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A novel Kunitz-type neurotoxin peptide that inhibited voltage-gated sodium channel was purified and characterized from the skin secretions of rufous-spotted torrent frog, Amolops loloensis. It has a 240-bp cDNA encoding an 79-amino acid residue (aa) precursor protein containing 6 half-cysteines. The precursor was proven to release a 57-aa mature peptide with amino acid sequence, DRNPICNLPPKEGFCLWMMRRSFFNPSKGRCDTFGYRGCGGNKNNFETPRACKEACG. The mature was named amotoxin. Amotoxin shares sequence homology with other Kunitz-type toxins and also has three cysteine bridges. Amotoxin showed an inhibitory ability against trypsin with an inhibitory constant (Ki) of 0.087 μM. To the best of our knowledge, this is the first gene-encoded neurotoxin found in Amolops loloensis. Recombinant amotoxin showed similar functional properties as the native amotoxin. The functional properties of amotoxin may provide insights into the ecological adaptation of amphibians and deepen our understanding about the biological function spectrum of amphibian skin peptides., Competing Interests: Declaration of competing interest The authors claim no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Several studies indicated that Odorrana grahami (O. grahami) skin contains abundant antimicrobial peptides, and the skin was recognized as hostile habitat for microorganisms. In this study, the microbial community was evaluated by 16S rRNA gene sequencing, and two associated bacterial isolates were obtained and characterized from the skin of O. grahami. Sixteen bacterial genera were identified from the O. grahami skin by uncultured clone sequences. The dominant groups were Comamonas, Bacillus and Morganella, and the genus Comamonas was the most abundant group (41.7% of the total) of the community. Fortunately, strains CW-25 T and CW-518 belonging to genus Comamonas were isolated by plating dilutions. The polyphasic taxonomy results indicated that strain CW-25 T was a member of Comamonas aquatica, it showed much higher antimicrobials resistance than the closest C. aquatica strains of LMG 2370 T , LMG5937 and LMG 6112 isolated from freshwater. Based on the polyphasic taxonomic studies and antimicrobials resistance characteristics, two subspecies of Comamonas aquatica subsp. aquatica nov. and Comamonas aquatica subsp. rana nov. were proposed. The super-antimicrobial resistance endows the strains of Comamonas aquatica subsp. rana inhabit the O. grahami skin, and the primary defense of O. grahami might be composed by the antimicrobial peptides and the native bacteria.

The diversity of defensive peptides from skin of amphibians has been demonstrated. These peptides may have resulted from the diversity of microorganisms encountered by amphibians. In this study, peptidomics and RNA sequencing analyses were used to study deeply the defensive peptides of the skin secretions from Polypedates megacephalus. A total of 99 defensive peptides have been identified from the skin secretions. Among these peptides, 3 peptides were myotropical peptides and 34 peptides classified as protease inhibitor peptides. 5 lectins, 8 antimicrobial peptides, 26 immunomodulatory peptides, 10 wound-healing peptides and 13 other bioactive peptides were identified as belonging to the innate immune system. One antimicrobial peptide Pm-amp1 showed high similarity to antimicrobial peptide marcin-18. This peptide was successfully expressed and showed moderate activity against four tested strains. These identified peptides highlight the extensive diversity of defensive peptides and provide powerful tools to understand the defense weapon of frog.

Albeit many bioactive peptides have been reported from amphibian skins, no anti-platelet peptide has been identified till to date. Here, an anti-platelet peptide, namely Zongdian platelet inhibitor (ZDPI), with the molecular weight of 1798.6 Da, was purified and characterized from skin secretions of the frog, Amolops loloensis. The amino acid sequence of ZDPI was determined as FRGCWLKNYSPRGCL-NH2 by combination methods of Edman degradation, mass spectrometry analysis and carboxypeptidase Y treatment revealing that it is composed of 15 amino acid residues with two cysteines formed an intra-molecular disulfide bridge and C-terminal amidation. cDNA encoding ZDPI precursor was cloned from skin cDNA library of A. loloensis. The precursor is composed of 63 amino acid (aa) residues including the predicted signal peptide (22 aa), an acidic spacer peptide (19 aa), and mature ZDPI. BLAST search indicates that ZDPI belongs to antimicrobial peptide family of ranacyclin, peptide leucine arginine or odorranain. It was found to inhibit ADP-induced platelet aggregation in a dose-dependent manner. At the concentration of 32 μg/ml, ZDPI completely inhibited platelet aggregation induced by ADP. To the best of our knowledge, this is the first report about an anti-platelet peptide from amphibian skin secretions. Considering its strong inhibitory ability on platelets and simple structure, ZDPI might be an excellent candidate or template to develop anti-thrombosis agent. In addition, the discovery of anti-platelet peptide in the frog skin increases biological function spectrum of amphibian skin peptides.

