Identification and functional characterization of three chicken cathelicidins with potent antimicrobial activity

Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain. Here we report that the entire chicken genome encodes three cathelicidins, namely fowlicidin-1 to -3, which are densely clustered within a 7.5-kb distance at the proximal end of chromosome 2p. Each fowlicidin gene adopts a fourexon, three-intron structure, typical for a mammalian cathelicidin. Phylogenetic analysis revealed that fowlicidins and a group of distantly related mammalian cathelicidins known as neutrophilic granule proteins are likely to originate from a common ancestral gene prior to the separation of birds from mammals, whereas other classic mammalian cathelicidins may have been duplicated from the primordial gene for neutrophilic granule proteins after mammals and birds are diverged. Similar to ovine cathelicidin SMAP-29, putatively mature fowlicidins displayed potent and salt-independent activities against a range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range of 0.4-2.0 microm for most strains. Fowlicidin-1 and -2 also showed cytotoxicity, with 50% killing of mammalian erythrocytes or epithelial cells in the range of 6-40 microm. In addition, two fowlicidins demonstrated a strong positive cooperativity in binding lipopolysaccharide (LPS), resulting in nearly complete blockage of LPS-mediated proinflammatory gene expression in RAW264.7 cells. Taken together, fowlicidin-1 and -2 are clearly among the most potent cathelicidins that have been reported. Their broad spectrum and salt-insensitive antibacterial activities, coupled with their potent LPS-neutralizing activity, make fowlicidins excellent candidates for novel antimicrobial and anti-sepsis agents.     

Molecular phylogeny of elapid snakes and a consideration of their biogeographic history

Evolutionary relationships among the major elapid clades, particularly the taxonomic position of the partially aquatic sea kraits (Latkauda) and the fully aquatic true sea snakes have been the subject of much debate. To discriminate among existing phylogenetic and biogeographic hypotheses, portions of both the 16S rRNA and cytochrome b mitochondrial DNA genes were sequenced from 16 genera and 17 species representing all major elapid snake clades from throughout the world and two non-elapid outgroups. This sequence data yielded 181 informative sites under parsimony. Parsimony analyses of the separate data sets produced trees of broad agreement although less well supported than the single most parsimonious tree resulting from the combined analyses. These results support the following hypotheses: (1) the Afro-Asian cobra radiation forms one or more sister groups to other elapids, (2) American and Asian coral snakes form a clade, corroborating morphological studies, (3) Bungarus forms a sister group to the hydrophiines comprised of Latkauda, terrestrial Australo-Papuan elapids and true sea snakes, (4) Latkauda and true sea snakes do not form a monophyletic group but instead each group shares an independent history with terrestrial Australo-Papuan elapids, corroborating previous studies, (5) a lineage of Melanesian elapids forms the sister group to Latkauda, terrestrial Australian species and true sea snakes. In agreement with previous morphologically based studies, the sequence data suggests that Bungarus and Latkauda represent transitional clades between the elapine 'palatine erectors' and hydrophiine 'palatine draggers'. Both intra and inter-clade genetic distances are considerable, implying that each of the major radiations have had long independent histories. I suggest an African, Asian, or Afro-Asian origin for elapids as a group, with independent Asian origins for American coral snakes and the hydrophiines.     

Phylogenetic relationships of elapid snakes based on cytochrome b mtDNA sequences

Published molecular phylogenetic studies of elapid snakes agree that the marine and Australo-Melanesian forms are collectively monophyletic. Recent studies, however, disagree on the relationships of the African, American, and Asian forms. To resolve the relationships of the African, American, and Asian species to each other and to the marine/Australo-Melanesian clade, we sequenced the entire cytochrome b gene for 28 elapids; 2 additional elapid sequences from GenBank were also included. This sample includes all African, American, and Asian genera (except for the rare African Pseudohaje), as well as a representative sample of marine/Australo-Melanesian genera. The data were analyzed by the methods of maximum-parsimony and maximum-likelihood. Both types of analyses yielded similar trees, from which the following conclusions can be drawn: (1) Homoroselaps falls outside a clade formed by the remaining elapids; (2) the remaining elapids are divisible into two broad sister clades, the marine/Australo-Melanesian species vs the African, American, and Asian species; (3) American coral snakes cluster with Asian coral snakes; and (4) the "true" cobra genus Naja is probably not monophyletic as the result of excluding such genera as Boulengerina and Paranaja.     

Proteins toxic to arthropods in the venom of elapid snakes.

It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals.This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica (= N. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation.It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms.     

