1 (four peptides), brevinin-2 (one peptide), ranatuerin-2 (one peptide) and chensinin-1 (two peptides), five AMPs from the four families (brevinin-1ZHa, brevinin-1ZHb, brevinin-2ZHa, ranatuerin-2ZHa and chensinin-1ZHa) were chemically synthesized, their antimicrobial and hemolytic activities were examined. The results indicated that the five AMPs possess different antimicrobial and hemolytic activities. Of these, brevinin-2ZHa exhibited the strongest and most broad-spectrum antimicrobial activity. Furthermore, scanning electron microscopy (SEM) experiment was carried out to investigate the potential antimicrobial mechanism of chensinin-1ZHa. The result indicated that chensinin-1ZHa may exert its function through disruption of the bacterial membrane.