As a group of intestinal hormones and neurotransmitters, cholecystokinins (CCKs) regulate and affect pancreatic enzyme secretion, gastrointestinal motility, pain hypersensitivity, digestion and satiety, and generally contain a DYMGWMDFG sequence at the C-terminus. Many CCKs have been reported in mammals. However, only a few have been reported in amphibians, such as Hyla nigrovittata, Xenopus laevis, and Rana catesbeiana, with none reported in urodele amphibians like newts and salamanders. Here, a CCK called CCK-TV was identified and characterized from the skin of the salamander Tylototriton verrucosus. This CCK contained an amino acid sequence of DYMGWMDF-NH2 as seen in other CCKs. A cDNA encoding the CCK precursor containing 129 amino acid residues was cloned from the cDNA library of T. verrucosus skin. The CCK-TV had the potential to induce the contraction of smooth muscle strips isolated from porcine gallbladder, eliciting contraction at a concentration of 5.0 x 10⁻¹¹ mol/L and inducing maximal contraction at a concentration of 2.0 x 10⁻⁶ mol/L. The EC50 was 13.6 nmol/L. To the best of our knowledge, this is the first report to identify the presence of a CCK in an urodele amphibian.

Delayed wound healing will result in the development of chronic wounds in some diseases, such as diabetes. Amphibian skins possess excellent wound-healing ability and represent a resource for prospective wound-healing promoting compounds. A potential wound-healing promoting peptide (CW49; amino acid sequence APFRMGICTTN) was identified from the frog skin of Odorrana grahami. It promotes wound healing in a murine model with a full-thickness dermal wound in both normal and diabetic animals. In addition to its strong angiogenic ability with respect to the upregulation of some angiogenic proteins, CW49 also showed a significant anti-inflammatory effect in diabetic wounds, which was very important for healing chronic wounds. CW49 had little effect on re-epithelialization, resulting in no significant effect on wound closure rate compared to a vehicle control. Altogether, this indicated that CW49 might accelerate diabetic wound healing by promoting angiogenesis and preventing any excessive inflammatory response. Considering its favorable traits as a small peptide that significantly promotes angiogenesis, CW49 might be an excellent candidate or template for the development of a drug for use in the treatment of diabetic wounds., (© 2014 FEBS.)

Although it is well known that wound healing proceeds incredibly quickly in urodele amphibians, such as newts and salamanders, little is known about skin-wound healing, and no bioactive/effector substance that contributes to wound healing has been identified from these animals. As a step toward understanding salamander wound healing and skin regeneration, a potential wound-healing-promoting peptide (tylotoin; KCVRQNNKRVCK) was identified from salamander skin of Tylototriton verrucosus. It shows comparable wound-healing-promoting ability (EC50=11.14 μg/ml) with epidermal growth factor (EGF; NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR) in a murine model of full-thickness dermal wound. Tylotoin directly enhances the motility and proliferation of keratinocytes, vascular endothelial cells, and fibroblasts, resulting in accelerated reepithelialization and granulation tissue formation in the wound site. Tylotoin also promotes the release of transforming growth factor β1 (TGF-β1) and interleukin 6 (IL-6), which are essential in the wound healing response. Gene-encoded tylotoin secreted in salamander skin is possibly an effector molecule for skin wound healing. This study may facilitate understanding of the cellular and molecular events that underlie quick wound healing in salamanders., (© FASEB.)

Two novel analgesic peptides (Analgesin-HJ, FWPVI-NH2 and Analgesin-HJ(I5T), FWPVT-NH2) were identified from the skin of the tree frog, Hyla japonica. There are 171 amino acid residues in the precursor encoding analgesin-HJs. The precursor contains 10 copies of mature peptide, which include 9 copies of analgesin-HJ and one copy of analgesin-HJ(I5T). Results from analgesic experiments using mice models including abdominal writhing induced by acetic acid, formalin-induced paw licking, and thermal pain test indicated that this two peptides exerted comparable analgesic activities with morphine. In addition, they had ability to inhibit inflammatory factor secretion induced by lipopolysaccharides (LPS). Considering their easy production, storage, transfer and potential analgesic activity, analgesin-HJs might be exciting leading compounds or templates for the development of novel analgesic agent. In addition, this study might facilitate to understand skin defensive mechanism of amphibians., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Cutaneous wound healing is a dynamic, complex, and well-organized process that requires the orchestration of many different cell types and cellular processes. Transforming growth factor β1 is an important factor that plays a key role during wound healing. Amphibian skin has been proven to possess excellent wound healing ability, whilst no bioactive substrate related to it has ever been identified. Here, a potential wound healing-promoting peptide (AH90, ATAWDFGPHGLLPIRPIRIRPLCG) was identified from the frog skin of Odorrana grahami. It showed potential wound healing-promoting activity in a murine model with full thickness dermal wound. AH90 promoted release of transforming growth factor β1 through activation of nuclear factor-κB and c-Jun NH2-terminal kinase mitogen-activated protein kinases signaling pathways, while inhibitors of nuclear factor-κB and c-Jun NH2-terminal kinase inhibited the process. In addition, the effects of AH90 on Smads family proteins, key regulators in transforming growth factor β1 signaling pathways, could also be inhibited by transforming growth factor β1 antibody. Altogether, this indicated that AH90 promoted wound healing by inducing the release of transforming growth factor β1. This current study may facilitate the understanding of effective factors involved in the wound repair of amphibians and the underlying mechanisms as well. Considering its favorable traits as a small peptide that greatly promoting generation of endogenous wound healing agents (transforming growth factor β1) without mitogenic effects, AH90 might be an excellent template for the future development of novel wound-healing agents., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Most of amphibians belonging to family Rhacophoridae live in arboreal habitats. A large number of antimicrobial peptides (AMPs) have been identified from amphibian skins. No antimicrobial peptide from Rhacophoridae amphibians has been reported. In this study, we purified and characterized a novel antimicrobial peptide, pleurain-a1-thel from skin secretions of the tree frog, Theloderma kwangsiensis. Its amino acid sequence was determined as RILTMTKRVKMPQLYKQIVCRLFKTC by Edman degradation, mass spectrometry analysis and cDNA cloning. There are two cysteines, which form an intra-molecular disulfide bridge, in the sequence of pleurain-a1-thel. Pleurain-a1-thel exerted potential antimicrobial activities against wide spectrum of microorganisms, including Gram-negative and -positive bacteria and fungi. It exerted little hemolytic activity in human or rabbit red cells. To the best of our knowledge, this is the first report of antimicrobial peptide from Rhacophoridae amphibians.

