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Isolation of dermatoxin from frog skin, an antibacterial peptide encoded by a novel member of the dermaseptin genes family
Abstract
A 32-residue peptide, named dermatoxin, has been extracted from the skin of a single specimen of the tree frog Phyllomedusa bicolor, and purified to homogeneity using a four-step protocol. Mass spectral analysis and sequencing of the purified peptide, as well as chemical synthesis and cDNA analysis were consistent with the structure: SLGSFLKGVGTTLASVGKVVSDQF GKLLQAGQ. This peptide proved to be bactericidal towards mollicutes (wall-less eubacteria) and Gram-positive eubacteria, and also, though to a lesser extent, towards Gram-negative eubacteria. Measurement of the bacterial membrane potential revealed that the plasma membrane is the primary target of dermatoxin. Observation of bacterial cells using reflected light fluorescence microscopy after DNA-staining was consistent with a mechanism of cell killing based upon the alteration of membrane permeability rather than membrane solubilization, very likely by forming ion-conducting channels through the plasma membrane. CD spectroscopy and secondary structure predictions indicated that dermatoxin assumes an amphipathic alpha-helical conformation in low polarity media which mimic the lipophilicity of the membrane of target microorganisms. PCR analysis coupled with cDNA cloning and sequencing revealed that dermatoxin is expressed in the skin, the intestine and the brain. Preprodermatoxin from the brain and the intestine have the same sequence as the skin preproform except for two amino-acid substitutions in the preproregion of the brain precursor. The dermatoxin precursor displayed the characteristic features of preprodermaseptins, a family of peptide precursors found in the skin of Phyllomedusa ssp. Precursors of this family have a common N-terminal preproregion followed by markedly different C-terminal domains that give rise to 19-34-residue peptide antibiotics named dermaseptins B and phylloxin, and to the D-amino-acid-containing opioid heptapeptides dermorphins and deltorphins. Because the structures and cidal mechanisms of dermatoxin, dermaseptins B and phylloxin are very different, dermatoxin extends the repertoire of structurally and functionally diverse peptides derived from the rapidly evolving C-terminal domains of precursors of the dermaseptins family.
... Unassigned to group [7 species] P. atelopoides — P. bicolor 21 Anastasi et al. (1970), Richter et al. (1990), Daly et al. (1992), Mignogna et al. 1992, Amiche et al. (1993), Amiche et al. (1994), Mor et al. (1994a, b); Charpentier et al. (1998); Fleury et al. (1998), Seon et al. (2000), Amiche et al. (2000), Pierre et al. (2000), Vanhoye et al. (2003), Chen et al. (2005b), UniProt (2009) ...
... Among the peptides of the FSAP family, the AMPs are the most diverse class. To date, the AMPs described in Phyllomedusa skin include seven distinct families (or subfamilies ) according to their sequence similarity and activity, e.g., Dermaseptin (Mor et al. 1991a), Dermatoxin (Amiche et al. 2000), Distinctin (Batista et al. 2001), Phylloseptin (Leite et al. 2005), Phylloxin (Pierre et al. 2000), Plasticin (Vanhoye et al. 2004), and Skin Polypeptide YY (Mor et al. 1994a, b, c). These peptides comprise the skin anti-infective passive defense barrier of these anurans. ...
... Another interesting feature was the presence of the C-terminal dipeptide -GQ on the P. bicolor dermatoxin-B1 (DRT-B1) (Amiche et al. 2000; Chen et al. 2005a). The dermatoxin is structured as a preproprotein of the dermaseptin family of peptide precursors (Table 3) and characterized by strongly conserved preproregions followed by C-terminal sequence domain of precursors of the dermaseptin family (Amiche et al. 2000). ...
... As previously mentioned, three novel dermatoxin variants were sequenced in P. jandaia (Table 7 and Fig. 8E). Dermatoxins have been previously found in P. bicolor (Amiche et al., 2000), P. sauvagii (Chen et al., 2005), P. tarsius (Prates et al., unpublished results), Agalychnis danicolor and Agalychnis annae (Wechselberger, 1998). It has been reported that DRT-TA1 (originally named as Dermaseptin-8) from P. tarsius, is active against the Gram-positive, and Gram-negative bacteria besides displaying hemolytic activity (Prates et al., unpublished results). ...
... It has been reported that DRT-TA1 (originally named as Dermaseptin-8) from P. tarsius, is active against the Gram-positive, and Gram-negative bacteria besides displaying hemolytic activity (Prates et al., unpublished results). DRT-B1 (formerly known as dermatoxin) was found to be particularly active against mollicutes and Gram-positive bacteria, although having lower activity upon Gram-negative bacteria (Amiche et al., 2000). The bacterial plasma membrane is thought to be the primary target of DRT-B1 due to the membrane depolarization observed upon the application of this peptide, being its cell killing mechanisms likely involved with the alteration of membrane permeability rather than membrane solubilization (Amiche et al., 2000). ...
... DRT-B1 (formerly known as dermatoxin) was found to be particularly active against mollicutes and Gram-positive bacteria, although having lower activity upon Gram-negative bacteria (Amiche et al., 2000). The bacterial plasma membrane is thought to be the primary target of DRT-B1 due to the membrane depolarization observed upon the application of this peptide, being its cell killing mechanisms likely involved with the alteration of membrane permeability rather than membrane solubilization (Amiche et al., 2000). DRT-S1 (CAI26288 -original name: Dermatoxin S). ...
The systematic investigation of the peptidic composition of the skin secretion of Phasmahyla jandaia, a phyllomedusine anuran endemic to the southern region of the Espinhaço range in Brazil, is herein reported. By means of de novo interpretation of tandem mass spectrometric data, Edman N-terminal sequencing and similarity searches, 57 peptides - including phylloseptins, dermaseptins stricto sensu, dermatoxins, hyposins, tryptophyllins, caerulein-related, bradykinin-related, bradykinin potentiating, tyrosine-rich, and opioid peptides - were sequenced. Moreover, five peptide families without significant similarity to other known molecules were verified. Differently from most Phyllomedusinae genera, the molecular diversity in the skin of representatives of Phasmahyla remained unprospected until now. Therefore, besides disclosing novel natural variants of number of bioactive peptides, the present study contributes to the understanding of the evolution of biochemical characters of the phyllomedusines.
... In fact, this observation is extended to nucleotide sequences of cDNAs within 3V-and 5V-noncoding regions. Dermatoxin and phylloxin are two prototype narrow-spectrum antimicrobial peptides identified in Phyllomedusa bicolor skin [14,15]. Despite significant differences in primary structure with each other and with dermaseptins, the organization, nucleotide sequence of cloned cDNAs and primary structures of signal peptides and acidic spacer domains of respective precursors are remarkably highly conserved [16]. ...
... The cDNA was subjected to 3V-RACE procedures to obtain full-length preprodermatoxin/preprophylloxin nucleic acid sequence data using a SMART-RACE kit (Clontech UK) essentially as described by the manufacturer. Briefly, the 3V-RACE reactions employed a NUP primer (supplied with the kit) and a sense primer (S1; 5V-TCTGAATTRYAAGMSCAR-ACATG-3V) that was designed to a highly conserved domain of the 5V-untranslated region of previously characterized homologous peptide cDNAs from Phyllomedusa bicolor [14,15]. The PCR cycling procedure was as follows. ...
... The nucleotide sequences of dermatoxin (DRT-S) and phylloxin (PLX-S) have been deposited in the EMBL Nucleotide Sequence Database under the accession codes AJ865344 and AJ865345, respectively. An EBI-BLAST search found that DRT-S and PLX-S nucleotide sequences showed 90% and 94% sequence identity, respectively, with the dermatoxin and phylloxin sequences from Phyllomedusa bicolor [14,15] (Fig. 2). However the EBI-BLAST search revealed that DRT-S and PLX-S open-reading frame amino acid sequences showed 81% and 95% sequence identity, respectively, with dermatoxin and phylloxin precursor sequences from Phyllomedusa bicolor [14,15] (Fig. 3). ...
Amphibian skin is a morphologically, biochemically and physiologically complex organ that performs the wide range of functions necessary for amphibian survival. Here we describe the primary structures of representatives of two novel classes of amphibian skin antimicrobials, dermatoxin and phylloxin, from the skin secretion of Phyllomedusa sauvagei, deduced from their respective precursor encoding cDNAs cloned from a lyophilized skin secretion library. A degenerate primer, designed to a highly conserved domain in the 5'-untranslated region of analogous peptide precursor cDNAs from Phyllomedusa bicolor, was employed in a 3'-RACE reaction. Peptides with molecular masses coincident with precursor-deduced mature toxin peptides were identified in LC/MS fractions of skin secretion and primary structures were confirmed by MS/MS fragmentation. This integrated experimental approach can thus rapidly expedite the primary structural characterization of amphibian skin peptides in a manner that circumvents specimen sacrifice whilst preserving robustness of scientific data.
... The South American hylid frogs of the Phyllomedusinae subfamily (family: Hylidae) also produce a rich array of linear a-helical antimicrobial peptides that are 19-34 residues long (Table 1). They include the dermaseptins B and dermaseptins S [30][31][32][33], phylloxin [34] and dermatoxin [35] from frogs of the Phyllomedusa genus and 24-33 residues peptides called dermaseptin-related peptides DRP-AA and DRP-PD from Agalychnis annae and Pachymedusa dacnicolor, respectively [36]. Analysis of cDNA clones of antimicrobial peptides from South American hylids [32,[34][35][36][37] and Asian, European and North American ranins [24,29,38,39] has indicated that they are all derived from a single family of precursor polypeptides with unique features [39]. ...
... They include the dermaseptins B and dermaseptins S [30][31][32][33], phylloxin [34] and dermatoxin [35] from frogs of the Phyllomedusa genus and 24-33 residues peptides called dermaseptin-related peptides DRP-AA and DRP-PD from Agalychnis annae and Pachymedusa dacnicolor, respectively [36]. Analysis of cDNA clones of antimicrobial peptides from South American hylids [32,[34][35][36][37] and Asian, European and North American ranins [24,29,38,39] has indicated that they are all derived from a single family of precursor polypeptides with unique features [39]. Precursors belonging to this family, designated the preprodermaseptin, have an N-terminal preprosequence of approximately 50 residues that is remarkably well conserved both within and between species, while the C-terminal sequence corresponding to antimicrobial peptides varies markedly. ...
... Gram-positive eubacteria (Aerococcus viridans, Bacillus megaterium, Staphylococcus aureus and Staphylococcus haemolyticus), Gram-negative eubacteria (E. coli B, Salmonella typhimurium, Salmonella enteritidis, Enterobacter cloacae, Klebsiella pneumoniae) and Saccaromyces cerevisiae were cultured as described previously [34,35]. The minimal inhibitory concentrations (MICs) of peptides were determined in 96-well microtitration plates by growing the bacteria in the presence of twofold serial dilutions of peptide. ...
The dermal glands of frogs produce antimicrobial peptides that protect the skin against noxious microorganisms and assist in wound repair. The sequences of these peptides are very dissimilar, both within and between species, so that the 5000 living anuran frogs may produce approximately 100 000 different antimicrobial peptides. The antimicrobial peptides of South American hylid frogs are derived from precursors, the preprodermaseptins, whose signal peptides and intervening sequences are remarkably conserved, but their C-terminal domains are markedly diverse, resulting in mature peptides with different lengths, sequences and antimicrobial spectra. We have used the extreme conservation in the preproregion of preprodermaseptin transcripts to identify new members of this family in Australian and South American hylids. All these peptides are cationic, amphipathic and alpha-helical. They killed a broad spectrum of microorganisms and acted in synergy. 42 preprodermaseptin gene sequences from 10 species of hylid and ranin frogs were analyzed in the context of their phylogeny and biogeography and of geophysical models for the fragmentation of Gondwana to examine the strategy that these frogs have evolved to generate an enormous array of peptide antibiotics. The hyperdivergence of modern antimicrobial peptides and the number of peptides per species result from repeated duplications of a approximately 150-million-year-old ancestral gene and accelerated mutations of the mature peptide domain, probably involving a mutagenic, error-prone, DNA polymerase similar to Escherichia coli Pol V. The presence of antimicrobial peptides with such different structures and spectra of action represents the successful evolution of multidrug defense by providing frogs with maximum protection against infectious microbes and minimizing the chance of microorganisms developing resistance to individual peptides. The hypermutation of the antimicrobial domain by a targeted mutagenic polymerase that can generate many sequence changes in a few steps may have a selective survival value when frogs colonizing a new ecological niche encounter different microbial predators.
