The role of environmental calcium in the extreme acid tolerance of northern banjo frog (Limnodynastes terraereginae) larvae.

Many aquatically respiring animals acutely exposed to low pH waters suffer inhibition of ion uptake and loss of branchial (gill) epithelial integrity, culminating in a fatal loss of body Na+. Environmental calcium levels ([Ca2+]e) are pivotal in maintaining branchial junction integrity, with supplemental Ca2+ reversing the negative effects of low pH in some animals. Tolerance of some naturally acidic environments by aquatic animals is further complicated by low [Ca2+]e, yet many of these environments are surprisingly biodiverse. How animals overcome the damaging actions of low pH and low environmental Ca2+ remains unknown. We examined the effects of [Ca2+]e on the response to low pH in larvae of the highly acid-tolerant frog Limnodynastes terraereginae. Acute exposure to low pH water in the presence of low (5 µmol l-1) [Ca2+]e increased net Na+ efflux. Provision of additional [Ca2+]e reduced net Na+ efflux, but the effect was saturable. Acclimation to both low and high (250 µmol l-1) [Ca2+]e improved the resistance of larvae to Na+ efflux at low pH. Exposure to the Ca2+ channel inhibitor ruthenium red resulted in an abrupt loss of tolerance in low pH-acclimated larvae. Acclimation to acidic water increased branchial gene expression of the intracellular Ca2+ transport protein calbindin, consistent with a role for increased transcellular Ca2+ trafficking in the tolerance of acidic water. This study supports a role for [Ca2+]e in promoting branchial integrity and highlights a potential mechanism via the maintenance of transcellular Ca2+ uptake in the acid tolerance of L. terraereginae larvae.

A novel peptide identified from skin secretions of Bombina maxima possesses LPS-neutralizing activity.

Lipopolysaccharide (LPS) is a major pathogenic factor in endotoxin shock or sepsis. Most antibiotics have little clinical anti-endotoxin activity, but some antimicrobial peptides (AMPs) have been shown to be effective in blocking LPS. We identified a novel peptide from the skin secretions of Bombina maxima (B. _maxima) by challenging the skin of frogs with an LPS solution. Peptide 2 has an amino acid sequence of LVGKLLKGAVGDVCGLLPIC. Peptide 2 possesses low hemolytic activity, low cytotoxicity against RAW 264.7 cells, and strong anti-inflammatory activity. Moreover, peptide 2 plays an anti-inflammatory role by inhibiting inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). A biolayer interferometry (BLI) assay indicated that peptide 2 binds to LPS with strong affinity and that this interaction has an affinity constant (KD) value of 1.05 × 10-9 M. A survival study showed that peptide 2 possesses potent LPS-neutralizing activity to protect LPS-treated mice from death. In conclusion, we have identified a potent peptide with LPS neutralizing activity, which lays a foundation for future research and development.

Connected to the spirit of the frog: An Internet-based survey on Kambô, the secretion of the Amazonian Giant Maki Frog (Phyllomedusa bicolor): Motivations for use, settings and subjective experiences.

BACKGROUND/AIM: Kambô is a name for the secretion of the Giant Maki Frog (Phyllomedusa bicolor), which has been used by indigenous cultures from the Amazonas basin and has recently become popular in alternative healing circles in Western countries, with a certain overlap with psychedelic self-exploration. METHODS: We carried out an online-based survey in English (54.92%) and German investigating motivations for using Kambô, settings in which rituals take place, and subjective experiences during and after the application. RESULTS: Participants (n = 386, mean age: 38.08 years, (standard deviation = 9.95)) were well-educated individuals with an increased lifetime prevalence of the use of ayahuasca (67.88%). A plethora of motivations for using Kambô was reported, including general healing, detoxification and spiritual growth. Acute effects included severe physical reactions and mild psychoactive effects, most surprisingly, the feeling of being connected to the frog's spirit (41.97%), whereas predominantly positive persisting psychological effects were reported. Few participants reported long-lasting physical (2.85%) or mental (1.81%) health problems which they attributed to Kambô. Of the participants, 87.31% reported an increase in personal well-being or life satisfaction, and 64.26% considered Kambô to have been at least of 'very much' spiritual significance for their lives. CONCLUSIONS: The majority of users claimed beneficial effects including more health-orientated behaviors, whereas only very few participants complained about new health problems which they ascribed to Kambô. In retrospect, Kambô was given a high personal and spiritual significance by many participants. Additional research is needed to determine in how far reported effects are modulated by setting and subjective expectations.