A 255-bp cDNA encoding an 84-amino acid residue (aa) precursor protein containing 8 half-cysteines was cloned from the skin of the frog, Ceratophrys calcarata. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 63-aa mature peptide with amino acid sequence, NVTPATKPTPSKPGYCRVMDELILCPDPPLSKDLCKNDSDCPGAQKCCYRTCIMQCLPPIFRE. The mature was named ceratoxin. Ceratoxin shares significant sequence similarity with the toxin family of waprins containing the whey acidic protein-type (WAP) four-disulfide core domain found in snake venoms. Antimicrobial and trypsin-inhibitory abilities of recombinant ceratoxin were tested. Recombinant ceratoxin showed strong antimicrobial activities against wide spectrum of microorganisms including Gram-negative and Gram-positive bacteria and fungi. It had no serine protease-inhibitory activity. The current results suggested that the snake venom-like waprin with antimicrobial activities in the frog skin plays a role in innate immunity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Antimicrobial peptide diversity has been found in some amphibians. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms encountered by amphibians. Peptidomics and genomics analyses were used to study antimicrobial peptide diversity in the skin secretions of the torrent frog, Amolops jingdongensis. Thirty-one antimicrobial peptides belonging to nine groups were identified in the skin secretions of this frog. Among them, there are two novel antimicrobial groups (jingdongin-1 and -2) with unique structural motifs. The other seven groups belong to known antimicrobial peptide families, namely brevinin-1, brevinin-2, odorranain-F, esculentin-2, temporin, amolopin-3, and ranacyclin. Combined with previous reports, more than 13 antimicrobial peptide groups have been identified from the genus Amolops. Most of these antimicrobial peptide groups are also found in amphibians belonging to the genus Rana or Odorrana which suggests a possible evolutionary connection among Amolops, Rana, and Odorrana. Two novel antimicrobial groups (jingdongin-1 and -2) were synthesized and their antimicrobial activities were assayed. Some of them showed strong antimicrobial abilities against microorganisms including Gram-negative and -positive bacteria, and fungi. The extreme diversity of antimicrobial peptides in the Amolops amphibians was demonstrated. In addition, several novel peptide templates were provided for antimicrobial agent design.

Five novel antimicrobial peptides (temporin-LK1, rugosin-LK1, rugosin-LK2, gaegurin-LK1, and gaegurin-LK2) are purified and characterized from Kuhl's wart frog skin secretions, Limnonectes kuhlii. They share obvious similarity to temporin, rugosin, and gaegurin antimicrobial peptide family, respectively. Their amino acid sequences were determined by Edman degradation and mass spectrometry, and further confirmed by cDNA cloning. Nine cDNA sequences encoding precursors of these five purified antimicrobial peptides and other four hypothetical antimicrobial peptides were cloned from the skin cDNA library of L. kuhlii. The deduced precursors are composed of a predicted signal peptide, an acidic spacer peptide, and a mature antimicrobial peptide. Most of them showed strong antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi. The current work identified and characterized three families of antimicrobial peptides from L. kuhlii skins and confirmed that the genus of Limnonectes amphibians share similar antimicrobial peptide families with the genus of Rana amphibians. In addition, a unique antimicrobial peptide (temporin-LK1) with 17 residues including four phenylalanines, which is significantly different from other temporins (16 residues, one or two phenylalanines), was identified in this work. Such unique structure might provide novel template or leading structure to design antimicrobial agents.

A novel bioactive peptide (polypedarelaxin 1) was identified from the skin secretions of the tree frog, Polypedates pingbianensis. Polypedarelaxin 1 is composed of 21 amino acid residues with a sequence of QGGLLGKVSNLANDALGILPI. Its primary structure was further confirmed by cDNA cloning and mass spectrometry analysis. Polypedarelaxin 1 was found to elicit concentration-dependent relaxation effects on isolated rat ileum. It has no antimicrobial and serine protease inhibitory activities. BLAST search revealed that polypedarelaxin 1 did not show similarity to known proteins or peptides. Especially, polypedarelaxin 1 do not contain conserved structural motifs of other amphibian myotropic peptides, such as bradykinins, bombesins, cholecystokinin (CCK), and tachykinins, indicating that polypedarelaxin 1 belongs to a novel family of amphibian myotropic peptide., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