... In Brazil, Syphaxin (Spx) and Syphaxin 1.5 were found in Leptodactylus syphax (Dourado et al., 2007) and Ryptophyllin (FPPWM-NH2) and Rohdei-litorin in Phyllomedusa rhodei (Barra et al., 1985;Montecucchi et al., 1984;Renda et al., 1985). Or in South America, 130 nmol Plasticin-L1 was found in Leptodactylus laticeps (Sousa et al., 2009), mixed Ocellatin-1 and Ocellaltin-4 in Leptodactylus (Sousa et al., 2009), Phyllomedusa sauvagei contains Dermorphin, Sauvagine and Dermaseptin I (Montecucchi et al., 1981;Mor & Nicolas, 1994) and a variety of substances such as dermaseptin I, dermaseptin B, dermatoxin, DS01, adenoregulin (with 33 amino acids) and PS-1 (Phylloseptins) were found in the Phyllomedusa family (Amiche et al., 1993;Amiche et al., 2000;Brand et al., 2002;Leite et al., 2005;Mor & Nicolas, 1994). The most prominent ones are octadecapeptidep-LR, esculentin-2P (E2P) and ranateurin-2P (R2P) isolated from R. pipiens (Canada) (Chinchar et al., 2001;Manika et al., 1998). ...
... ContinuedAmiche et al., 1993;Amiche et al., 2000;Brand et al., 2002;Leite et al., 2005;Mor & Nicolas, 1994) ...
... 7 Alm molecules are active against Gram-positive bacteria and fungi. 6,8 The membraneolytic activity of Alm depends on temperature, peptide concentration, peptide/lipid (P/L) molar ratio, and lipid bilayer composition. The Alm peptides can either bind to the lipid bilayer surface (inactive state) or insert into the membrane core (active state). ...
... 9−11 It is widely accepted that in the inserted state, the Alm molecules act as individual staves forming a water-filled barrel-like pore. 7,8,12 Alm has been extensively studied in recent years. He et al. applied neutron in-plane scattering to show that inserted Alm monomers form large, well-defined pores in dilaurylphosphatidylcholine lipid membranes. ...
... The antibacterial activity of ALM has already been recognized in the 1960s by Meyer and Reusser [8]. ALM is active against Gram-positive bacteria and fungi ( Table 3) [8] [78] [79], but seems ineffective against Gram-negative bacteria, which is probably due the lipopolysaccharides (LPS) of the outer membrane of Gram-negative bacteria forming a strong diffusion barrier against hydrophobic molecules such as peptaibols [66g]. However, Amiche et al. [78] found that the growth of Sinorhizobium meliloti (a Gram-negative soil bacterium) is inhibited by ALM (MIC ¼ 25 mm). ...
... ALM is active against Gram-positive bacteria and fungi ( Table 3) [8] [78] [79], but seems ineffective against Gram-negative bacteria, which is probably due the lipopolysaccharides (LPS) of the outer membrane of Gram-negative bacteria forming a strong diffusion barrier against hydrophobic molecules such as peptaibols [66g]. However, Amiche et al. [78] found that the growth of Sinorhizobium meliloti (a Gram-negative soil bacterium) is inhibited by ALM (MIC ¼ 25 mm). Obligatory anaerobic Gram-positive rumen bacteria proved to be sensitive to ALM, and in certain cases their ability to produce volatile fatty acids was reduced [79]. ...
The discovery of ALM, the archetype of the peptaibol family,
opened a new chapter in peptide research. During the past 40 years, the number of
known compounds within this peptide family has been growing exponentially. Besides
ALMs, the peptaibols known to date [2] [6] [84] [130] include (in alphabetical order)
aibellin, ampullosporins, antiamoebins, atroviridins, bergofungins, boletusin, cephaibols,
cervinins, chrysospermins, clonostachin, emerimicins, harzianins, helioferins,
heptaibin, hypelcins, hypomurocins, lipopubescins, lipostrigocins, longibrachins, paracelsins,
peptaibolin, peptaivirins, polysporins, pseudokonins, samarosporins, saturnisporins,
stilbellins, stilboflavins, suzukacillin, trichobrachins, trichobrevins, trichocellins,
trichocompactin, trichocryptins, trichodecenins, trichoferins, trichofumins, trichogin,
trichokindins, trichokonins, tricholongins, trichopolyns, trichorovins, trichorozins,
trichorzianines, trichorzins, trichosporins, trichostrigocins, trichostromaticins, trichotoxins,
trichovirins, trikoningins, tylopeptins, and zervamicins.
The primary-structure concept for ALM evolved in parallel with the technical
improvement of the chemical instrumentation. The determination of structural and
conformational properties of ALM and its analogues, as well as the characterization of
the channel structures formed by ALM and its ALM analogues, proved useful to
identify the key residues playing relevant roles in ALM-bilayer interactions, channel
formation, and channel stabilization. Overall, the results of these experimental and
theoretical investigations can contribute to a better understanding of the structural
basis of channel formation and function. Furthermore, molecular-modeling studies of ALM bundles embedded in lipid bilayers may provide valuable insights that could be
applied to examine the structural and dynamic features of other complex ion channels.
The efficiency of antibacterial chemotherapy is increasingly challenged by the
emergence of pathogenic strains exhibiting high levels of antibiotic resistance. It is very
important to search for novel compounds produced by living organisms. Peptide
antibiotics such as ALMs and other peptaibols are, therefore, in the focus of extensive
research efforts. Detailed knowledge of their properties may provide important
information about their potential applicability as drugs against pathogens.
ALM may become a valuable tool to unravel the early events of plant-defense
reactions under well-defined conditions. Due to its ability of membrane permeabilization,
ALM also has the potential to become a widely used, important agent to study
enzymes under conditions that resemble their native environment in intact cells,
mitochondria, chloroplasts, peroxisomes, or the endoplasmic reticulum.
Although the primary structure, conformation, as well as a series of biological
activities are already known in the case of ALM and a number of other peptaibols,
there is lackof information about the regulation of their biosynthesis. However, it can
be predicted that the potential applicability of these compounds in biotechnology and
industry will encourage research efforts aimed at studying the regulation of their
biosynthetic pathways.
... ), Mor and Nicolas (1994a), Chen and Shaw (2003), Chen et al. (2005a), UniProt (2009) P. tarsius 12 Silva et al. (2000), UniProt (2009) Unassigned to group [7 species] P. atelopoides — P. bicolor 21 Anastasi et al. (1970), Richter et al. (1990), Daly et al. (1992), Mignogna et al. 1992, Amiche et al. (1993), Amiche et al. (1994), Mor et al. (1994a, b); Charpentier et al. (1998); Fleury et al. (1998), Seon et al. (2000), Amiche et al. (2000), Pierre et al. (2000), Vanhoye et al. (2003), Chen et al. (2005b), UniProt (2009) ...
... Among the peptides of the FSAP family, the AMPs are the most diverse class. To date, the AMPs described in Phyllomedusa skin include seven distinct families (or subfamilies ) according to their sequence similarity and activity, e.g., Dermaseptin (Mor et al. 1991a), Dermatoxin (Amiche et al. 2000), Distinctin (Batista et al. 2001), Phylloseptin (Leite et al. 2005), Phylloxin (Pierre et al. 2000), Plasticin (Vanhoye et al. 2004), and Skin Polypeptide YY (Mor et al. 1994a, b, c). These peptides comprise the skin anti-infective passive defense barrier of these anurans. ...
Screening for new bioactive peptides in South American anurans has been pioneered in frogs of the genus Phyllomedusa. All frogs of this genus have venomous skin secretions, i.e., a complex mixture of bioactive peptides against potential predators and pathogens that presumably evolved in a scenario of predator-prey interaction and defense against microbial invasion. For every new anuran species studied new peptides are found, with homologies to hormones, neurotransmitters, antimicrobials, and several other peptides with unknown biological activity. From Vittorio Erspamer findings, this genus has been reported as a "treasure store" of bioactive peptides, and several groups focus their research on these species. From 1966 to 2009, more than 200 peptide sequences from different Phyllomedusa species were deposited in UniProt and other databases. During the last decade, the emergence of high-throughput molecular technologies involving de novo peptide sequencing via tandem mass spectrometry, cDNA cloning, pharmacological screening, and surface plasmon resonance applied to peptide discovery, led to fast structural data acquisition and the generation of peptide molecular libraries. Research groups on bioactive peptides in Brazil using these new technologies, accounted for the exponential increase of new molecules described in the last decade, much higher than in any previous decades. Recently, these secretions were also reported as a rich source of multiple antimicrobial peptides effective against multidrug resistant strains of bacteria, fungi, protozoa, and virus, providing instructive lessons for the development of new and more efficient nanotechnological-based therapies for infectious diseases treatment. Therefore, novel drugs arising from the identification and analysis of bioactive peptides from South American anuran biodiversity have a promising future role on nanobiotechnology.
... Phylloxin is a prototype narrow-spectrum antimicrobial peptide identified in P. bicolor skin [10]. Despite significant differences in primary structure to dermaseptins, the organization, nucleotide sequence of cloned cDNA, and primary structures of signal peptide and acidic spacer domains of the phylloxin precursor are remarkably highly-conserved [2] [10]. Here, we report the use of our recently-described molecular cloning technique to acquire nucleotide sequences of both phylloxin precursor cDNA (from polyadenylated mRNA) and of the phylloxin gene (from genomic DNA) using cDNA libraries generated from a single sample of lyophilized skin secretion (a " surrogate " tissue) obtained by non-invasive, non-lethal means. ...
... Phylloxin is a prototype narrow-spectrum antimicrobial peptide identified in P. bicolor skin [10]. Despite significant differences in primary structure to dermaseptins, the organization, nucleotide sequence of cloned cDNA, and primary structures of signal peptide and acidic spacer domains of the phylloxin precursor are remarkably highly-conserved [2,10]. Here, we report the use of our recently-described molecular cloning technique to acquire nucleotide sequences of both phylloxin precursor cDNA (from polyadenylated mRNA) and of the phylloxin gene (from genomic DNA) using cDNA libraries generated from a single sample of lyophilized skin secretion (a "surrogate" tissue) obtained by non-invasive, non-lethal means. ...
Phylloxin is a novel prototype antimicrobial peptide from the skin of Phyllomedusa bicolor. Here, we describe parallel identification and sequencing of phylloxin precursor transcript (mRNA) and partial gene structure (genomic DNA) from the same sample of lyophilized skin secretion using our recently-described cloning technique. The open-reading frame of the phylloxin precursor was identical in nucleotide sequence to that previously reported and alignment with the nucleotide sequence derived from genomic DNA indicated the presence of a 175 bp intron located in a near identical position to that found in the dermaseptins. The highly-conserved structural organization of skin secretion peptide genes in P. bicolor can thus be extended to include that encoding phylloxin (plx). These data further reinforce our assertion that application of the described methodology can provide robust genomic/transcriptomic/peptidomic data without the need for specimen sacrifice.
... En la piel de los anfibios un gran número de glándulas segregan un conjunto de péptidos antimicrobianos y otros compuestos bioactivos para protegerse de las infecciones. Los péptidos son sintetizados y almacenados en glándulas granulares, son liberados usualmente como respuesta a factores de estrés como depredación o a la presencia de una infección (Amiche et al., 2000;Shi et al., 2016). ...