Manual mass spectrometry de novo sequencing of the anionic host defense peptides of the Cuban Treefrog Osteopilus septentrionalis.

RATIONALE: Host defense peptides accumulated in the skin glands of the animals constitute the basis of the adaptive and immune system of amphibians. The peptidome of the Cuban frog Osteopilus septentrionalis was established using tandem mass spectrometry as the best analytical tool to elucidate the sequence of these peptides. METHODS: Manual interpretation of complementary collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), and electron transfer dissociation (ETD) tandem mass spectra recorded with an Orbitrap Elite mass spectrometer in liquid chromatography/mass spectrometry (LC/MS) mode was used to sequence the peptide components of the frog skin secretion, obtained by mild electrostimulation. RESULTS: Although the vast majority of amphibian peptides discovered so far are cationic, surprisingly only anionic peptides were identified in the skin secretion of the Cuban frog Osteopilus septentrionalis. Mass spectrometry allowed the sequences to be established of 16 representatives of new peptide families: septenins 1 and septenins 2. The highest sequence coverage when dealing with these anionic peptides was obtained with CID normalized collision energy 35 and HCD normalized collision energy 28. CONCLUSIONS: Mirror-symmetrical peptides are sequenced using N-terminal acetylation. Acetylated Ser is reliably distinguished from isomeric Glu by the loss of ketene from b-ions containing the corresponding residue. Calculations of the physicochemical and structural properties of the discovered anionic septenins 1 and 2 allowed the mechanism of their interaction with microbe cells to be postulated.

Identification and functional characterisation of Esculentin-2 HYba peptides and their C-terminally amidated analogs from the skin secretion of an endemic frog.

Here, we report the identification, functional characterisation, and the effect of C-terminal amidation on the activity profile of two novel Esculentin-2 peptides (Esculentin-2 HYba1 and Esculentin-2 HYba2). The parent peptides and their analogs exhibited potent activity against the tested Gram-positive and Gram-negative bacteria. The effect of amidation was evident in the activity profile of fish pathogens and killing kinetics. The analogs showed a 10-fold decrease in MIC, and the killing time was reduced to 10-15 minutes. The hemolytic potential was unaltered upon amidation. The selectivity index revealed that these peptides are more selective to bacteria than mammalian cells. Cytotoxicity against Hep3B cells reveals their potential to destroy cancer cells; they showed potential inhibition compared to anticancer drug silymarin. The study also highlights the need for further truncations and modifications of esculentin peptides for developing them as lead drug molecules.

Concentration ratios of 238U and 226Ra for insects and amphibians living in the vicinity of the closed uranium mine at Ningyo-toge, Japan.

There is still a scarcity of data on the transfer of naturally occurring radionuclides to wildlife in various ecosystems. In the present study, concentration ratios (CRwo-media) of 238U and 226Ra were obtained for grasshoppers, frogs and newts in terrestrial and freshwater ecosystems. Soil, water and animal samples were collected for 2 years in the vicinity of the closed uranium mine at Ningyo-toge, Japan. Three sites with different 238U and 226Ra levels were of interest: (i) pond and its shore (PO); (ii) low-level stream and its shore near overburden dump (OD); and (iii) uranium mill tailings pond and its shore (MP). The activity concentrations in both soil and water were PO ≈ OD < MP for 238U, and PO < OD < MP for 226Ra. Regarding the wildlife, 238U was able to be determined for all samples, but the detection of 226Ra was observed only for part of the samples. The means and standard deviations of CRwo-soil or CRwo-water were then calculated and may indicate the insignificant dependence of CRwo-media on environmental conditions characterized by the tested sites. The present data on CRwo-media were compared to the corresponding data or surrogate data from the IAEA's database, showing both agreement and discrepancy. Our data contribute to enhancing the available data for those radionuclides and animals. In particular, the transfer to amphibians, one of the main links in common food webs, is reported here for the first time.

Identification of Arenin, a Novel Kunitz-Like Polypeptide from the Skin Secretions of Dryophytes arenicolor.