A novel myotropic peptide, polypedatein, was purified and characterized from the skin secretions of the tree frog, Polypedates pingbianensis. Its primary structure, TLLCKYFAIC, was determined by Edman degradation and mass spectrometry. Polypedatein was subjected to bioassays including myotropic, antimicrobial, and serine protease inhibitory activities, which are related with many amphibian skin bioactive peptides. It was found to elicit concentration-dependent contractile effects on isolated rat ileum. cDNA clones encoding the precursor of polypedatein were isolated by screening a skin cDNA library of P. pingbianensis and then sequenced. The amino acid sequence deduced from the cDNA sequences matches well with the result from Edman degradation. BLAST search revealed that the sequence of polypedatein did not show similarity to known protein or peptide sequences. Especially, polypedatein does not contain conserved structural motifs of other amphibian myotropic peptides, such as bradykinins, bombesins, cholecystokinin (CCK), and tachykinins, indicating that polypedatein belongs to a novel amphibian myotropic peptide family. The signal peptide of the precursor encoding polypedatein shows significant sequence identity to that of other amphibian skin defensive peptides, such as antimicrobial peptides, bradykinins, lectins, and serine protease inhibitors, suggesting that polypedatein belongs to a novel amphibian myotropic peptide family. Polypedatein is also the first bioactive peptide from the genus of the frog, Polypedates., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

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.

Tree frogs produce a variety of skin defensive chemicals against many biotic and abiotic risk factors for their everyday survival. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testings, 27 peptides or proteins belonging to 9 families, which act mainly as defensive functions, were identified and characterized from skin secretions of the tree frog, Hyla simplex. They are: (1) a novel family of peptides with EGF- and VEGF-releasing activities; (2) a novel family of analgesic peptides; (3) a family of neurotoxins acting on sodium channel; (4) a snake venom-like presynaptically active neurotoxin; (5) a snake venom-like neurotoxin targeting cyclic nucleotide-gated ion channels; (6) a tachykinin-like peptide, which is the first report from tree frogs; (7) two antimicrobial peptides; (8) a alpha-1-antitrypsin-like serpin; and (9) a wasp venom-like toxin with serine protease inhibitors activity. Families of 1, 2, 4, 5, and 8 proteins or peptides are first reported in amphibians. The chemical array in the tree frog skin shares some similarities with snake venoms. Most of these components in this tree frog help defend against predators, heal wounds, or attenuate suffering.

Two novel antimicrobial peptides with similarity to brevinin-2 family are purified and characterized from the skin secretions of the frog, Rana nigrovittata. Their amino acid sequences were determined as GAFGNFLKGVAKKAGLKILSIAQCKLSGTC (brevinin-2-RN1) and GAFGNFLKGVAKKAGLKILSIAQCKLFGTC (brevinin-2-RN2), respectively, by Edman degradation. Different from brevinin-2, which is composed of 33 amino acid residues (aa), both brevinin-2-RN1 and -RN2 contain 30 aa. Five cDNA sequences (Genbank accession numbers, EU136465-9) encoding precursors of brevinin-2-RN1 and -RN2 were screened from the skin cDNA library of R. nigrovittata. These precursors are composed of 72 aa including a predicted signal peptide, an acidic spacer peptide, and a mature brevinin-2-RN. Both brevinin-2-RN1 and -RN2 showed strong antimicrobial activities against gram-positive and gram-negative bacteria and fungi. The current work identified and characterized two novel antimicrobial peptides with unique primary structure., (Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.)

The question of how amphibians can protect themselves from reactive oxygen species when exposed to the sun in an oxygen-rich atmosphere is important and interesting, not only from an evolutionary viewpoint, but also as a primer for researchers interested in mammalian skin biology, in which such peptide systems for antioxidant defense are not well studied. The identification of an antioxidant peptide named antioxidin-RL from frog (Odorrana livida) skin in this report supports the idea that a peptide antioxidant system may be a widespread antioxidant strategy among amphibian skins. Its ability to eliminate most of the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical tested within 2 s, which is much faster than the commercial antioxidant factor butylated hydroxytoluene, suggests that it has a potentially large impact on redox homeostasis in amphibian skins. Cys10 is proven to be responsible for its rapid radical scavenging function and tyrosines take part in the binding of antioxidin-RL to radicals according to our nuclear magnetic resonance assay., ((c) 2010 Elsevier Inc. All rights reserved.)

While investigating antimicrobial peptide diversity of Amolops loloensis, five novel antimicrobial peptides belonging to two families were identified from skin secretions of this frog. The first family including two members is esculentin-2-AL (esculentin-2-ALa and -ALb); the second family including three members is temporin-AL (temporin-ALd to -ALf). The family of esculentin-2-AL is composed of 37 amino acid residues (aa); the family of temporin-AL is composed of 16, 13 and 10 aa, respectively. All of these antimicrobial peptides showed antimicrobial activities against tested microorganisms. cDNAs encoding precursors of esculentin-2-ALs and temporin-ALs were cloned from the skin cDNA library of A. loloensis. All the precursors share similar overall structures. There is a typical prohormone processing signal (Lys-Arg) located between the acidic propiece and the mature peptide. The antimicrobial peptide family of esculentin-2 is firstly reported in the genus of Amolops. Combined with previous reports, a total of four antimicrobial peptide families have been identified from the genus of Amolops; three of them are also found in the genus of Rana. These results suggest the possible evolutionary connection between the genera Amolops and Rana.

