A new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.     

Amphibian tachykinin precursor

The precursor of amphibian tachykinin has not been found although more than 30 tachykinins have been isolated from amphibians since 1964. In this report, two tachykinin-like peptides are identified from the skin secretions of the frog, Odorrana grahami. Their amino acid sequences are DDTEDLANKFIGLM-NH(2) (named tachykinin OG1) and DDASDRAKKFYGLM-NH(2) that is the same with ranamargarin found in Rana margaretae, respectively, with a conserved FXGLM-NH(2) C-terminal consensus motif. By cDNA cloning, their precursors were screened from the skin cDNA library of O. grahami. The precursors are composed of 61 amino acid (aa) residues including a signal peptide followed by an acidic spacer peptide and one copy of mature tachykinin-like peptide. Their overall structure is different from structures of other tachykinin precursors such as human protachykinin 1 precursor containing 143 aa including one copy of substance P (SP) and neurokinin A (NKA), and ascidian tachykinin 1 precursor containing 164 aa including two copies of tachykinin-like peptides. The current results demonstrate that the biosynthesis mode of tachykinins in amphibians is different from other animals.     

雌二醇对大仓鼠胁腺抑制作用的研究

通过研究发现雌二醇对雌性大仓鼠(Cricetulus triton)胁腺的大小和分泌活动存在明显的抑制作用。利用生理手术, 形成3组不同内分泌状况的鼠: 卵巢切除(OF)、正常(IF)、和卵巢切除后又置入雌二醇硅胶管 (OEF) 的雌性大仓鼠; 经过放射免疫方法测定表明由OF、IF到OEF 鼠血液雌二醇的浓度显著增高(P <0.01), 胁腺的重量、长度、宽度和厚度也显著减小(P< 0.05或P <0.01); 利用气相色谱对胁腺分泌物的分析发现, 从OF、IF到OEF鼠的胁腺分泌物的种类逐渐减少 , 有些成分尽管在 3 组不同生理状况的鼠中都存在, 但含量依次明显减少。以上结果表明雌二醇不仅能够抑制胁腺大小的增长, 而且也抑制了胁腺的分泌活动,从而减少了胁腺分泌物或其中某些成分的分泌量。     

Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins

Amphibian skins act as the first line against noxious aggression by microorganisms, parasites, and predators. Anti-microorganism activity is an important task of amphibian skins. A large amount of gene-encoded antimicrobial peptides (AMPs) has been identified from amphibian skins. Only a few of small protease inhibitors have been found in amphibian skins. From skin secretions of 5 species (Odorrana livida, Hylarana nigrovittata, Limnonectes kuhlii, Odorrana grahami, and Amolops loloensis) of Ranidae frogs, 16 small serine protease inhibitor peptides have been purified and characterized. They have lengths of 17-20 amino acid residues (aa). All of them are encoded by precursors with length of 65-70 aa. These small peptides show strong trypsin-inhibitory abilities. Some of them can exert antimicrobial activities. They share the conserved GCWTKSXXPKPC fragment in their primary structures, suggesting they belong to the same families of peptide. Signal peptides of precursors encoding these serine protease inhibitors share obvious sequence similarity with those of precursors encoding AMPs from Ranidae frogs. The current results suggest that these small serine protease inhibitors are the common defensive compounds in frog skin of Ranidae as amphibian skin AMPs.     

A novel bradykinin-like peptide from skin secretions of rufous-spotted torrent frog, Amolops loloensis

A bradykinin-like peptide has been isolated from skin secretions of rufous-spotted torrent frog, Amolops loloensis. This bradykinin-like peptide was named amolopkinin. Its primary structure, RAPVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Amolopkinin is composed of 12 amino acid residues and is related to bradykinin composed of nine amino acid residues, identified from the skin secretions of Odorrana schmackeri. Amolopkinin was found to elicit concentration-dependent contractile effects on isolated guinea pig ileum. cDNA clones encoding the precursor of amolopkinin were isolated by screening a skin cDNA library of A. loloensis and then sequenced. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that a deficiency of an18-nucleotide fragment (TCAAGAATGATCAGACGC in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in absence of a di-basic site for trypsin-like proteinases and an unusual - APV - insertion in the N-terminal part of amolopkinin. This is the first report of a bradykinin-like peptide comprised of bradykinin with an insertion in its N-terminal part. Our results demonstrate the hypervariability of amphibian bradykinin-like peptides, as well as the diversity of antimicrobial peptides in amphibians.     