Amphibian skin secretions are enriched with complex cocktails of bioactive molecules such as proteins, peptides, biogenic amines, alkaloids guanidine derivatives, steroids and other minor components spanning a wide spectrum of pharmacological actions exploited for centuries in folk medicine. This study presents evidence on the protein profile of the skin secretions of the canyon tree frog, Dryophytes arenicolor. At the same time, it presents the reverse-phase liquid chromatography isolation, mass spectrometry characterization and identification at mRNA level of a novel 58 amino acids Kunitz-like polypeptide from the skin secretions of Dryophytes arenicolor, arenin. Cell viability assays performed on HDFa, CaCo2 and MCF7 cells cultured with different concentrations of arenin showed a discrete effect at low concentrations (2, 4, 8 and 16 µg/mL) suggesting a multi-target interaction in a hormetic-like dose-response. Further work is required to investigate the mechanisms underlying the variable effect on cell viability produced by different concentrations of arenin.

Monoaminergic integration of diet and social signals in the brains of juvenile spadefoot toads.

Social behavior often includes the production of species-specific signals (e.g. mating calls or visual displays) that evoke context-dependent behavioral responses from conspecifics. Monoamines are important neuromodulators that have been implicated in context-dependent social behavior, yet we know little about the development of monoaminergic systems and whether they mediate the effects of early life experiences on adult behavior. We examined the effects of diet and social signals on monoamines early in development in the plains spadefoot toad (Spea bombifrons), a species in which diet affects the developmental emergence of species recognition and body condition affects the expression of adult mating preferences. To do so, we manipulated the diet of juveniles for 6 weeks following metamorphosis and collected their brains 40 min following the presentation of either a conspecific or a heterospecific call. We measured levels of monoamines and their metabolites using high pressure liquid chromatography from tissue punches of the auditory midbrain (i.e. torus semicircularis), hypothalamus and preoptic area. We found that call type affected dopamine and noradrenaline signaling in the auditory midbrain and that diet affected dopamine and serotonin in the hypothalamus. In the preoptic area, we detected an interaction between diet and call type, indicating that diet modulates how the preoptic area integrates social information. Our results suggest that the responsiveness of monoamine systems varies across the brain and highlight preoptic dopamine and noradrenaline as candidates for mediating effects of early diet experience on later expression of social preferences.

Dietary Accumulation of Inorganic Selenium by a Larval Amphibian (Hyla chrysoscelis) and Influence on Accumulation of Background Mercury.

Larval gray tree frogs (Hyla chrysoscelis) were exposed to inorganic Se (SeO2) added to the diet (10.2 and 86.3 ug/g dw) and monitored for accumulation and effect. Background concentrations of Hg were also measured in food and carcasses to assess possible effects of Se on Hg accumulation. Selenium was accumulated in a dose dependent manner, and life stages did not differ. No effects of Se exposure were observed on survival, growth, or time to metamorphosis. Mercury concentrations in carcasses, resulting from background concentrations in food, were significantly affected by the presence of Se. In the high Se treatment, Hg concentrations were significantly decreased relative to those in the low Se treatment and the control. Our study suggests that exposure to inorganic Se as SeO2 at the concentrations tested do not elicit adverse biological effects, but exposure to relatively high concentrations of Se may reduce accumulation of Hg from food.

Bufalin, a bufanolide steroid from the parotoid glands of the Chinese toad, inhibits L-type Ca2+ channels and contractility in rat ventricular myocytes.

Bufalin is a bufanolide steroid compound in Chan Su. Chan Su is a traditional Chinese medicine prepared from the dried white secretion of the auricular and skin glands of toads and has been used as an oriental drug. However, the effect of bufalin on cardiac function and its underlying cellular mechanisms remain unclear. Here, we explore the cellular mechanisms of bufalin on myocardial protection via the whole-cell patch-clamp recording and video-based edge detection system. Exposure to bufalin resulted in a concentration-dependent blockade of ICa-L , with the half-maximal inhibitory concentration (IC50 ) of 60 µm and the maximal inhibitory effect of 71.50 ± 2.67%. Bufalin at 100 µm reduced cell shortening by 33.83 ± 4.01%. Bufalin restrained L-type Ca2+ channels conductance, and contractility in rat ventricular myocytes. Thus, the protective effects of bufalin on the heart may be determined by the inhibitory effect on ICa-L and the negative inotropic action caused by the decrease of intracellular Ca2+ in rat myocardial cells.