While investigating the innate defense of brackish water-living amphibian and its comparison with freshwater-living amphibians, two novel 12-residue antimicrobial peptides were purified from the skin secretions of the crab-eating frog, Fejervarya cancrivora which typically inhabits brackish water of mangrove forests of Southeast Asia. These two antimicrobial peptides, tigerinin-RC1 and -RC2 share significant structural similarity with tigerinins found in the skin of Indian frog, Hoplobatrachus tigerinus. cDNAs encoding tigerinin-RC1 and -RC2 were also cloned from the skin cDNA library of F. cancrivora. Tigerinin-RC precursors are composed of 71 amino acid residues including a signal peptide, acidic spacer peptide, which are very similar to other amphibian antimicrobial peptide precursors and mature tigerinin-RC. The current results confirmed that both amphibians inhabiting freshwater and brackish water share the same antimicrobial peptide family to exert innate defense. Furthermore, the current work was also the first report of precursor and cDNA cloning of the tigerinin antimicrobial peptide family.

It is generally agreed that reactive oxygen species (ROS) contribute to skin aging, skin disorders, and skin diseases. Skin possesses an extremely efficient antioxidant system. This antioxidant activity is conferred by two systems: antioxidant enzymes and small molecules that can scavenge ROS by donating electrons. No gene-encoded secreted ROS scavengers have been reported. Amphibian skin is a multifunctional organ acting in defense, respiration, and water regulation, although it seems susceptible. Amphibian skins are easily harmed by biological or non-biological attacks such as microorganism infection or radiation injury. Among vertebrates, skins of amphibian are exposed to more dangers of radiation injury than others. Radiation toxicity occurs by directly attacking the genetic material and/or by generating ROS. In addition, amphibian skin respiration and inflammatory response also induce ROS generation. It is rational to hypothesize that amphibian skins should have potent free radical scavenging and radioprotective ability for their survival. Rana pleuraden is distributed in Southwest of China; it lives in the subtropical plateau (altitude around 2300 m) where there is strong ultraviolet radiation and long duration of sunshine. By peptidomics and genomics approaches, a large amount of antioxidant peptides belonging to 11 different groups with variable structures were isolated from the skin secretions of R. pleuraden. Their free radical scavenging and anti-inflammatory abilities were studied. All of these peptide share highly homologous preproregions, although mature antioxidant peptides have very divergent primary structures, suggesting the possibility of a common ancestor. Some peptides were also found to have multifunctional properties, such as combined antioxidant, anti-inflammatory, and antimicrobial activities. According to our knowledge, no gene-encoded specific antioxidant peptides have been reported except metallothionein. Our work possibly reveals a new skin antioxidant system. The current work also provides a large amount of peptide candidates with medical-pharmaceutical significance.

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Sichuan region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the rufous-spotted torrent frog, Amolops loloensis. Members of the new peptide family named amolopins are composed of 18 amino acids with a unique sequence, for example, NILSSIVNGINRALSFFG. By BLAST search, amolopins did no show similarity to any known peptides. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined with beta-sheet in water, Tris-HCl or Tris-HCl-SDS. Several cDNAs encoding amolopins were cloned from the skin cDNA library of A. loloensis. The precursors of amolopin are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin 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 amolopins 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.

A novel antimicrobial peptide named odorranain-NR was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLSGILGAGKHIVCGLTGCAKA. Odorranain-NR was classified into a novel family of antimicrobial peptide although it shared similarity with amphibian antimicrobial peptide family of nigrocin. Odorranain-NR has an unusual intramolecular disulfide-bridged hexapeptide segment that is different from the intramolecular disulfide-bridged heptapeptide segment at the C-terminal end of nigrocins. Furthermore, the -AKA fragment at the C-terminal of odorranain-NR is also different from nigrocins. Three different cDNAs encoding two odorranain-NR precursors and only one mature odorranain-NR was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms except Escherichia coli (ATCC25922). Its antimicrobial mechanisms were investigated by transmission electron microcopy. Odorranain-NR exerted its antimicrobial functions by various means depending on different microorganisms.

Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, Bombina microdeladigitora. The cDNAs encoding BMSIs were cloned from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI 1 and BMSI 2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI 1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 microM and 0.15 microM, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures.

Background: In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive., Principal Findings: A naturally existing 72-kDa complex of non-lens betagamma-crystallin (alpha-subunit) and trefoil factor (beta-subunit), named betagamma-CAT, was identified from frog Bombina maxima skin secretions. Its alpha-subunit and beta-subunit (containing three trefoil factor domains), with a non-covalently linked form of alphabeta(2), show significant sequence homology to ep37 proteins, a group of non-lens betagamma-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. betagamma-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. betagamma-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K(+) efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the alpha-subunit with Western blotting. Furthermore, betagamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of betagamma-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC(50) 10 nM) and apoptosis. betagamma-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited betagamma-CAT induced vacuole formation, significantly inhibited betagamma-CAT induced cell detachment, suggesting that betagamma-CAT endocytosis is important for its activities., Conclusions/significance: These findings illustrate novel cellular functions of non-lens betagamma-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.