Peptidomics and genomics analysis of novel antimicrobial peptides from the frog,Rana nigrovittata

Much attention has been paid on amphibian peptides for their wide-ranging pharmacological properties, clinical potential, and gene-encoded origin. More than 300 antimicrobial peptides (AMPs) from amphibians have been studied. Peptidomics and genomics analysis combined with functional test including microorganism killing, histamine-releasing, and mast cell degranulation was used to investigate antimicrobial peptide diversity. Thirty-four novel AMPs from skin secretions of Rana nigrovittata were identified in current work, and they belong to 9 families, including 6 novel families. Other three families are classified into rugosin, gaegurin, and temporin family of amphibian AMP, respectively. These AMPs share highly conserved preproregions including signal peptides and spacer acidic peptides, while greatly diversified on mature peptides structures. In this work, peptidomics combined with genomics analysis was confirmed to be an effective way to identify amphibian AMPs, especially novel families. Some AMPs reported here will provide leading molecules for designing novel antimicrobial agents.     

Multiple bombesin-like peptides with opposite functions from skin of Odorrana grahami

Bombesin-like peptides (BLPs) are a family of neuroendocrinic peptides that mediate a variety of biological activities. Three mature BLPs from the skin secretions of the frog Odorrana grahami were purified. Several bombesin-like peptide cDNA sequences encoding precursors of BLPs were identified from the skin cDNA library of O. grahami. This is the maximal diversity of BLPs ever found in animals. Five mature BLPs (B1-B5) based on the amino acid sequences derived from the cDNA cloning were synthesized. In the in vitro myotropic contraction experiment, all synthesized BLPs displayed a stimulating effect toward rat stomach strips, except B4 and B5 which showed the opposite effect, suggesting that certain BLPs may act as antagonists of bombesin receptors while most other BLPs act as agonists. This finding will facilitate the finding of novel bombesin receptors and novel ligands of bombesin receptors. The diversity of amphibian BLPs and their precursors were also analyzed and results suggest that amphibian BLPs and corresponding precursors of various sizes and processing patterns can be used as markers of taxonomic and molecular phylogenetics. The remarkable similarity of preproregions gives rise to very different BLPs and 3'-terminal regions in distantly related frog species, suggesting that the corresponding genes form a multigene family originating from a common ancestor. The diversification of BLP loci could thus be part of an evolutionary strategy developed by amphibian species as a result of shifts to novel ecological niches when environmental factors change rapidly.     

Circ_0025039 acts an oncogenic role in the progression of non-small cell lung cancer through miR-636-dependent regulation of CORO1C

Non-small cell lung cancer remains the leading cause of cancer-related death worldwide. Circular RNA plays vital roles in NSCLC progression. This study is designed to reveal the role of circ_0025039 in NSCLC cell malignancy. The RNA expression of circ_0025039, microRNA-636 (miR-636), and coronin 1C was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by Western blot analysis or immunohistochemistry assay. Cell proliferation, migration, invasion, tube formation ability, sphere formation capacity, and apoptosis were investigated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine, transwell assay, tube formation assay, sphere formation assay, and flow cytometry analysis, respectively. Mouse model assay was conducted to reveal the effect of circ_0025039 silencing on tumor formation in vivo. The interaction between miR-636 and circ_0025039 or CORO1C was identified through dual-luciferase reporter and RNA pull-down assays. The expression of circ_0025039 and CORO1C was significantly increased, while miR-636 was decreased in NSCLC tissues and cells compared with controls. Circ_0025039 depletion repressed NSCLC cell proliferation, migration, invasion, tube-forming capacity, and sphere formation ability, but induced cell apoptosis. The neoplasm formation was repressed after circ_0025039 silencing. Additionally, circ_0025039 acted as a sponge for miR-636, which was found to target CORO1C. Importantly, the contribution of circ_0025039 to NSCLC progression was mediated by miR-636/CORO1C axis. Circ_0025039 silencing repressed NSCLC malignant progression by reducing CORO1C expression through miR-636, showing the possibility of circ_0025039 as a therapeutic target for NSCLC.

     

A small trypsin inhibitor from the frog of Odorrana grahami

A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found.