In vivo efficacy of anuran trypsin inhibitory peptides against staphylococcal skin infection and the impact of peptide cyclization.

Staphylococcus aureus is a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weak in vitro inhibitory activities against S. aureus, but several had strong antibacterial activities against S. aureus in an in vivo murine wound infection model. pYR, an immunomodulatory peptide from Rana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg(-1). Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

Effect of dietary carotenoids on vitamin A status and skin pigmentation in false tomato frogs (Dyscophus guineti).

Several species in captivity develop nutritional diseases including vitamin A deficiency; cases of this disease have been documented in amphibians, which may be linked to an insectivorous diet lacking in vitamin A or carotenoids. Adults and young of Dyscophus guineti were fed three diets over 9 weeks to evaluate effects on carotenoids and vitamin A status and skin pigmentation. Feeder crickets were either supplemented with soy oil (control, CON), soy oil enriched with ß-carotene (BC) or mixed carotenoids (MIX) by direct injection of known dosages. Vitamin A from feeder crickets (measured as retinol) was higher in insects supplemented with both BC and MIX; (P=0.0001) and plasma retinol concentrations were significantly higher in frogs fed MIX (P<0.02). Results suggest that both false tomato frogs and feeder crickets could receive some provitamin A activity through consumption of diets supplemented with ß-carotene, and xanthophylls like lutein and zeaxanthin. Pigmentation was evaluated weekly through the use of visual color charts, as well as quantitatively using a hand-held spectrophotometer. MIX diets had a significant effect on skin color values (P<0.0001), as well as on lightness (P=0.0005) and hue (P=0.0022). Results indicated that frogs fed with BC changed to yellower colors, and frogs fed with MIX changed to oranger colors. Visual color chart observations also scored significantly different between CON and MIX diets (P<0.05); the animals fed MIX also appeared oranger according to the qualitative observations. Dietary supplements with carotenoids resulted in color changes and higher circulating retinol concentrations in false tomato frogs. These pigments may provide provitamin A activity in diets, thus may support improved nutrition and health of captive-fed insectivorous amphibians.

Efficacy of six frog skin-derived antimicrobial peptides against colistin-resistant strains of the Acinetobacter baumannii group.

The emergence of Acinetobacter sp. strains resistant to all antibacterial agents including colistin necessitates the development of new types of antimicrobial agents. Six cationic α-helical frog skin-derived peptides (CPF-AM1, PGLa-AM1, B2RP-ERa, [E4K]alyteserin-1c, [D4K]B2RP and [G4K]XT-7) were selected for this study on the basis of potent growth-inhibitory activity against Gram-negative bacteria and low haemolytic activity against human erythrocytes. All peptides were active against a range of colistin-susceptible [minimum inhibitory concentration (MIC)≤2 µg/mL] and colistin-resistant (MIC≥64 µg/mL) clinical isolates of multidrug-resistant strains of Acinetobacter baumannii and Acinetobacter nosocomialis. The most potent peptides against the colistin-resistant strains were [D4K]B2RP and [E4K]alyteserin-1c (MIC=4-16 µg/mL for both). The MIC values of these peptides against the colistin-susceptible strains were in the same range. The frog peptides show potential for development into drugs to treat infections caused by pandrug-resistant Gram-negative pathogens.

Auditory selectivity for acoustic features that confer species recognition in the tungara frog.

In anurans, recognition of species-specific acoustic signals is essential to finding a mate. In many species, behavioral tests have elucidated which acoustic features contribute to species recognition, but the mechanisms by which the brain encodes these species-specific signal components are less well understood. The túngara frog produces a `whine' mating call that is characterized by a descending frequency sweep. However, much of the signal is unnecessary for recognition, as recognition behavior can be triggered by a descending two-tone step that mimics the frequency change in a portion of the whine. To identify the brain regions that contribute to species recognition in the túngara frog, we exposed females to a full-spectrum whine, a descending two-tone step that elicits recognition, the reversed two-tone step that does not elicit recognition, or no sound, and we measured expression of the neural activity-dependent gene egr-1 in the auditory brainstem and thalamus. We found that the behavioral relevance of the stimuli was the best predictor of egr-1 expression in the laminar nucleus of the torus semicircularis but not elsewhere. That is, the laminar nucleus responded more to the whine and the two-tone step that elicits recognition than to the reversed two-tone step. In contrast, in other brainstem and thalamic nuclei, whines induced egr-1 expression but tones did not. These data demonstrate that neural responses in the laminar nucleus correspond to behavioral responses of females and they suggest that the laminar nucleus may act as a feature detector for the descending frequencies characteristic of conspecific calls.