Several algesic agents including bradykinin and tachykinin have been identified from skin secretions of amphibians. They may act in defensive roles against aggressors. In this study, a novel peptide named Amolos with an amino acid sequence of FLPIVGAKL and isolated from skin secretion of the frog Amolops loloensis, is shown to strengthen nociceptive responses induced by inflammatory factors and strongly inhibit the contraction of isolated ileum. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. These two types of responses seem to be a part of the defensive functions against predators or aggressors. The current results suggest that pharmacological molecules in amphibian skins not only act as innate defense mechanisms against microorganisms but also exert other defensive physiological functions against other aggressors.

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.

Peptidomics and genomics analyses were used to study an anti-infection array of peptides of amphibian skin. 372 cDNA sequences of antimicrobial peptides were characterized from a single individual skin of the frog Odorrana grahami that encode 107 novel antimicrobial peptides. This contribution almost triples the number of currently reported amphibian antimicrobial peptides. The peptides could be organized into 30 divergent groups, including 24 novel groups. The diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal. The patterns of diversification suggest that point mutations as well as insertion, deletion, and "shuffling" of oligonucleotide sequences were responsible for the diversity. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms. These diverse peptides exhibited both diverse secondary structure and "host defense" properties. Such extreme antimicrobial peptide diversity in a single amphibian species is amazing. This has led us to reconsider the strong capability of innate immunity and molecular genetics of amphibian ecological diversification and doubt the general opinion that 20-30 different antimicrobial peptides can protect an animal because of the relatively wide specificity of the peptide antibiotics. The antimicrobial mechanisms of O. grahami peptides were investigated. They exerted their antimicrobial functions by various means, including forming lamellar mesosome-like structures, peeling off the cell walls, forming pores, and inducing DNA condensation. With respect to the development of antibiotics, these peptides provide potential new templates to explore further.

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.

There are around 27 species of Amolops amphibian distributed in South-east of Asia. Seven antimicrobial peptides (AMPs) belonging to two different families were purified from skin of rufous-spotted torrent frog, Amolops loloensis, and designated brevinins-ALa, b, c, and d, and temporins-ALa, b, and c. The brevinins-AL family which is structurally related to brevinins-1 from skin secretions of the European frog, Rana brevipoda, is composed of 24 amino acids and has an intra-disulfide bridge at the C-terminus. The temporins-AL family, composed of 13 or 16 amino acid residues, is related with temporins from the skin secretions of R. temporaria. The findings of this study will facilitate the solutions to the taxonomic questions of the ranid genus Amolops and Staurois. In the work of this paper, both brevinins-ALb and temporin-Ma induced mast cell degranulation and histamine release, and had cytotoxic activity toward solid tumor cell line HepG(2). Brevinins-ALb also exerted strong hemolytic activity while temporin-Ma had no such activity.

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.

PR-bombesin is a bombesin-like peptide derived from the skin of the Chinese red belly toad, Bombina maxima. The 8-residue segment of N-terminal of RP-bombesin, comprising four prolines and three basic residues, is extensively different from other bombesin-like peptides. Since sequence of Pro-Arg-Pro generally plays an important role in the antimicrobial activity of proline-rich antimicrobial peptides, the componential feature of PR-bombesin indicates that it may have antimicrobial activity. In this paper, we presented the first evidence that bombesin-like peptides possess direct antimicrobial activities as some neuropeptides. It was determined by CD spectroscopy that PR-bombesin adopted a combination of random coil and beta-sheet structure, suggesting RP-bombesin is a new member of antimicrobial peptides having beta structure but without disulfide bonds. Current results also supported that PR-bombesin plays a direct defensive role besides its neuro-endocrological functions.

Two antimicrobial peptides manifested a broad spectrum of antimicrobial activity against various microorganisms have been isolated from skin secretions of Rana grahami. These antimicrobial peptides were named grahamin 1 and grahamin 2. Their primary structures are GLLSGILGAGKNIVCGLSGLC and GLLSGILGAGKHIVCGLSGLC, respectively, determined by Edman degradation and mass spectrometry. They are structurally related to nigrocins identified from skin secretions of the dark-spotted frog, Rana nigromaculata. The cDNA clones encoding the precursor of grahamins were screened and sequenced from the skin cDNA library of R. grahami. The amino sequences deduced from the cDNA sequences match well with the results from Edman degradation. As other antimicrobial peptides from Rana species, grahamins contain a C-terminal loop region delineated by an intra-disulfide bridge named Rana box. Based on structural comparison of grahamin with other known antimicrobial peptides, grahamins could be classified into the family of antimicrobial peptides containing a single intra-disulfide bridge.

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B.maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1:1 molar ratio. The equilibrium dissociation constants (KD) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys53-Cys62), which comprises the scissile bond Arg58(P1)-His59(P1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

Amphibian skin contains rich neuropeptides. In the present study, a novel neuromedin U (NmU) analog was isolated from skin secretions of Chinese red belly toad Bombina maxima. Being 17-amino acids long, its primary structure was established as DSSGIVGRPFFLFRPRN-NH2, in which the C-terminal 8-residue segment (FFLFRPRN) is the same as that of rat NmU, while the N-terminal part DSSGIVGRP shows a great sequence variation compared with those of NmU peptides from different resources. The peptide, named Bm-NmU-17, was found to elicit concentration-dependent contractile effects on smooth muscle of rat uterus horns. The cDNA structure of the peptide, as obtained by a 3'-RACE strategy and subsequently cloning from a skin cDNA library, was found to contain a coding region of 438 nucleotides. The encoded precursor is composed of 145 amino acids with a single copy of Bm-NmU-17 located towards the C-terminus. The sequence of the peptide is preceded by a dibasic site (Lys-Arg) and followed by the sequence of Gly-Arg-Lys, providing the sites of cleavage and releasing of the mature peptide.