In the present Thesis, bioinformatic analysis of potential anticancer small proteins from amphibia was carried out.
... (Erspamer et al., 1993). In addition to these peptides, other peptides such as adenoregulins and dermaseptins have been found with antibacterial, antifungal and anticancer properties (Amiche, Seon, Wroblewski, & Nicolas, 2000;Cao, Zhou, Ma, Luo, & Wei, 2005;van Zoggel et al., 2012). These properties stimulate some users' imagination and further support the perception of Kambo as a vaccine against many disorders, including infectious diseases. ...
A healing ritual has emerged in the West, based on the appropriation of elements from an indigenous Amazonian ritual involving a psychoactive secretion from the skin of a tree frog (Phyllomedusa bicolor) called Kambo. Kambo contains a plethora of bioactive peptides. It is applied via a heat-induced blister, referred to as a vaccination. The administration of Kambo leads to a quick onset of nausea, vomiting and a number of autonomic symptoms, including edema in the face (frog-face), palpitations and hypotension. These physiological effects of Kambo are analyzed as supporting therapeutic processes based in symbolic and transpersonal healing dynamics. Kambo induces a profound parasympathetic state with an intense internal orientation that evokes a number of physiological and emotional processes. This neoshamanistic ritual therapy uses these reactions to engage symbolic healing processes where intense physiological changes produced by Kambo support the experienced symbolism of what is referred to as being “infected by bad influences,” and subsequently being “cleansed” and “vaccinated against them.” Kambo healing involves core transpersonal principles of ecopsychology and ecotherapy that engage the transformative potency of nature in the form of the jungle frog’s venom and produces personal transformation and self-actualization through the intrinsic meanings provided by purging and intensified relations with nature.
... Among these peptides, AMPs are a class of well-characterized immune defense molecules. To date, some distinct families of AMPs have been characterized from different species of tree frogs, e.g., dermaseptin (Charpentier et al. 1998), dermatoxin (Amiche et al. 2000), plasticin (Vanhoye et al. 2004), and aurein (Boland and Separovic 2006). These peptides comprise an active defensive barrier in the tree frog skin, but no tree frog cathelicidin antimicrobial peptide has been reported. ...
As of February 2017, approximately 7639 amphibian species have been described in the AmphibiaWeb database. However, only 20 cathelicidin-like antimicrobial peptides have been identified to date from 10 amphibian species. Half of these peptides were identified from genome sequences and have not yet been functionally characterized. In this study, a novel cathelicidin-like peptide designated cathelicidin-PP was purified from the skin of tree frog Polypedates puerensis. Cathelicidin-PP is a 32 residue peptide of sequence ASENGKCNLLCLVKKKLRAVGNVIKTVVGKIA. Circular dichroism spectroscopy indicated that cathelicidin-PP mainly adopts a β-sheet structure in membrane-mimetic solutions. Cathelicidin-PP exhibits potent antimicrobial activity against bacteria and fungi, especially Gram-negative bacteria. Meanwhile, it shows low cytotoxicity toward mammalian cells. Scanning electron microscopy analysis indicated that cathelicidin-PP kills bacteria through the disruption of the bacterial cell membrane integrity. Furthermore, cathelicidin-PP exerts significant anti-inflammatory functions by inhibiting the lipopolysaccharide (LPS)-mediated generation of nitric oxide and pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1β, and interleukin-6. The MAPKs (ERK, JNK, and p38) and NF-κB signaling pathways are involved in the anti-inflammatory effect. Cathelicidin-PP caused partial neutralization of LPS in a dose-dependent manner. Quantitative PCR indicated that infection of tree frogs with bacteria causes increased expression of cathelicidin-PP in immune-related tissues. Taken together, cathelicidin-PP is the first identified cathelicidin-like peptide from tree frogs. Our findings demonstrate that in addition to direct bactericidal capacity, cathelicidin-PP also possesses immunomodulatory properties, including partial neutralization of LPS, and inhibiting the production of inflammatory cytokines.
... Indeed, the extremely conserved sequences have allowed the design of primers able to target this region and have been instrumental in the discovery of a large number of related peptides. These peptides have been classified in the following families: dermaseptins sensu stricto, dermatoxins, phylloxins, phylloseptins, plasticins, medusins, caerin-related peptides and orphan peptides [8][9][10][11][12][13][14][15][16][17][18]. ...
Biological significance:
Through the combination of molecular cloning, Edman degradation sequencing, tandem mass spectrometry and MALDI-TOF MS we have identified a new family of 15 antimicrobial peptides in the skin secretion of Cruziohyla calcarifer. The novel family is named "Cruzioseptins" and contains cationic amphipathic peptides of 20-32 residues. They have a broad range of antimicrobial activity that also includes effective antifungals with low haemolytic activity. Therefore, C. calcarifer has proven to be a rich source of novel peptides, which could become leading structures for the development of novel antibiotics and antifungals of clinical application.
... Amphibian skins contain a well-developed chemical defense system comprised of gene-encoded antimicrobial peptides and those with a wide range of pharmacological activities [1,2]. These peptides are synthesized and stored in the granular glands and are released when required, usually in response to a stressor, such as a predator attack or infection [3]. A large number of antimicrobial peptides have been studied and it has been shown that among different amphibian species, the expression of antimicrobial peptides is genetically determined and that they exhibit highly-conserved structures in their signal peptide sequences. ...
The dermaseptin antimicrobial peptide family contains members of 27-34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested.
... The study of different environments throughout the world has yielded a lot of antimicrobial agents that are of great value for the treatment of many infectious diseases. Animals are considerable to these researches, since they defend themselves against pathogenic microorganisms via production and secretion of antimicrobial components [3,4]. On the other hand, their intestinal tract is continuously exposed to bacteria present in the external environment. ...
Introduction and objective: Nowadays, widespread application of antibiotics results in resistant microorganisms all over the world. Thus screening researches for the products with antimicrobial activity have been lead to probe natural sources of antimicrobial agents to find new pharmaceutical products. Here we describe the antagonistic activity of a bacterium isolated from digestive system of Iran endemic slug, Parmacella iberica, its biochemical identification and phylogenetic relationships and extraction of antimicrobial substance. Materials and methods: Samples from digestive system of slugs were immediately transferred to broth medium. Antimicrobial agent-producing bacteria were isolated using serial dilution method on nutrient agar. One of them was selected for more investigation. Disk diffusion test was used against some laboratory standard strains to screen the antimicrobial potential. Identification of bacteria was done with 16S ribosomal DNA amplification and standard biochemical tests. Results: Phylogenetic analysis of 16S rDNA gene of this bacterium showed similarity to Pseudomonas aeruginosa confirmed by the result of biochemical tests. The results of the antibiogram assay implied that the antimicrobial substance is broad-spectrum. This substance is nonpolar since it is extracted by different solvents. The cell growth measurements revealed that this compound was produced at first logarithmic phase. Conclusion: High antimicrobial effects of the bacterium are evident. Due to increscent need to new antibiotics, purification and identification of this antimicrobial compound seem to be necessary for introducing new medicinal source. Significance and impact of the study: The isolation of bacterial strain producing antimicrobial agent with the broad spectrum antimicrobial properties is proposed.
... The study of different environments throughout the world has yielded a lot of antimicrobial agents that are of great value for the treatment of many infectious diseases. Animals are considerable to these researches, since they defend themselves against pathogenic microorganisms via production and secretion of antimicrobial components [3,4]. On the other hand, their intestinal tract is continuously exposed to bacteria present in the external environment. ...
Introduction and objective: Nowadays, widespread application of antibiotics results in resistant microorganisms all over the world. Thus screening researches for the products with antimicrobial activity have been lead to probe natural sources of antimicrobial agents to find new pharmaceutical products. Here we describe the antagonistic activity of a bacterium isolated from digestive system of Iran endemic slug, Parmacella iberica, its biochemical identification and phylogenetic relationships and extraction of antimicrobial substance. Materials and methods: Samples from digestive system of slugs were immediately transferred to broth medium. Antimicrobial agent-producing bacteria were isolated using serial dilution method on nutrient agar. One of them was selected for more investigation. Disk diffusion test was used against some laboratory standard strains to screen the antimicrobial potential. Identification of bacteria was done with 16S ribosomal DNA amplification and standard biochemical tests. Results: Phylogenetic analysis of 16S rDNA gene of this bacterium showed similarity to Pseudomonas aeruginosa confirmed by the result of biochemical tests. The results of the antibiogram assay implied that the antimicrobial substance is broad-spectrum. This substance is nonpolar since it is extracted by different solvents. The cell growth measurements revealed that this compound was produced at first logarithmic phase. Conclusion: High antimicrobial effects of the bacterium are evident. Due to increscent need to new antibiotics, purification and identification of this antimicrobial compound seem to be necessary for introducing new medicinal source. Significance and impact of the study: The isolation of bacterial strain producing antimicrobial agent with the broad spectrum antimicrobial properties is proposed.
... The antibacterial peptide dermatoxin was successfully extracted from the skin of the tree frog Phyllomedusa bicolor in 2000. This peptide exhibits bactericidal effects on Gram-positive and Gram-negative eubacteria (Amiche, Seon, Wroblewski, & Nicolas, 2000). Six peptides that demonstrate antimicrobial activities were also isolated from an extract of freezedried skin of the Japanese mountain brown frog Rana ornativentris (Kim, Iwamuro, Knoop, & Conlon, 2001). ...
The extraction process of polysaccharides from Rana chensinensis skin was optimized by using a Box–Behnken design. The optimum extraction conditions were as follows: extraction time, 4.96 h; extraction temperature, 100 °C; ratio of water to raw material, 60; and extraction frequency, 1. Under these conditions, the experimental polysaccharide yield was 2.03 ± 0.14%, which agreed with the predicted yield. The purified polysaccharide RCSP II was successfully obtained by diethylaminoethanol–Sepharose and Sepharose CL-6B column chromatography. In vitro experiments showed that RCSP II exhibited a strong scavenging activity against superoxide anion and 1,1-diphenyl-2-picrylhydrazyl radicals but a weak scavenging activity against hydroxyl radicals. RCSP II also showed a strong reducing capacity. Thus, this polysaccharide can be used as a natural antioxidant in functional foods or medicines.
... Although alamethicin is less effective against Gram-negative bacteria, possibly due to the lipopolysaccharide barrier present in the bacterium's outer membrane, it can still inhibit growth at higher concentrations. Alamethicin at 25 M was found to inhibit the growth of Sinorhizobium meliloti, a Gram-negative bacterium [20]. ...
... Anuran skin is a very complex organ that plays important roles in mechanical protection (Amiche et al. 2000), respiration (Van Driessche & Hillyard 1985), and ion and water balance (Stiffler 1994;Sørensen & Larsen 1996;Jørgensen 1993;. It is comprised of two major parts: a mucus-covered epidermis and underlying dermis. ...
Mucins are the major glycoprotein secretions of mucous glands and display important functions in amphibian skin such as regulation
of water homeostasis and mechanical and chemical protection. In the present study, we evaluated the glycoconjugate contents
of developing mucous glands on dorsal regions of metamorphosing Bufo viridis (Amphibia: Anura) tadpoles using an alcian blue-PAS panel and lectin histochemistry. All the conical cells of mucous glands
showed weak positivity for alcian blue in 0.025 M MgCl2 at pH 5.7 but only a few cells were positive for 0.3 M MgCl2 at the same pH. In addition, all the conical cells of mucous glands were negative for alcian blue at pH 2.5. In lectin histochemistry,
conical cells reacted strongly with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and peanut agglutinin (PNA), weakly with Maackia amurensis leucoagglutinin (MAL). These results suggest that they express predominantly mannose, galactose and partially α(2→3)-linked sialic acid containing glycoconjugates. We concluded that dorsal mucous glands of metamorphosing Bufo viridis tadpoles contain at least two different conical cell types and glycoconjugate heterogeneity of mucous glands may be related
with different functions of mucins.