Anticancer potential of animal venoms and toxins.

Anticancer drug development from natural resources are ventured throughout the world. Animal venoms and toxins a potential bio resource and a therapeutic tool were known to man for centuries through folk and traditional knowledge. The biodiversity of venoms and toxins made it a unique source of leads and structural templates from which new therapeutic agents may be developed. Venoms of several animal species (snake, scorpion, toad, frog etc) and their active components (protein and non protein toxins, peptides, enzymes, etc) have shown therapeutic potential against cancer. In the present review, the anticancer potential of venoms and toxins from snakes, scorpions, toads and frogs has been discussed. Some of these molecules are in the clinical trials and may find their way towards anticancer drug development in the near future. The implications of combination therapy of natural products in cancer have been discussed.

Immunohistochemical localization of calbindin-D28k and calretinin in the brainstem of anuran and urodele amphibians.

Calbindin-D28k (CB) and calretinin (CR) are calcium binding proteins present in distinct sets of neurons; they act as buffers regulating the concentration of intracellular calcium. CB and CR immunohistochemistry was studied in the brainstem of anuran and urodele amphibians in combination with other markers (choline acetyltransferase, tyrosine hydroxylase, and nitric oxide synthase), which served to clarify the localization and signature of many cell groups. CR labeled the retinorecipient layers of the optic tectum, and CB and CR labeled distinct tectal cell populations. The two proteins were largely complementary in the torus semicircularis and marked auditory and lateral line sensory regions, depending on the species. CB and CR in the mesencephalic and isthmic tegmentum specified the boundaries of basal and medial longitudinal bands. In the cerebellum, CB labeled Purkinje cells in all species, whereas CR was mainly found in fibers and labeled Purkinje cells only in Rana. In the parabrachial region, CB and CR allowed the distinction of the laterodorsal tegmental nucleus, isthmic nucleus, locus coeruleus, and rostral octavolateral nuclei. The distribution of CB- and CR-immunoreactive cells in the reticular formation and central gray was consistent with the current models of brainstem segmentation in amphibians. CR was found in the auditory fibers and nuclei in Rana and in mechanosensory lateral line fibers in Xenopus and urodeles, whereas CB mainly labeled vestibular fibers and nuclei in all species. These results highlight the anatomical complexity of the amphibian brainstem and help in an understanding of its regional organization that is not cytoarchitectonically evident.

Differential roles of p38-MAPK and JNKs in mediating early protection or apoptosis in the hyperthermic perfused amphibian heart.

In the present study the activation of p38 mitogen-activated protein kinase (p38-MAPK) and c-Jun N-terminal kinases (JNKs) by hyperthermia was investigated in the isolated perfused Rana ridibunda heart. Hyperthermia (42 degrees C) was found to profoundly stimulate p38-MAPK phosphorylation within 0.5 h, with maximal values being attained at 1 h [4.503(+/-0.577)-fold relative to control, P<0.01]. JNKs were also activated under these conditions in a sustained manner for at least 4 h [2.641(+/-0.217)-fold relative to control, P<0.01]. Regarding their substrates, heat shock protein 27 (Hsp27) was maximally phosphorylated at 1 h [2.261(+/-0.327)-fold relative to control, P<0.01] and c-Jun at a later phase [3 h: 5.367(+/-0.081)-fold relative to control, P<0.001]. Hyperthermia-induced p38-MAPK activation was found to be dependent on the Na+/H+ exchanger 1 (NHE1) and was also suppressed by catalase (Cat) and superoxide dismutase (SOD), implicating the generation of reactive oxygen species (ROS). ROS were also implicated in the activation of JNKs by hyperthermia, with the Na+/K+-ATPase acting as a mediator of this effect at an early stage and the NHE1 getting involved at a later time point. Finally, JNKs were found to be the principal mediators of the apoptosis induced under hyperthermic conditions, as their inhibition abolished poly(ADP-ribose) polymerase (PARP) cleavage after 4 h at 42 degrees C. Overall, to our knowledge, this study highlights for the first time the variable mediators implicated in the transduction of the hyperthermic signal in the isolated perfused heart of an ectotherm and deciphers a potential salutary effect of p38-MAPK as well as the fundamental role of JNKs in the induced apoptosis.