Antimicrobial peptides secreted by the skin of many amphibians play an important role in innate immunity. From two skin cDNA libraries of two individuals of the Chinese red belly toad (Bombina maxima), we identified 56 different antimicrobial peptide cDNA sequences, each of which encodes a precursor peptide that can give rise to two kinds of antimicrobial peptides, maximin and maximin H. Among these cDNA, we found that the mean number of nucleotide substitution per non-synonymous site in both the maximin and maximin H domains significantly exceed the mean number of nucleotide substitution per synonymous site, whereas the same pattern was not observed in other structural regions, such as the signal and propiece peptide regions, suggesting that these antimicrobial peptide genes have been experiencing rapid diversification driven by Darwinian selection. We cloned and sequenced seven genes amplified from skin or liver genomic DNA. These genes have three exons and share the same gene structure, in which both maximin and maximin H are encoded by the third exon. This suggests that alternative splicing and somatic recombination are less likely to play a role in creating the diversity of maximins and maximin Hs. The gene trees based on different domain regions revealed that domain shuffling or gene conversion among these genes might have happened frequently.

Two novel bioactive peptides were purified from skin secretions of the toad Bombina maxima. The partial N-terminal sequences of these two peptides were determined by automated Edman degradation. This allowed the cloning of full-length cDNAs encoding these two peptides from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences indicate that both peptides are composed of 77 amino acids. A FASTA search in the databanks revealed that they exhibit 86-91% sequence identity with Bv8, a peptide originally isolated from skin secretions of Bombina variegata. They were thus named as Bv8-like peptide 1 (Bv8-LP1) and Bv8-like peptide 2 (Bv8-LP2), respectively. Sequence differences between Bv8-LP1 and 2 were due to six amino acid substitutions at positions 6, 11, 23, 24, 62 and 63. Bv8-LP1 and 2 differed from Bv8 with eleven and seven amino acid substitutions, respectively. Like Bv8, Bv8-LP1 and 2 possessed contractile activity on isolated guinea pig ileum. Additionally, they stimulated contraction of rabbit aortic rings in a dose-dependent manner at nanomolar concentrations.

A novel 28-amino acid peptide, termed bombinakinin-GAP, was purified and characterized from skin secretions of the toad Bombina maxima. Its primary structure was established as DMYEIKQYKTAHGRPPICAPGEQCPIWV-NH(2), in which two cysteines form a disulfide bond. A FASTA search of SWISS-PROT databank detected a 32% sequence identity between the sequences of the peptide and a segment of rat cocaine- and amphetamine-regulated transcript (CART). Intracerebroventricular (i.c.v.) administration of the peptide induced a significant decrease in food intake in rats, suggesting that it played a role in the control of feeding by brain. Analysis of its cDNA structure revealed that this peptide is coexpressed with bombinakinin M, a bradykinin-related peptide from the same toad. Bombinakinin-GAP appears to be the first example of a novel class of bioactive peptides from amphibian skin, which may be implicated in feeding behavior.

Water-soluble skin secretions of salamander Tylototriton verrucosus, first described by Anderson in 1871, were studied for their biological and enzymatic activities. They were found to be toxic to mice with an intraperitoneal LD50 of 11.5 mg/kg. Using Sephadex G-75 gel filtration, it was proven that the toxic components of the secretions are proteins with molecular weights ranging from 30,000 to 50,000 Da. The secretions of T. verrucosus display a wide spectrum of antimicrobial activities and also contain both proteolytic activity and trypsin inhibitory activity. In contrast, neither hemolytic nor hemorrhagic activities were found. The secretions were determined to have phospholipase A2 activity; however, no acetylcholine esterase activity was detectable under the assay conditions.

Two groups of antimicrobial peptides have been isolated from skin secretions of Bombina maxima. Peptides in the first group, named maximins 1, 2, 3, 4 and 5, are structurally related to bombinin-like peptides (BLPs). Unlike BLPs, sequence variations in maximins occurred all through the molecules. In addition to the potent antimicrobial activity, cytotoxicity against tumor cells and spermicidal action of maximins, maximin 3 possessed a significant anti-HIV activity. Maximins 1 and 3 were toxic to mice with LD(50) values of 8.2 and 4.3 mg/kg, respectively. Peptides in the second group, termed maximins H1, H2, H3 and H4, are homologous with bombinin H peptides. cDNA sequences revealed that one maximin peptide plus one maximin H peptide derived from a common larger protein.

A novel bombesin-related peptide was isolated from skin secretions of Chinese red belly toad Bombina maxima. Its primary structure was established as pGlu-Lys-Lys-Pro-Pro-Arg-Pro-Pro-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH(2.) The amino-terminal (N-terminal) 8-residue segment comprising four prolines and three basic residues is extensively different from bombesins from other Bombina species. The peptide was thus named proline rich bombesin (PR-bombesin). PR-bombesin was found to elicit concentration-dependent contractile effects in the rat stomach strip, with both increased potency and intrinsic activity as compared with those of [Leu(13)]bombesin. Analysis of different bombesin cDNA structures revealed that an 8 to 14- nucleotide fragment replacement in the peptide coding region (TGGGGAAT in the cDNAs of multiple bombesin forms from Bombina orientalis and CACCCCGGCCACCC in the cDNA of PR-bombesin) resulted in an unusual Pro-Pro-Arg-Pro-Pro motif in the N-terminal part of PR-bombesin.