... Gram-positive eubacteria (S. aureus) and Gram-negative eubacteria (E. coli 363 and K. pneumoniae) were cultured as described previously [29]. The minimal inhibitory concentrations (MICs) of peptides were determined in 96-well microtitration plates by growing the bacteria in the presence of 2-fold serial dilutions of peptide. ...
Cell penetrating peptides (CPPs) are usually short, highly cationic peptides that are capable of crossing the cell membrane and transport cargos of varied size and nature in cells by energy- and receptor-independent mechanisms. An additional potential is the newly discovered anti-tumor activity of certain CPPs, including RW16 (RRWRRWWRRWWRRWRR) which is derived from penetratin and is investigated here. The use of CPPs in therapeutics, diagnosis and potential application as anti-tumor agents increases the necessity of understanding their mode of action, a subject yet not totally understood. With this in mind, the membrane interaction and perturbation mechanisms of RW16 with both zwitterionic and anionic lipid model systems (used as representative models of healthy vs tumor cells) was investigated using a large panoply of biophysical techniques. It was shown that RW16 autoassociates and that its oligomerization state highly influences its membrane interaction. Overall a stronger association and perturbation of anionic membranes was observed, especially in presence of oligomeric peptide, when compared to zwitterionic ones. This might explain, at least in part, the anti-tumor activity and so the selective interaction with cancer cells whose membranes have been shown to be especially anionic. Hydrophobic contacts between the peptide and lipids were also shown to play an important role in the interaction. That probably results from the tryptophan insertion into the fatty acid lipid area following a peptide flip after the first electrostatic recognition. A model is presented that reflects the ensemble of results.
... Indeed, killing can occur synergistically with other AMPs and conventional antibiotics, helping overcome some barriers that resistant bacteria have against currently used antibiotics (Marr et al., 2006). Until then, many efforts have been carried out in order to use the AMPs in the development of new infection-fighting drugs applicable to new treatments of nosocomial infections and multidrug-resistant infections ( Amiche et al., 2000), due to the skill of the AMPs to kill multidrug resistant strains of microorganism by a mechanism unlikely to induce antibioticresistance. The development of new antimicrobials based on AMPs hold promises to medicine at the end of the classical antibiotic age by the emergence of the multidrugresistant microorganisms (Alanis, 2005;Arias & Murray, 2009;Nordmann et al., 2011). ...
... Current knowledge of anuran bioactive peptides suggests that they are only partially amenable to phylogenetic interpretation. Most of the peptides isolated from phyllomedusines have been divided into several families on the basis of amino acid sequence and pharmacological characteristics (see Erspamer, 1994 for a review; Chen et al., 2004 for a further refinement; and Amiche et al., 2000;Leite et al., 2005;and Thompson et al., 2007 for new families of antibiotic peptides), but there are various peptides that have not been associated with any of these families (e.g. Gebhard et al., 2004). ...
The leaf or monkey frogs of the hylid subfamily Phyllomedusinae are a unique group of charismatic anurans. We present a molecular phylogenetic analysis that includes 45 of the 60 species of phyllomedusines using up to 12 genes and intervening tRNAs. The aims were to gain a better understanding of the phylogenetic position of Phrynomedusa, test the monophyly and explore the relationships among several putative lineages (Hylomantis, the H. buckleyi Group, Phasmahyla, the four species groups of Phyllomedusa, and the species of Phyllomedusa that remain unassigned to any group), and to examine the implications of our phylogeny for the evolution of several characters in phyllomedusines. The analyses resulted in a well-supported phylogenetic hypothesis that provides a historical framework for a discussion of the evolution of characters associated with reproductive biology, gliding behaviour, the physiology of waterproofing, and bioactive peptides. Implications include an earlier origin for eggless capsules than for leaf-folding behaviour during amplexus, two independent origins of gliding, and an earlier origin of reduction in evaporative water loss than uricotelism, which is a result that originally was predicted on the basis of physiology alone. Furthermore, our results support the prediction that bioactive peptides from different peptide families are to be expected in all species of Phyllomedusinae. Hylomantis (as recently redefined) is shown to be paraphyletic and the synonymy of Agalychnis is revised to remedy this problem by including both Hylomantis and Pachymedusa.
© The Willi Hennig Society 2009.
... The frogs were anesthetized with pentobarbital, immersed in liquid nitrogen, and kept frozen until further processing (the animals were cared for in accordance with the guidelines outlined in the "Guide to the Care and Use of Experimental Animals", and the use of the animals was reviewed and approved by the local animal care review committee at the institution where the experiments were carried out). The skin of frogs was removed on dry ice and homogenized as described previously (Amiche et al. 2000). Total RNAs were extracted by the TRIzol method (Invitrogen, Shanghai, China) and were then purified via Transcript RNA clean-up kit (TianGen, Beijing, China) according to the manufacturer's instructions. ...
The skin glands of Ranidae are a rich source of antimicrobial peptides. In this study, the genomic RNA of Rana dybowskii was extracted from its skin while under Rana grylio virus stress. Five new cDNA sequences encoding 5 mature peptides, Ranatuerin-2YJ (GLMDIFKVAVNKLLAAGMNKPRCKAAHC), Dybowskin-YJb (IIPLPLGYFAKKP), Dybowskin-YJa (IIPLPLGYFAKKKKKKDPVPLDQ), Temperin-YJa (VLPLLETCSMTCWENNQTFGK), and Temperin-YJb (VLPLVGNLLNDLLGK), were obtained by reverse transcription polymerase chain reaction with a pair of degenerate primers designed according to the conserved terminal sequences of cDNA encoding antimicrobial peptide precursors of genus Rana. The antimicrobial activities of the peptides were analyzed, and the results demonstrated that all these peptides showed a significant anti-Rana grylio virus activity, and the virus was gradually cleared with the increase in gene expression. Among the 5 peptides obtained in this work, Ranatuerin-2YJ also showed a broad-spectrum anti-Gram-positive bacteria and anti-Gram-negative bacteria activity with a minimal inhibitory concentration of 22.5 µg/mL and 7.64% hemolysis activity, both of which were significantly lower (p < 0.05) than that of the other peptides. Moreover, Ranatuerin-2YJ was widely distributed in the skin, liver, spleen, and blood of R. dybowskii, while the other 4 peptides could only be cloned from the skin, indicating that the Ranatuerin-2YJ in vivo plays an important role in the protection against pathogen invasion.
... Indeed, killing can occur synergistically with other AMPs and conventional antibiotics, helping overcome some barriers that resistant bacteria have against currently used antibiotics (Marr et al., 2006). Until then, many efforts have been carried out in order to use the AMPs in the development of new infection-fighting drugs applicable to new treatments of nosocomial infections and multidrug-resistant infections (Amiche et al., 2000), due to the skill of the AMPs to kill multidrug resistant strains of microorganism by a mechanism unlikely to induce antibioticresistance . The development of new antimicrobials based on AMPs hold promises to medicine at the end of the classical antibiotic age by the emergence of the multidrugresistant microorganisms (Alanis, 2005; Arias & Murray, 2009; Nordmann et al., 2011). ...
... Cysteins participating to a disulfide bridge are in italics. ⁎Nomenclature for the orphan peptides awaits discovery of novel members [6,51,52,67,84,86,90,929394959697. enzyme has operated in hylids and ranids within the peptide progenitor sequence of antimicrobial peptide loci, but not in the signal peptide and acidic propiece domains. ...
Skin secretions of hylid frogs show amazing levels of interspecific and intraspecific diversity and are comprised of a cocktail of genetically-related, but markedly diverse antimicrobial peptides that are grouped into a superfamily, termed the dermaseptins, comprising several families: dermaseptins (sensu stricto), phylloseptins, plasticins, dermatoxins, phylloxins, hyposins, caerins, and aureins. Dermaseptin gene superfamily evolution is characterized by repeated gene duplications and focal hypermutations of the mature peptide coding sequence, followed by positive (diversifying) selection. We review here molecular mechanisms leading to these vast combinatorial peptide libraries, and structural and functional properties of antimicrobial peptides of the dermaseptin and plasticin families, as well as those of dermaseptin S9, an amyloidogenic peptide with antimicrobial and chemoattractant activities.
... South American and Neotropical frogs of the Phyllomedusinae subfamily (family Hylidae) produce a rich array of linear antimicrobial peptides that adopt an amphipathic ␣-helical structure. They include dermaseptins B and S (24-34 residues), phylloxin (19 residues), and Evolution of Amphibian Antimicrobial Peptides 859 dermatoxin (32 residues) from the genus Phyllomedusa (Amiche et al. 1994;Mor and Nicolas 1994;Charpentier et al. 1998;Amiche et al. 2000;Pierre et al. 2000) and peptides of 24-33 residues called dermaseptin-related peptides AA and PD from Agalychnis and Pachymedusa (Wechselberger 1998). ...
Antimicrobial peptides are expressed in the skin of amphibians and are used to prevent infection by microorganisms. Frog species store distinct collections of antimicrobial peptides that show variation in size, charge, conformation, and bactericidal activity, and so the evolution of antimicrobial peptide gene families may reflect the adaptive diversification of these loci. We examined the molecular evolution of antimicrobial peptide transcripts from hylid and ranid frog species. Our results show that after the gene family arose in the common ancestor of the Hylidae and Ranidae, before the divergence of these families in the Mesozoic, it subsequently diversified within these groups with numerous duplication events and divergence of loci. Moreover, we provide evidence that suggests that members of the antimicrobial peptide gene family have been subject to diversifying selection within both propiece and mature domains of hylids and solely within the mature domain of ranids. Finally, our results suggest that coordinated and compensatory amino acid replacements have occurred within the acidic propiece and cationic mature domain of hylid antimicrobial peptide precursors, as has been observed for mammalian defensin genes, but not among those of ranid precursors.
... Serial doubling dilutions of the three native and synthetic chrysophsins were carried out following the protocol described above, and the minimal lethal concentration (MLC) against E. coli and B. subtilis was defined as the lowest peptide concentration that caused % 99% killing of the bacteria [28]. All assays were performed in duplicate. ...
We report here the isolation of three isoforms of a novel C-terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major. Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides. Three peptides, named chrysophsin-1, chrysophsin-2, and chrysophsin-3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C-terminal RRRH sequence. The alpha-helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy. The synthetic peptides displayed broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens. The three peptides were also hemolytic. Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell-like cells at the base of the secondary lamellae in red sea bream gills. Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell-like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills.
Phyllomedusa bicolor (Phyllomedusidae), popularly known as the kambô in Brazil, is a tree frog that is widely distributed in South American countries and is known for producing a skin secretion that is rich in bioactive peptides, which are often used in indigenous rituals. The biological effects of the skin secretion were observed in the first studies with indigenous communities. Over the last six decades, researchers have been studying the chemical composition in detail, as well as the potential pharmacological applications of its constituents. For this reason, indigenous communities and health agents fear the misuse of the kambô, or the inappropriate use of the species, which can result in health complications or even death of users. This article seeks to provide a transdisciplinary review that integrates knowledge regarding the biology of P. bicolor, ethnoknowledge about the ritual of the kambô, and the chemistry and pharmacology of the skin secretion of this species, in addition to medical aspects of the indiscriminate use of the kambô. Furthermore, this review seeks to shed light on perspectives on the future of research related to the kambô.