Odorranalectin is a small peptide lectin with potential for drug delivery and targeting.

BACKGROUND: Lectins are sugar-binding proteins that specifically recognize sugar complexes. Based on the specificity of protein-sugar interactions, different lectins could be used as carrier molecules to target drugs specifically to different cells which express different glycan arrays. In spite of lectin's interesting biological potential for drug targeting and delivery, a potential disadvantage of natural lectins may be large size molecules that results in immunogenicity and toxicity. Smaller peptides which can mimic the function of lectins are promising candidates for drug targeting. PRINCIPAL FINDINGS: Small peptide with lectin-like behavior was screened from amphibian skin secretions and its structure and function were studied by NMR, NMR-titration, SPR and mutant analysis. A lectin-like peptide named odorranalectin was identified from skin secretions of Odorrana grahami. It was composed of 17 aa with a sequence of YASPKCFRYPNGVLACT. L-fucose could specifically inhibit the haemagglutination induced by odorranalectin. (125)I-odorranalectin was stable in mice plasma. In experimental mouse models, odorranalectin was proved to mainly conjugate to liver, spleen and lung after i.v. administration. Odorranalectin showed extremely low toxicity and immunogenicity in mice. The small size and single disulfide bridge of odorranalectin make it easy to manipulate for developing as a drug targeting system. The cyclic peptide of odorranalectin disclosed by solution NMR study adopts a beta-turn conformation stabilized by one intramolecular disulfide bond between Cys6-Cys16 and three hydrogen bonds between Phe7-Ala15, Tyr9-Val13, Tyr9-Gly12. Residues K5, C6, F7, C16 and T17 consist of the binding site of L-fucose on odorranalectin determined by NMR titration and mutant analysis. The structure of odorranalectin in bound form is more stable than in free form. CONCLUSION: These findings identify the smallest lectin so far, and show the application potential of odorranalectin for drug delivery and targeting. It also disclosed a new strategy of amphibian anti-infection.

Components of the peptidome and transcriptome persist in lin wa pi: the dried skin of the Heilongjiang brown frog (Rana amurensis) as used in traditional Chinese medicine.

Although the ancient practice of traditional Chinese medicine (TCM) utilizes predominantly herbal ingredients, many of which are now the subject of intense scientific scrutiny, significant quantities of animal tissue-derived materials are also employed. Here we have used contemporary molecular techniques to study the material known as lin wa pi, the dried skin of the Heilongjiang brown frog, Rana amurensis, that is used commonly as an ingredient of many medicines, as a general tonic and as a topical antimicrobial/wound dressing. Using a simple technology that has been developed and validated over several years, we have demonstrated that components of both the skin granular gland peptidome and transcriptome persist in this material. Interrogation of the cDNA library constructed from the dried skin by entrapment and amplification of polyadenylated mRNA, using a "shotgun" primer approach and 3'-RACE, resulted in the cloning of cDNAs encoding the precursors of five putative antimicrobial peptides. Two (ranatuerin-2AMa and ranatuerin-2AMb) were obvious homologs of a previously described frog skin peptide family, whereas the remaining three were of sufficient structural novelty to be named amurins 1-3. Mature peptides were each identified in reverse phase HPLC fractions of boiling water extracts of skin and their structures confirmed by MS/MS fragmentation sequencing. Components of traditional Chinese medicines of animal tissue origin may thus contain biologically active peptides that survive the preparation procedures and that may contribute to therapeutic efficacy.

Biosynthesis of a D-amino acid in peptide linkage by an enzyme from frog skin secretions.

d-amino acids are present in some peptides from amphibian skin. These residues are derived from the corresponding L-amino acids present in the respective precursors. From skin secretions of Bombinae, we have isolated an enzyme that catalyzes the isomerization of an L-Ile in position 2 of a model peptide to D-allo-Ile. In the course of this reaction, which proceeds without the addition of a cofactor, radioactivity from tritiated water is incorporated into the second position of the product. The amino acid sequence of this isomerase could be deduced from cloned cDNA and genomic DNA. After expression of this cDNA in oocytes of Xenopus laevis, isomerase activity could be detected. Polypeptides related to the frog skin enzyme are present in several vertebrate species, including humans.