A novel trypsin inhibitor was identified and purified from skin secretions of Chinese red-belly toad Bombina maxima. The partial N-terminal 29 amino acid residues of the peptide, named BMTI, were determined by automated Edman degradation. This allowed the cloning of a full-length cDNA encoding BMTI from a cDNA library prepared from the toad skin. The deduced complete amino acid sequence of BMTI indicates that mature BMTI is composed of 60 amino acids. A FASTA search in the databanks revealed that BMTI exhibits 81.7% sequence identity with BSTI, a trypsin/thrombin inhibitor from European toad Bombina bombina skin secretions. Sequence differences between BMTI and BSTI were due to 11 substitutions at positions 2, 9, 25, 27, 36-37, 39, 41-42, 50 and 56. BMTI potently inhibited trypsin with a K(i) value of 0.06 microM, similar to that of BSTI. However, unlike BSTI, which also inhibited thrombin with a K(i) value of 1 microM, no inhibitory effect of BMTI on thrombin was observed under the assay conditions.

Psoriasis is histologically characterized by keratinocytes (KC) hyperproliferation, inflammation, and increased angiogenesis, but the pathological factor responsible for these symptoms is unknown. Here, a neuroendocrine peptide (prokineticin 2, PK2), is highly expressed in human and mouse psoriatic skins but no significant change in other autoimmune diseases, suggesting that PK2 is a psoriasis-specific factor. Bacterial products significantly up-regulated PK2, implying that infection induces PK2 over-expression. PK2 promoted KC and macrophage to produce interleukin-1 (IL-1), the central player of inflammation and psoriasis, which acts on adjacent fibroblast to induce inflammatory cascades and KC hyperproliferation. IL-1 feeds back on macrophages to induce PK2 production to perpetuate PK2-IL-1 positive feedback loop. PK2 also promoted angiogenesis, another psoriatic symptom. In mouse models, PK2 over-expression aggravated psoriasis while its knock-down inhibited pathological development. The results indicate that PK2 over-production perpetuates psoriatic symptoms by creating PK-2-IL-1 vicious loop. PK2 is a central player in psoriasis and a promising psoriasis-specific target.

Antimicrobial peptides have been widely identified from amphibian skins except salamanders. A novel antimicrobial peptide (CFBD) was isolated and characterized from skin secretions of the salamander, Cynops fudingensis. The cDNA encoding CFBD precursor was cloned from the skin cDNA library of C. fudingensis. The precursor was composed of three domains: signal peptide of 17 residues, mature peptide of 41 residues and intervening propeptide of 3 residues. There are six cysteines in the sequence of mature CFBD peptide, which possibly form three disulfide-bridges. CFBD showed antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Escherichia coli. This peptide could be classified into family of β-defensin based on its seqeuence similarity with β-defensins from other vertebrates. Evolution analysis indicated that CFBD was close to fish β-defensin. As far as we know, CFBD is the first β-defensin antimicrobial peptide from salamanders. [ABSTRACT FROM AUTHOR]

Abstract: Anntoxin is the first gene-encoded neurotoxin identified from amphibians, which is a 60-residue neurotoxin peptide, acting as an inhibitor of tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel (VGSC). Sodium channels have been considered as therapeutic targets for pain. Several animal models of persistent inflammatory and neuropathic pain (tail-flick test, hot plate test, acetic acid-induced writhing test, formalin-induced paw licking, carrageenan-induced paw edema) were used to test analgesic functions of recombinant anntoxin (r-anntoxin). In all these animal models, r-anntoxin showed strong analgesic functions. R-anntoxin obviously inhibited secretions of both tumor necrosis factor alpha (TNF-α) and cyclooxygenase-2 (COX-2). Histopathological study indicated that r-anntoxin reduced the edematous epidermis induced by carrageenan. All these results indicate that r-anntoxin has strong analgesic and anti-inflammatory activities. [Copyright &y& Elsevier]

Abstract: More than ten bradykinin-related peptides and their cDNAs have been identified from amphibians, but their genes are unknown. In present study, four cDNAs encoding one, two, four and six copies of bradykinin-related peptides were cloned from the frog (Odorrana grahami) skin cDNA library, respectively. Three bradykinin-related peptides (bradykinin, Thr6-bradykinin, Leu5Thr6-bradykinin) were deduced from these four cDNA sequences. Based on the cDNA sequence, the gene sequence encoding an amphibian bradykinin-related peptide from O. grahami was determined. It is composed of 7481 base pairs including two exons and two introns. The first exon codes signal peptide and the second exon codes acidic spacer peptide and Thr6-bradykinin. The promoter region of the bradykinin gene contains several putative recognition sites for nuclear factors, such as SRY, GATA-1, LYF-1, DeltaE, CDXA, NKX-2.5, MIF1 and S8. The current work may facilitate to understand the regulation and possible functions of amphibian skin bradykinin-related peptides. [Copyright &y& Elsevier]