Indonesia has about 450 frog species which is approximately 20% of frog species in the world. Among frog species found in Indonesia are Fejervarya limnocharis dan Limnonectes macrodon belonging to family Dicroglossidae. Frog skin secretion is considered to have a potency to be used as an alternative source of antibacterial agent against Streptococcus pneumoniae multidrug resistant (MDR). The aims of the present study were to analyze antibacterial activity of skin secretions of F. limnocharis and L. macrodon against S. pneumoniae multidrug resistant (MDR) and conduct molecular phylogenetic analysis of the frog used to ensure classification of frog species. The release of skin secretion was stimulated using epinephrine injection. Antibacterial activity of the skin secretions was tested using the well and paper disc methods. Results showed that skin secretions of F. limnocharis have antibacterial activity against S. pneumoniae multidrug resistant (MDR) SPN1307. The activity, however, was lower compared to that of chloramphenicol in both well and paper disc methods. On the other hand, skin secretions of L. macrodon failed to inhibit the growth of S. pneumoniae multidrug resistant (MDR) SPN1307. Molecular phylogenetic analysis was carried out on F. limnocharis based on DNA sequence of a partial fragment of mitochondrial cytochrome oxidase subunit I (COI) gene. Results showed that the frog F. limnocharis is closely related (97%) to Fejervarya sp from Bali. Skin secretions of F. limnocharis, therefore, has the potency to be developed as a source of antibacterial agents against S. pneumoniae multidrug resistant (MDR) SPN1307.
Peptaibols are peptide antibiotics that typically feature an N-terminal acetyl cap, a C-terminal aminoalcohol, and a high proportion of α-aminoisobutyric acid (Aib) residues. To establish how each feature might affect the membrane-activity of peptaibols, biomimetic Aib foldamers with different lengths and terminal groups were synthesised. Vesicle assays showed that long foldamers (eleven Aib residues) with hydrophobic termini had the highest ionophoric activity. C-terminal acids or primary amides inhibited activity, while replacement of an N-terminal acetyl with an azide group made little difference. Crystallography showed that N3Aib11CH2OTIPS folded into a 3<10> helix 2.91 nm long, which is close to the bilayer hydrophobic width. Planar bilayer conductance assays showed discrete ion channels only for N-acetylated foldamers. However long foldamers with hydrophobic termini had the highest antibacterial activity, indicating that ionophoric activity in vesicles was a better indicator of antibacterial activity than the observation of discrete ion channels.
A grafting techniques or using various synthetic and biological dressing also widely used to protect the wound area. There are 8 peptides with differential antimicrobial activities contained in Odorrana hosii’s skin secretion. However, to our best knowledge no study has been scientifically conducted to reveal the value off this species on wound healing. Primarily, the aim of this study was to look at the potential use of O. hosii’s skin as a biological dressing in wound healing management. This study assessed the wound healing in rat compared between wound grafted with O. hosii’s skin and wound treated with normal saline dressing. Histological examination was done to assess the wound healing activities after 14 days. The result shown, both wounds which were treated with O. hosii’s skin and untreated wound heal completely on day 14 as the epidermis and dermis completely close. Histologically, the percentage of neutrophils, macrophages and fibroblasts, were reduced on day 14. However, wounded skin, which was treated with O. hosii’s skin, had better healing quality as more new tissues and hair follicle regrowth compared with the untreated wound. It is suggested that poison gland in the O. hosii’s skin did not harm the wounded rat skin, instead, poison that act as defensive mechanism can help the species to fight the pathogen on the wound.
The chemical communication systems constitute an essential element in the establishment of intra- or inter-species relationships in marine environment, weaving a dense network of relations between individuals, in ecosystem. The invertebrates lacking of an immune system and usually sessile produce this type of bioactive metabolites playing a crucial role in the answer to the environmental pressures like the predation and the defence against potentially pathogens organisms. The aim of this work was to identify antimicrobial peptides in commercially bivalve and gastropod marine molluscs. Thus, the search for antimicrobial molecules from peptidic nature was undertaken in acid extracts of bivalve molluscs Cerastoderma edule, Ruditapes philippinarum, Ostrea edulis, and gastropods Crepidula fornicata, Buccinum undatum and Littorina littorea and from Crassostrea gigas hemolymph. The extracts were pre-purified by Solid Phase Extraction C18 (SPE) and elution was ensured by three successive steps of 10%, 40% and 80% of ACN-0.1% TFA. The antibacterial activities were assayed by determination of the CMI against a panel of target bacteria including Gram+ and Gram- bacteria. In parallel, antiviral activities were assayed in vitro against Herpes simplex virus type 1 and Vero cells by cell viability. The species C. edule, L. littorea and C. gigas proved to be the most effective and non cytotoxic species. A partial characterization of the activity detected in these species allowed determining the protenaceous nature of the active molecules. The purification of antimicrobial peptides realised on the C. gigas hemolymph led us to the identification of a peptide which structure lets foresee a bacterial origin. The hypothese of an association between C. gigas and bacteria led us from non axenic oysters culture to search for antagonist bacteria in C. gigas hemolymph and has conduced to isolate 2 Vibrio spp. and 3 Pseudoalteromonas spp. The Pseudoalteromonas spp. hCg 5 strain, allowed to partially characterized an active compound. Whole of these results suggests that the bacteria associated with the immune system could play an essential function of defence in bivalves.
Cell-penetrating peptides (CPPs) are able to efficiently transport cargos acrosscell membranes in a receptor- and energy-independent manner, without being cytotoxic to cells and thus present a great potential in drug delivery and diagnosis. The understanding of the cellular internalization and membrane interaction mechanisms is thus fundamental for their pharmaceutical development. In this study, two Arginine-rich CPPs derived from penetratin have been investigated: the peptides RW16 (RRWRRWWRRWWRRWRR) andRW9 (RRWWRRWRR). Firstly, a complete biophysical study of the peptide/lipid (P/L)interactions of RW16 has been accomplished and a preferential interaction for anionic lipids was demonstrated. Secondly, peptides derived from RW9 have been synthesized where tryptophan residues have been systematically replaced by phenylalanine. Cellular internalization and P/L interactions have been characterized, and the importance of the number and position of tryptophan has been highlighted.
Resistance to antibiotics is becoming a very serious problem, with so-called superbugs exhibiting resistance to nearly all conventional antibiotic drugs. Consequently, these organisms often cause severe illness and even death. Alternatives to conventional antibiotics are antimicrobial peptides (AMPs). These widely expressed short peptides, which have been isolated from insects, plants, marine organisms and mammals, including humans, show strong antimicrobial activity against both Gram-negative and Gram-positive bacteria. Most AMPs act by disrupting the bacterial membrane through "Barrel-stave", "Toroidal pore", "carpet" mechanism. In addition, AMPs may prevent septic shock through strongly binding lipopolysaccharides and lipoteichoic acid located on the bacterial membrane. The action mechanisms of AMP to minimize the likelihood developing resistance to the peptides would be particular advantage. For these reasons, we anticipate that AMPs will replace conventional antibiotic drugs in a variety of contexts.
Amphibians exhibit various, characteristic adaptations related to their “incomplete” shift from the aquatic to the terrestrial habitat. In particular, the integument was subject to a number of specialised modifications during the evolution of these animals. In this review, we place special emphasis on endogenous host-defence skin peptides from the cuteanous granular glands anuran amphibians (frogs and toads). The overview on the two broad groups of neuroactive and antimicrobial peptides (AMPs) goes beyond a simple itemisation in that we provide a new perspective into the evolution and function of anuran AMPs. Briefly, these cationic, amphipathic and α-helical peptides are traditionally viewed as being part of the innate immune system, protecting the moist skin against invading microorganisms through their cytolytic action. However, the complete record of anuran species investigated to date suggests that AMPs are distributed sporadically (i.e., non-universally) across Anura. Together with the intriguing observation that virtually all anurans known to produce neuropeptides in their granular glands also co-secrete cytolytic peptides, we call the traditional role for AMPs as being purely antimicrobial into question and present an alternative scenario. We hypothesise AMPs to assist neuroactive peptides in their antipredator role through their cytolytic action increasing the delivery of the latter to the endocrine and nervous system of the predator. Thus, AMPs are more accurately viewed as cytolysins and their contribution to the immune system is better regarded as an accessory benefit.
Background: Amphibian skins possess various antibacterial compounds that are effective against some microbial pathogens and are mostly released in response to environmental stress. In fact, the skin of Rana ridibunda, a large green frog, is a rich source of antimicrobial compounds that can be developed for therapeutic use. In the present study, the skin extract of Iranian Rana ridibunda was evaluated for its antimicrobial, hemolytic and cytototoxic activities.
Methods: The frog specimens were collected from Minoodasht located in Golesten province in Iran, during 2009. Subsequently, their skins were removed and the intended compounds were extracted. The crude extract was partially purified by gel filtration chromatography. The antimicrobial effects of skin extract were assessed against various microorganisms such as Escherchia coli, methicillin-resistant and -sensitive Staphyloccus aureus, vancomycin-resistant and -susceptible Enteroccus fecalis, Pseudomonas aeroginosa and Candida albicans. In addition, its minimum inhibition concentration, cytotoxic and hemolytic activities were determined.
Results: The crude extract of Rana ridibunda skin had valuable antimicrobial effects against methicillin-resistant and -susceptible S. aureus in comparison with E.coli and vancomycin-resistant and -susceptible E. fecalis. Besides, no antimicrobial activities were seen against P. aeroginosa or C. albicans. Moreover, the hemolytic and cytotoxic activities of the skin extract were minimal.
Conclusion: The antimicrobial activity of Iranian Rana ridibunda was comparable to those isolated from other Rana species. In conclusion, the skin extract of Rana ridibunda had the potential for a new therapeutic agent against the emerging drug-resistant bacteria, particularly methicillin-resistant and -sensitive S. aureus.
A growing number of cationic antimicrobial peptides have been isolated from the skin of hylid frogs belonging to the Phyllomedusinae subfamily. The amino acid sequences of these peptides are currently located in several databases under identifiers with no consistent system of nomenclature to describe them. In order to provide a workable terminology for antimicrobial peptides from Phyllomedusid frogs, we have made a systematic effort to collect, analyze, and classify all the Phyllomedusid peptide sequences available in databases. We propose that frogs belonging to the Phyllomedusinae subfamily should be described by the species names set out in Amphibian Species of the World: http://research.amnh.org/herpetology/amphibia/index.php, American Museum of Natural History, New York, USA. Multiple alignments analysis of at least 80 antimicrobial peptides isolated from 12 Phyllomedusinae species were distributed in seven distinct peptide families including dermaseptin, phylloseptin, plasticin, dermatoxin, phylloxin, hyposin and orphan peptides, and will be considered as the name of the headgroup of each family. The parent peptide's name should be followed by the first upper letter of the species for orthologous peptides and publication date determines priority. For example, the abbreviation B for bicolor and H for hypochondrialis. When two species begin with the same letter, two letters in upper case should be used (the first letter followed by the second or the third letter and so on). For example, the abbreviation DI for distincta, DU for duellmani, VA for vaillanti and VN for vanzolinii. Paralogous peptides should bear letter(s) in upper case followed by numbers.
It is well-known that there is a large amount of antimicrobial peptides in amphibian skins but few antimicrobial peptides are found in amphibian brains. Twenty-two and four antimicrobial peptides were purified and characterized from the brain homogenate of Bombina maxima and B. microdeladigitora, respectively. One hundred fifty-eight cDNA clones encoding 79 antimicrobial peptides were isolated from brain cDNA libraries of B. maxima and B. microdeladigitora. These antimicrobial peptides belong to two peptide groups (maximin and maximin-H). Twenty of them are identical to previously reported antimicrobial peptides (maximin 1-8, 10, 11, maximin H1, 3-5, 7, 9, 10, 12, 15, 16) from B. maxima skin secretions. Fifty-nine of them are novel antimicrobial peptides. Some of these antimicrobial peptides showed strong antimicrobial activities against tested microorganism strains including Gram-positive and -negative bacteria and fungi. The current diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal brains. The extreme diversity may give rise to interest to prospect the actual functions of antimicrobial peptides in amphibian brains.
Previous studies led to the isolation from skin extracts of Oki Tago's brown frog, Rana tagoi okiensis of five antimicrobial peptides belonging to the brevinin-1 (brevinin-1TOa), temporin (temporin-TOa and -TOb), and ranatuerin-2 (ranatuerin-2TOa and -2TOb) families, and bradykinin (BK) identical to mammalian BK. Using the reverse-transcription polymerase chain reaction (RT-PCR), we have now cloned from skin total RNA preparations cDNAs encoding biosynthetic precursors of brevinin-1TOa and brevinin-1TOb (containing the substitution Gly(1)-->Val), temporin-TOa and -TOb, and ranatuerin-2TOa and -2TOb. In addition, three cDNA clones encoding preprobradykinins were obtained that contained either one, two, or three tandem repeats of the sequence of BK followed by the sequence of [Thr(6)]-BK. In tissue expression analyses, preprobrevinin-1, preprotemporin, and preproranatuerin-2 gene transcripts were detected at higher levels in brain compared with peripheral tissues (heart, small intestine, kidney, liver lung, skeletal muscle, stomach, and testis). RT-PCR of brain RNA resulted in the amplification of cDNAs encoding ranatuerin-2TOc and ranatuerin-2TOd that contained the amino acid substitutions Lys(6)-->Arg and Ala(14)-->Thr, respectively compared with ranatuerin-2TOb. cDNAs encoding preprobrevinin-1TOa and preprotemporin-TOa were amplified from brain RNA as well as a second preprotemporin cDNA that contained a 10-nucleotide insertion that introduced a frame shift resulting in a premature stop codon. A cDNA encoding a novel peptide, DK25 (DVNDLKNLCAKTHNLLPMCAMFGKK) was amplified from brain RNA but neither DK25 nor its putative post-translationally modified form, DF22-amide (DVNDLKNLCAKTHNLLPMCAMF.NH(2)) displayed antimicrobial or hemolytic activities.
Four variants of the highly hemolytic antimicrobial peptide Pin2 were chemically synthesized with the aim to investigate the role of the proline residue in this peptide, by replacing it with the motif glycine-valine-glycine [GVG], which was found to confer low hemolytic activity in a spider antimicrobial peptide. The proline residue in position 14 of Pin2 was substituted by [V], [GV], [VG] and [GVG]. Only the peptide variant with the proline substituted for [GVG] was less hemolytic compared to that of all other variants. The peptide variant [GVG] kept its antimicrobial activity in Muller-Hilton agar diffusion assays, whereas the other three variants were less effective. However, all Pin2 antimicrobial peptide variants, were active when challenged against a Gram-positive bacteria in Muller-Hilton broth assays suggesting that chemical properties of the antimicrobial peptides such as hydrophobicity is an important indication for antimicrobial activity in semi-solid environments.
SWISS mice, edible frogs and goldfish i.p. injected with zymosan (Z groups) develop peritoneal inflammation connected with a massive intraperitoneal accumulation of leukocytes, which is significantly diminished in mice and fish (but not frogs) by supplementation of zymosan with morphine (ZM groups). In order to check the putative role of resident peritoneal macrophages in morphine-modulated zymosan-induced peritonitis, some animals were depleted of resident macrophages by repeated i.p. injections of clodronate-liposomes (CL) followed by Z or ZM injection. In SWISS mice such CL-induced removal ofMac-3-positive cells (macrophages) resulted in an enhanced influx and prolonged accumulation of polymorphonuclear leukocytes (PMNs) in CL-Z and CL-ZM groups in comparison with their counterparts with intact macrophages. Nevertheless, supplementation of zymosan with morphine inhibited the early stages of peritonitis in CL-treated animals as it did in untreated mice. This indicates that intact peritoneal macrophages of SWISS mice are important for limiting PMN accumulation, perhaps mainly through the release of IL-10, but are not critical for the induction of anti-inflammatory effects of morphine during the early stages of peritonitis. Unexpectedly, macrophage depletion in CL-treated frogs and fish resulted in a lack of a typical peritonitis in both Z and ZM groups of these ectothermic animals.
The flank skin of the premetamorphic stage of the green toad, Bufo viridis viridis, was studied by transmission electron microscopy. The skin was typically larval amphibian, made up of a highly fibrous and vascular dermis to the inside and a highly cellular epidermis to the outside with a distinct basal lamina separating the two layers. The epidermis is three-cells thick. The epidermal cells, mostly keratinocytes, are closely packed together and are attached to each other through desmosomes and interdigitations. The basal keratinocytes are anchored to the basal lamina through hemidesmosomes. In addition to the keratinocytes, some other types of cells known in the amphibian larval skin were found. These cells include mucus-secreting, mitochondria-rich cells, Merkle cells and flask cells. Anew type of cells, the dark cells, are described in this paper. The dark cell rests on the basal lamina. It is a ramified cell with a number of cytoplasmic processes intervening in between the keratinocytes. The cytoplasm is strikingly dark and rich with polysomes and granular endoplasmic reticulum.
Mycoplasmas, which are bacteria that are devoid of a cell wall and which belong to the class Mollicutes, are pathogenic for humans and animals and are frequent contaminants of tissue cell cultures. Although contamination of cultures with mycoplasma can easily be monitored with fluorescent dyes that stain DNA and/or with molecular probes, protection and decontamination of cultures remain serious challenges. In the present work, we investigated the susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to the membrane-active peptides alamethicin, dermaseptin B2, gramicidin S, and surfactin by growth inhibition and lethality assays. In the absence of serum, the four peptides killed mycoplasmas at minimal bactericidal concentrations that ranged from 12.5 to 100 microM, but in all cases the activities were decreased by the presence of serum. As a result, under standard culture conditions (10% serum) only alamethicin and gramicidin S were able to inhibit mycoplasma growth (MICs, 50 microM), while dermaseptin B2 and surfactin were ineffective. Furthermore, 8 days of treatment of HeLa cell cultures experimentally contaminated with either mycoplasma species with 70 microM enrofloxacin cured the cultures of infection, whereas treatment with alamethicin and gramicidin S alone was not reliable because the concentrations and treatment times required were toxic to the cells. However, combination of alamethicin or gramicidin S with 70 microM enrofloxacin allowed mycoplasma eradication after 30 min or 24 h of treatment, depending on the mycoplasma and peptide considered. HeLa cell cultures experimentally infected with mycoplasmas should prove to be a useful model for study of the antimycoplasma activities of antibiotics and membrane-active peptides under conditions close to those found in vivo.
To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1-8)-magainin 2 (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.
With the exception of cyclostomes, all vertebrates share the common immune strategy of adaptive, highly specific immunity, based on the products of recombination-activating genes and recombined noninherited receptors for antigens. In addition, they have retained ancient vectors of innate immunity, such as antimicrobial peptides, which are widespread in all eukaryotic organisms and show a high degree of structural homology across most animal taxa. Recently, these substances have become the objects of intensive study for their outstanding bioactive properties with the aim to be applied as very efficient antibiotics, antimicrobials, and even cancerostatics in clinical practice.
The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.
Two hemolytic peptides were isolated from the skin secretion of the Brazilian Hylidae frog Hyla biobeba, using reversed-phase chromatographic procedures. Hylins b1 and b2 exhibit short linear polypeptide chains, a large number of hydrophobic residues, amidated C termini and hemolytic properties. These two novel peptides are the first examples of bombinins H-like peptides isolated from anurans species not related to Bombina species.
We have shown that supplementation of proinflammatory agent with a high dose of morphine not only abolishes inflammation-related pain symptoms but also inhibits influx of leukocytes to the inflamed peritoneal cavity. Present investigations focused on effects of morphine on proopiomelanocortin and prodynorphin systems during zymosan-induced peritonitis. Males of SWISS mice were ip injected with zymosan (Z, 40 mg/kg) or zymosan with morphine (ZM, 20 mg/kg). At time 0 (controls) and 4 and 24h after stimulation, peritoneal leukocytes (PTLs) were counted, PTL levels of opioid peptides (beta-endorphin and dynorphin) measured by radioimmunoassays, while mRNAs coding their respective precursors (POMC and PDYN) and receptors (MOR and KOR) determined by QRT-PCR. Influx of inflammatory PTLs, mainly PMNs, was significantly delayed by morphine co-injection. Total levels of beta-endorphin and dynorphin corresponded with PTL numbers, while levels per cell were similar in all groups except of beta-endorphin, decreased in ZM at 4h. Levels of both peptides in peritoneal fluid were increased in Z and ZM groups at 4h, while at 24h only in case of beta-endorphin in Z group. POMC was increased only in ZM group at 4h of peritonitis, while PDYN in both Z and ZM groups at the same time. MOR mRNA was increased 24h after injection in Z and ZM groups, while KOR mRNA was similar in all groups except of decrease in Z at 24h. In conclusion, endogenous opioids and their receptors are involved in zymosan-induced peritonitis and affected in various ways by morphine co-injection.
Nonrandom patterns associated with adaptively evolving genes can shed light on how selection and mutation produce rapid changes in sequences. I examine such patterns in two independent families of antimicrobial peptide genes: those in frogs, which are known to have evolved under positive selection, and those in flatfishes, which I show have also evolved under positive selection. I address two recently proposed hypotheses about the molecular evolution of antimicrobial peptide genes. The first is that the mature peptide region is replicated by an error-prone polymerase that increases the mutation rate and the transversion/transition ratio compared to the signal sequence of the same genes. The second is that mature peptides evolve in a coordinated fashion with their propieces, such that a change in net charge in one molecular region prompts an opposite change in charge in the other region. I test these hypotheses using alternative methods that minimize alignment errors, correct for phylogenetic nonindependence, reduce sequence saturation, and account for differing selection pressures on different regions of the gene. In both gene families I show that divergence at both synonymous and nonsynonymous sites within the mature peptide region is enhanced. However, in neither gene family is there evidence of an increased mutational transversion/transition ratio or coordinated evolution. My observations are consistent with either an elevated mutation rate in an adaptively evolving gene region or widespread selection on "silent" sites. These hypotheses challenge the assumption that mutations are random and can be measured by the synonymous substitution rate.
Mortalities related to the detection of Ostreid Herpesvirus 1 (OsHV-1) have been previously reported in France among larvae and spat of the Pacific oyster Crassostrea gigas. Adult oysters appear less sensitive to herpesvirus infections, although OsHV-1 has been detected in adults without signs of disease or mortality. This suggests that the virus is able to persist in its host and that adult oysters may be able to control OsHV-1 infection. Little is known about antiviral substances in invertebrates. The present work concerns the research of antiviral substances in adult oyster C. gigas, where putative antiviral activities were monitored using 3 strategies: (1) in metabolites with variable polarity, (2) in peptidic extracts and (3) in crude haemolymph. In vitro antiviral assays were based on inhibition of Herpes simplex virus type 1 (HSV-1) replication in Vero cell monolayers. All extracts presented no cytotoxicity. Antiviral activity was detected in the fresh filtered haemolymph (EC50:425 microg ml(-1)) and seasonal variation of the haemolymph antiviral activity was monitored.
Analysis of antimicrobial activities that are present in the skin secretions of the South American frogPhyllomedusa bicolor revealed six polycationic (lysine-rich) and amphipathic α-helical peptides, 24–33 residues long, termed dermaseptins B1 to
B6, respectively. Prepro-dermaseptins B all contain an almost identical signal peptide, which is followed by a conserved acidic
propiece, a processing signal Lys-Arg, and a dermaseptin progenitor sequence. The 22-residue signal peptide plus the first
3 residues of the acidic propiece are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin
genes. The 25-residue amino-terminal region of prepro-dermaseptins B shares 50% identity with the corresponding region of
precursors ford-amino acid containing opioid peptides or for antimicrobial peptides originating from the skin of distantly related frog species.
The remarkable similarity found between prepro-proteins that encode end products with strikingly different sequences, conformations,
biological activities and modes of action suggests that the corresponding genes have evolved through dissemination of a conserved
“secretory cassette” exon.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
Skin of the frog Phyllomedusa sauvagei contains a cDNA sequence that codes for the selective mu-receptor peptide dermorphin and a new heptapeptide we have designated as dermorphin gene-associated peptide (DGAP). Investigation of the opioid receptor binding characteristics of synthetic DGAP and [D-Met2]DGAP revealed that the latter peptide had high affinity and selectivity for delta-type opioid receptors in rat brain synaptosomes. The IC50 values for DGAP on mu- and delta-receptors were only 28 microM and 670 nM, respectively, while that for [D-Met2]DGAP was 0.80 nM for delta-receptors and greater than 1 microM for mu-receptors yielding a very high delta selectivity ratio (SR) of 1345. In comparison, the SR values for [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Pen2,5]enkephalin, ligands which are considered to be specific for delta-receptors, were 20, 42, and 301, respectively. Dermorphin, which contains a D-Ala2 residue and is a selective mu-receptor ligand (Lazarus, L.H., Guglietta, A., Wilson, W.E., Irons, B.J., and de Castiglione, R. (1989) J. Biol. Chem. 264, 354-362), exhibits a SR of 0.0055 similar to that for the conventional mu-agonist [D-Ala2,NMePhe4,Gly-ol]enkephalin (0.0040). This finding that frog skin cDNA contains the information to code for dermorphin and DGAP, or the presumed [D-Met2]DGAP molecule, which are among the most selective high affinity opioid ligands described for mu- and delta-receptors, may permit new insight into the design of future opioid receptor agonists and antagonists.
The physiological significance of the occurrence of sequence similar antimicrobial peptides in frog skin, as the bombinins in Bombina, the magainins in Xenopus, and the dermaseptins in Phyllomedusa, is a major unanswered question. Dermaseptins s1, s2, s3, s4, and s5, a family of cationic (lysine-rich), amphipathic antifungal peptides of 28-34 residues were thus synthesized, purified to homogeneity, and evaluated for their growth-inhibition activity in vitro against various pathogenic microorganisms. Although all five of these peptides shared a similar spectrum of lytic activity against the filamentous fungi that are responsible for opportunistic lethal infections that follow the immunodeficiency syndrome or the use of immunosuppressive agents, they exhibited marked differences in their potencies to arrest the growth of Gram-positive and Gram-negative pathogenic bacteria and yeasts. Likewise, whereas dermaseptins s1 and s5 were devoid of hemolytic activity, dermaseptin s4 caused lysis of erythrocytes at micromolar concentrations. The dermaseptins exhibited dramatic synergy of action upon combination, resulting in some cases in a 100-fold increase in antibiotic activity of the mixture over the activity of the peptides separately. Shortening the peptide chain of dermaseptin s3 to dermaseptin s3-(1-16)-NH2 did not affect the antimicrobial potency of the peptide. Further reduction of the chain length yielded peptide derivatives gradually showing reduced activity. Surprisingly, however, analogs of dermaseptin s3 as shorter as 10-12 residues in length remained fully active against Enterococcus faecalis, Cryptococcus neoformans, and against Aeromonas caviae, the causal agent of red-leg disease in amphibians. Overall, these results suggest that, despite 40% sequence similarities, the dermaseptins have distinct spectra of anti-microbial activity and may act in concert to circumvent host invasion by providing frogs with a better shielding against a broad array of microorganisms. They also demonstrate the potential usefulness of short analogs of these peptides as potential candidates for biorational design of germicides.
The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.
Three cytolytic peptides, termed brevinin-1E, brevinin-2E, and esculentin, were isolated from skin secretions of the European frog Rana esculenta (Simmaco, M., Mignogna, G., Barra, D., and Bossa, F. (1993) FEBS Lett. 324, 159-161). Nucleotide sequence analysis of cDNAs coding for the corresponding precursors revealed that in all of them a single copy of the sequence of the mature peptide is present preceded by a dibasic cleavage site and followed by a stop codon. The signal peptides of these precursors show a clear homology to the corresponding region of the precursor of dermorphin, a neuropeptide occurring in the skin of amphibians of the subfamily Phyllomedusinae. Ten new peptides, ranging in size from 24 to 46 residues, all possessing an intramolecular disulfide bridge located at the carboxyl-terminal end, were isolated from skin secretions of R. esculenta. These peptides can be grouped into four subfamilies on the basis of their distinctive structural and/or functional properties. All of these new peptides have antimicrobial and/or hemolytic activities typical for the respective subfamily. In addition, we demonstrate that esculentin-1 also inhibits the growth of Pseudomonas aeruginosa, Candida albicans, and Saccharomyces cerevisiae.
Dermaseptin, a 34-amino acid residue cationic peptide, was recently shown to inhibit the growth of pathogenic fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. To improve our understanding of the mechanism by which dermaseptin exerts its potent antimicrobial action, a series of either NH2- or COOH-terminally truncated analogs was synthesized. These analogs were evaluated for their ability to inhibit the growth of various pathogenic agents in culture medium. Dermaseptin exerted a lytic action upon bacteria, protozoa, yeasts, and filamentous fungi at micromolar concentrations. No inhibition of proliferation was observed with human KB cells, and dermaseptin did not lyse guinea pig lymphocytes or rabbit erythrocytes at doses up to 200 micrograms/ml. Shortening the peptide chain of dermaseptin to dermaseptin-(3-34) slightly reduced the activity of the peptide, while further reduction of the chain length to residues 14-34, 16-34, 20-34, and 28-34 yielded peptide derivatives devoid of antimicrobial activity. On the other hand, lengthening the peptide chain starting from residues 1-4 to residues 1-8 and 1-18 led to a progressive recovery of the activity of the parent molecule. Whereas the central core of dermaseptin (residues 10-19) was virtually inactive, alteration of the COOH-terminal carboxylic group of dermaseptin-(1-18) to a carboxamide yielded a peptide exhibiting enhanced antimicrobial potency, yet displaying even less in vitro toxicity compared with dermaseptin. Overall, the data indicate that molecular elements responsible for the exceptional antimicrobial potency of dermaseptin are to be traced to the NH2-terminal alpha-helical amphipathic segment spanning residues 1-18 of the molecule. Dermaseptin-(1-18)-NH2 may therefore be considered as a useful and highly tractable tool for identifying key features responsible for membrane permeabilization and as a starting point for the design of new therapeutic agents.
Amphibian skin secretions contain many biologically active compounds, such as biogenic amines, complex alkaloids, or peptides. Within the latter class of molecules, a large number of peptide antibiotics has been isolated and characterized from different amphibian species. Antimicrobial peptides are considered the effector molecules of innate immunity, acting as a first line of defense against bacterial infections, by perturbing the phospholipid bilayer of the target cell membrane. These gene-encoded molecules are synthesized as inactive precursors and in several cases their proparts were shown to have highly conserved structures. It has also been demonstrated that the promoter regions of inducible peptide antibiotics are often regulated by the transcriptional control machinery NF-kappa B/I kappa B alpha. In amphibia of Rana and Bombina genera, inhibition of transcription of the genes encoding antimicrobial peptides has been obtained by glucocorticoid treatment, which causes an increase of I kappa B alpha synthesis. Moreover, determination of the structure of a number of genes coding for antimicrobial peptides in amphibia has actually shown that their promoter regions contain recognition sites for nuclear factors.
The skin secretions of female and male Litoria splendida have been monitored monthly over a three-year period using HPLC and electrospray mass spectrometry. Two minor peptides are present only in the skin secretion of the male. The first of these is the female-attracting aquatic male sex pheromone that we have named splendipherin, a 25 amino acid peptide (GLVSSIGKALGGLLADVVKSKGQPA-OH). This pheromone constitutes about 1% of the total skin peptides during the breeding season (January to March), dropping to about 0.1% during the period June to November. Splendipherin attracts the female in water at a concentration of 10 211 ±10 29 m, and is species specific. The second peptide is a wide-spectrum antibiotic of the caerin 1 group, a 25 residue peptide (GLLSVLGSVAKHVLPHVVPVIAEKL-NH 2) named caerin 1.10. The neuropeptides of L. splendida are also seasonally variable, the change identical for both the female and male. During the period October to March, the sole neuropeptide present in skin secretions is caerulein [pEQDY(SO 3)TGWMDF-NH 2 ]; this is active on smooth muscle and is also an analgaesic. During the southern winter (June to September), more than half of the caerulein is hydrolysed to [pEQDYTGWMDF-NH 2 ], a peptide that shows no smooth muscle activity. In place of caerulein, a new peptide, Phe8 caerulein [pEQDY(SO 3)TGWFDF-NH 2 ], becomes a major component of the skin secretion. Perhaps this seasonal change is involved in thermoregulation, that is, with the initiation and maintenance of the inactive (hibernation) phase of the animal.
We cloned the genes of two members of the dermaseptin family, broad-spectrum antimicrobial peptides isolated from the skin of the arboreal frog Phyllomedusa bicolor. The dermaseptin gene Drg2 has a 2-exon coding structure interrupted by a small 137-bp intron, wherein exon1 encoded a 22-residue hydrophobic signal peptide and the first three amino acids of the acidic propiece; exon2 contained the 18 additional acidic residues of the propiece plus a typical prohormone processing signal Lys–Arg and a 32-residue dermaseptin progenitor sequence. The dermaseptin genes Drg2 and Drg1g2 have conserved sequences at both untranslated ends and in the first and second coding exons. In contrast, Drg1g2 comprises a third coding exon for a short version of the acidic propiece and a second dermaseptin progenitor sequence. Structural conservation between the two genes suggests that Drg1g2 arose recently from an ancestral Drg2-like gene through amplification of part of the second coding exon and 3′-untranslated region. Analysis of the cDNAs coding precursors for several frog skin peptides of highly different structures and activities demonstrates that the signal peptides and part of the acidic propieces are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The organization of the genes that belong to this family, with the signal peptide and the progenitor sequence on separate exons, permits strikingly different peptides to be directed into the secretory pathway. The recruitment of such a homologous `secretory' exon by otherwise non-homologous genes may have been an early event in the evolution of amphibian.
The structure of caerin 1.1, a novel peptide with biological activity from Australian tree frogs, is determined on the basis of mass spectrometric data; the peptide's primary sequence prevents it from achieving a complete α-helix.
Seven peptides have been isolated from the parotoid and rostral glands of the Australian green tree frog Litoria splendida. These include caerulein (a known hypotensive toxin), and six new compounds named caerin 1.1 (M, 2582), 1.1.1 (2412), 1.1.2 (2299), 2.1 (2392), 3.1 (2382) and caeridin 1 (1139). The primary structures of the peptides have been determined by FAB mass spectrometric methods, and confirmed by automated sequencing. The major peptides have been synthesised using t-BOC (N-tert-butoxycarbonyl)N-protection and a p-methylbenzhydrylamine support.
A 37-residue cationic antimicrobial peptide named mesentericin Y 10537 was purified to homogeneity from cell-free culture supernatant of the Gram-positive bacterium Leuconostoc mesenteroides. The complete amino acid sequence of the peptide, KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW, has been established by automated
Edman degradation, mass spectrometry, and solid phase synthesis. Mesentericin Y 10537 contains a single intramolecular disulfide bond that forms a 6-membered ring within the molecule. Mesentericin Y 10537 was synthesized by the solid phase method. The synthetic replicate was shown to be indistinguishable from the natural peptide
with respect to electrophoretic and chromatographic properties, mass spectrometry analysis, automated amino acid sequence
determination, and antimicrobial properties. At nanomolar concentrations, synthetic mesentericin Y 10537 is active against Gram+ bacteria in the genera Lactobacillus and Carnobacterium. Most interestingly, the peptide is inhibitory to the growth of the food-borne pathogen Listeria. CD spectra of mesentericin Y 10537 in low polarity medium, which mimic the lipophilicity of the membrane of target organisms, indicated 30–40% α-helical conformation,
and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning over
residues 17–31. To reveal the molecular basis of the specificity of mesentericin Y 10537 targetting and mode of action, NH2- or COOH-terminally truncated analogs together with point-substituted analogs were synthesized and evaluated for their ability
to inhibit the growth of Listeria ivanovii. In sharp contrast with broad spectrum α-helical antimicrobial peptides from vertebrate animals, which can be shortened to
14–18 residues without deleterious effect on potency, molecular elements responsible for anti-Listeria activity of mesentericin Y 10537 are to be traced at once to the NH2-terminal tripeptide KYY, the disulfide bridge, the putative α-helical domain 17–31, and the COOH-terminal tryptophan residue
of the molecule. It is proposed that the amphipathic helical domain of the peptide interacts with lipid bilayers, leading
subsequently to alteration of the membrane functions, whereas residues 1–14 form part of a recognition structure for a membrane-bound
receptor, which may be critical for peptide targetting. Because mesentericin Y 10537 is easy to synthesize at low cost, it may represent a useful and tractable tool as a starting point for the design of more
potent analogs that may be of potential applicability in foods preservation.
The deltorphins are a class of highly selective δ-opioid heptapeptides from the skin of the Amazonian frogs Phyllomedusa sauvagei and P. bicolor. The first of these fascinating peptides came to light in 1987 by cloning of the cDNA of from frog skins, while the other members of this family were identified either by cDNA or isolation of the peptides. The distinctive feature of deltorphins is the presence of a naturally occurring d-enantiomer at the second position in their common N-terminal sequence, Tyr–d-Xaa–Phe, comparable to dermorphin, which is the prototype of a group of μ-selective opioids from the same source. The d-amino acid and the anionic residues, either Glu or Asp, as well as their unique amino acid compositions are responsible for the remarkable biostability, high δ-receptor affinity, bioactivity and peptide conformation.This review summarizes a decade of research from many laboratories that defined which residues and substituents in the deltorphins interact with the δ-receptor and characterized pharmacological and physiological activities in vitro and in vivo. It begins with a historical description of the topic and presents general schema for the synthesis of peptide analogues of deltorphins A, B and C as a means to document the methods employed in producing a myriad of analogues. Structure–activity studies of the peptides and their pharmacological activities in vitro are detailed in abundantly tabulated data. A brief compendium of the current level of knowledge of the δ-receptor assists the reader to appreciate the rationale for the design of these analogues. Discussion of the conformation of these peptides addresses how structure leads to further hypotheses regarding ligand–receptor interaction. The review ends with a broad discussion of the potential applications of these peptides in clinical and therapeutic settings.
The predicted amino acid sequence of the biosynthetic precursor of dermorphin, a highly potent and nearly specific μ-opioid peptide from amphibian skin, contains four repeats of the dermorphin progenitor sequence and one single copy of a different heptapeptide sequence. We have developed a specific enzyme immunoassay and used synthetic peptides to detect and purify the new predicted heptapeptide 2.4 μg/g dry skin) from the skin of the Phyllomedusa sauvagei frog from which dermorphin was originally isolated. The identity of the novel pro-dermorphin related peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, was established by co-chromatography with synthetic peptides on reverse-phase HPLC, immunological analysis, gas-phase sequencing, mass spectrometry and by pharmacological assays. Opioid-binding assays in vitro demonstrated that both the natural and synthetic heptapeptides displayed exceptionally high selectivity and affinity towards the δ-opioid receptors. Because of its origin and its δ-opioid (enkephalin) activity and specificity, this novel D-amino acid containing peptide is named dermenkephalin.
4'-6-DIAMIDINO-2-PHENYLINDOLE (DAPI), which was first synthesised by Dann et al.1 as a trypanocide related to Berenil, has been shown to possess useful DNA binding properties2. Thus, DAPI will bind differentially to yeast mitochondrial and nuclear DNA forming highly fluorescent complexes and enhancing the separation of the two DNAs in caesium chloride gradients2. DAPI can also be used as a highly specific fluorescent stain for both nuclear and mitochondrial DNA in yeast. It seems to be highly sensitive and probably permits detection of a single yeast mitochondrial DNA molecule (D.H.W., and D. J. Fennell, unpublished).
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Two unique antimicrobial peptides named brevinin-1 and -2 were isolated from the skin of the frog, Rana brevipoda porsa. Both of the peptides did not have any structural homology with bombinin nor magainin; the frog skin derived-antimicrobial peptides isolated from Bombina and Xenopus, nor even with other known antimicrobial peptides of non-amphibian origin. The minimum inhibitory concentration of brevinin-1 against the growth of St. aureus and E. coli was determined to be 8 micrograms/ml and 34 micrograms/ml while that of brevinin-2 was 8 micrograms/ml and 4 micrograms/ml, respectively, indicating the difference of the two peptides in the antimicrobial selectively on Gram-positive and Gram-negative bacteria.
Three equivocal amino acid sequences were synthesized that are predicted to be alpha-helical from amino acid preference but are found to be primarily beta-strand from x-ray diffraction of their respective proteins. In some solvent systems we recover the alpha-helical structure predicted by amino acid preference, whereas in other systems we mimic the interior of the protein and produce a beta-strand. These results are experimental proof that the environment is important in determining the secondary structure formed by an amino acid sequence; therefore schemes that predict secondary structure from amino acid sequence alone can never be totally successful.
Amphibian skin represents a formidable factory and storehouse of biogenic amines, potent alkaloid neurotoxins, and active peptides.
We present the structure of four precursors for [D-Ala2]deltorphins I and II as deduced from cDNAs cloned from skin of the frog Phyllomedusa bicolor. These contain the genetic information for one copy of [D-Ala2]deltorphin II and zero, one, or three copies of [D-Ala2]deltorphin I. In each case, the D-alanine of the end product is encoded by a normal GCG codon for L-alanine. In addition, the existence of three peptides related to dermorphin was predicted from the amino acid sequence of the precursors. These peptides were synthesized with a D-alanine in position 2 and their pharmacological properties were tested. Two of them, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH, were found to have roughly the same affinity and selectivity for mu-type opioid receptors as dermorphin.
With a cDNA library prepared from skin of Phyllomedusa sauvagei, the sequence of the precursor of dermorphin was elucidated recently. The sequence suggested the existence of another peptide, distantly related to dermorphin. Two variants of this peptide have now been synthesized containing either L- or D-methionine as the second amino acid. The peptide containing the D-methionine exhibited high-affinity and selectivity for delta opioid receptors in the mouse vas deferens and in rat brain homogenates. Moreover, using the synthetic peptide as marker, we could isolate small quantities of the corresponding natural peptide containing D-methionine as the second amino acid from skin extracts of Phyllomedusa sauvagei. The name deltorphin is proposed for this new peptide and its sequence is Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2.
Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.
Previously, we described a family of novel broad spectrum antimicrobial peptides, magainins, from the skin of Xenopus laevis. In this report we show that at least two other Xenopus peptides, present in the skin and its secretions, PGLa and a peptide released from the xenopsin precursor, exhibit antimicrobial properties comparable to the magainins. The identification of these newer members provides insight into the structural diversity of vertebrate antimicrobial peptides.
A D-alanine-containing peptide termed dermorphin, with potent opiate-like activity, has been isolated from skin of the frog Phyllomedusa sauvagei. Complementary DNA (cDNA) libraries were constructed from frog skin messenger RNA and screened with a mixture of oligonucleotides that contained the codons complementary to five amino acids of dermorphin. Clones were detected with inserts coding for different dermorphin precursors. The predicted amino acid sequences of these precursors contained homologous repeats of 35 amino acids that included one copy of the heptapeptide dermorphin. In these cloned cDNAs, the alanine codon GCG occurred at the position where D-alanine is present in the end product. This suggests the existence of a novel post-translational reaction for the conversion of an L-amino acid to its D-isomer.
Two nonapeptides were isolated from the toxin of the orange-bellied toad Bombina variegata. Their amino acid sequences were: ser-ala-lys-gly-leu-ala-glu-his-phe; gly-ala-lys-gly-leu-ala-glu-his-phe.
The three-dimensional structures of alpha-helices can be represented by two-dimensional projections which we call helical wheels. Initially, the wheels were employed as graphical restatements of the known structures determined by Kendrew, Perutz, Watson, and their colleagues at the University of Cambridge and by Phillips and his coworkers at The Royal Institution. The characteristics of the helices, discussed by Perutz et al. (1965), and Blake et al. (1965), can be readily visualized by examination of these wheels. For example, the projections for most helical segments of myoglobin, hemoglobin, and lysozyme have distinctive hydrophobic arcs. Moreover, the hydrophobic residues tend to be clustered in the n +/- 3, n, n +/- 4 positions of adjacent helical turns. Such hydrophobic arcs are not observed when the sequences of nonhelical segments are plotted on the wheels. Since the features of these projections are also distinctive, however, the wheels can be used to divide sequences into segments with either helical or nonhelical potential. The sequences of insulin, cytochrome c, ribonuclease A, chymotrypsinogen A, tobacco mosaic virus protein, and human growth hormone were chosen for application of the wheels for this purpose.
According to the signal hypothesis, a signal sequence, once having initiated export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. It has long been known that some part of the cleavage specificity resides in the last residue of the signal sequence, which invariably is one with a small, uncharged side-chain, but no further specific patterns of amino acids near the point of cleavage have been discovered so far. In this paper, some such patterns, based on a sample of 78 eukaryotic signal sequences, are presented and discussed, and a first attempt at formulating rules for the prediction of cleavage sites is made.
Dermorphin, a heptapeptide with very potent opiate-like activity, has been isolated from methanol extracts of the skin of the South American frog Phyllomedusa sauvagei. The amino acid sequence of the peptide is: H-Try-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2. Dermorphin presents striking differences from the known enkephalins; it offers a surprising example of a peptide from Vertebrata containing a D-amino acid residue in its sequence.
Gaegurins, a family of peptide antibiotics with sizes ranging from 24 to 37 amino acids, have recently been purified from Rana rugosa skin (Park, J.M., Jung, J.-E. and Lee, B.J. (1994) Biochem. Biophys. Res. Commun. 205, 948-954). Two complete cDNAs encoding gaegurins 4 and 5 were isolated from a library constructed with the frog skin mRNAs. Each clone contained a single open reading frame that encodes a gaegurin precursor polypeptide. The deduced amino acid sequences revealed that the precursors have a unique tripartite structure: a putative signal sequence at the NH2-terminus followed by an acidic spacer region rich in glutamic and aspartic acids, and a mature gaegurin peptide at the COOH-terminus. Similar modes of organization were also found in antimicrobial or opioid peptide precursors of other frog species, although their mature peptides show little sequence homology. The family of peptides with this characteristic now expands. Northern analysis revealed that gaegurins are extensively expressed in the skin tissue, but not in liver and muscle.
A novel protein family, showing a conserved proregion and a variable C-terminal antimicrobial domain, and named cathelicidin, has been identified in mammalian myeloid cells. The conserved proregion shows sequence similarity to members of the cystatin superfamily of cysteine proteinase inhibitors. Cathelicidins are stored in the cytoplasmic granules of neutrophil leukocytes and release the antimicrobial peptides upon leukocyte activation. Some of these peptides can assume an alpha-helical conformation, others contain one or two disulfide bonds, still others are Pro- and Arg-rich, or Trp-rich. In addition to bacterial killing, some of these peptides exert additional functions related to host defense such as LPS-neutralization and promotion of wound healing.
Three antimicrobial peptides were isolated from the skin of Rana rugosa. The major component, designated rugosin A, consisted of 33 amino acid residues and had structural homology (45%) with brevinin-2 of Rana porosa brevipoda. This peptide strongly inhibited the growth of gram-positive bacteria (e.g. Staphylococcus aureus 209P). The second peptide (rugosin B), a minor component, also had 33 amino acid residues, but was less homologous (33%) with brevinin-2. This peptide exhibited a striking antimicrobial activity against both gram-negative (e.g., Escherichia coli NIHJ) and gram-positive bacterial species. The third one, named rugosin C, composed of 37 amino acid residues, exhibited an antimicrobial activity against gram-positive bacteria. All three peptides had an intramolecular disulfide bond at the C-terminus.