Ecological and historical legacies on global diversity gradients in marine elapid snakes

Global diversity gradients have been extensively investigated for several biological groups. However, little is known whether the diversity drivers of clades that underwent major environmental transition (e.g., from land to sea) are equivalent across these different environmental settings. Here, we ask if the pattern of diversity of marine elapid snakes is determined by factors analogous to those previously found for terrestrial lineages. Through a model selection framework, we compare the effect of factors that represent five ecological and historical hypotheses. We found that both ecological and historical factors play significant roles, but habitat structure, which can be linked to historical climatic changes, was better supported. This result agrees with that previously found for terrestrial elapids, despite the different environmental pressures in terrestrial and marine contexts. Our findings suggest an equivalence of the underlying process of diversity gradient within the same lineage from land and sea, irrespective of the different physiological, spatial and historical constraints.     

Cloning and characterisation of novel cystatins from elapid snake venom glands

Snake venoms contain a complex mixture of polypeptides that modulate prey homeostatic mechanisms through highly specific and targeted interactions. In this study we have identified and characterised cystatin-like cysteine-protease inhibitors from elapid snake venoms for the first time. Novel cystatin sequences were cloned from 12 of 13 elapid snake venom glands and the protein was detected, albeit at very low levels, in a total of 22 venoms. One highly conserved isoform, which displayed close sequence identity with family 2 cystatins, was detected in each elapid snake. Crude Austrelaps superbus (Australian lowland copperhead) snake venom inhibited papain, and a recombinant form of A. superbus cystatin inhibited cathepsin L ≅ papain > cathepsin B, with no inhibition observed for calpain or legumain. While snake venom cystatins have truncated N-termini, sequence alignment and structural modelling suggested that the evolutionarily conserved Gly-11 of family 2 cystatins, essential for cysteine protease inhibition, is conserved in snake venom cystatins as Gly-3. This was confirmed by mutagenesis at the Gly-3 site, which increased the dissociation constant for papain by 10⁴-fold. These data demonstrate that elapid snake venom cystatins are novel members of the type 2 family. The widespread, low level expression of type 2 cystatins in snake venom, as well as the presence of only one highly conserved isoform in each species, imply essential housekeeping or regulatory roles for these proteins.     

Identification and characterization of integrin-binding sialoprotein (IBSP) genes in reptile and amphibian

Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.     

Histology,histochemistry, and emptying mechanism of the venom glands of some elapid snakes

The venom glands of several species of elapid snakes are described. The main venom gland consists of many tubules which usually contain large amounts of secretion product. The accessory gland surrounds the entire venom duct and is usually composed of uniform mucous epithelium. The epithelium lining the tubules of the accessory gland of Naja naja is composed of two distinct types of cells. Histochemical tests indicate that the main venom gland reacts with mercury bromphenol blue and PAS but not with alcian blue. The accessory gland reacts with PAS and alcian blue, and not with mercury bromphenol blue. Treatment of sections with sialidase demonstrates the presence of a sialomucin in the accessory gland. Stimulation of the muscles associated with the venom gland offers an indication of the venom expulsion mechanism of Bungarus caeruleus. A comparison of the venom apparatus of elapid and viperid snakes emphasizes marked differences in the internal anatomy of the venom glands, muscles associated with the gland, and arrangement of glandular components. The morphological differences and dissimilar venom expulsion mechanisms support the recent view of the polyphyletic origin of venomous snakes.     

Generalization of predator recognition: Velvet geckos display anti-predator behaviours in response to chemicals from non-dangerous elapid snakes

Many prey species detect chemical cues from predators and modify their behaviours in ways that reduce their risk of predation. Theory predicts that prey should modify their anti-predator responses according to the degree of threat posed by the predator. That is, prey should show the strongest responses to chemicals of highly dangerous prey, but should ignore or respond weakly to chemicals from non-dangerous predators. However, if anti-predator behaviours are not costly, and predators are rarely encountered, prey may exhibit generalised antipredator behaviours to dangerous and non-dangerous predators. In Australia, most elapid snakes eat lizards, and are therefore potentially dangerous to lizard prey. Recently, we found that the nocturnal velvet gecko Oedura lesueurii responds to chemicals from dangerous and non-dangerous elapid snakes, suggesting that it displays gen-eralised anti-predator behaviours to chemicals from elapid snakes. To explore the generality of this result, we videotaped the be-haviour of velvet geckos in the presence of chemical cues from two small elapid snakes that rarely consume geckos: the nocturnal golden-crowned snake Cacophis squamulosus and the diurnal marsh snake Hemiaspis signata. We also videotaped geckos in tri-als involving unsceted cards (controls) and cologne-scented cards (pungency controls). In trials involving Cacophis and Hemi-aspis chemicals, 50% and 63% of geckos spent long time periods (> 3 min) freezing whilst pressed flat against the substrate, re-spectively. Over half the geckos tested exhibited anti-predator behaviours (tail waving, tail vibration, running) in response to Ca-cophis (67%) or Hemiaspis (63%) chemicals. These behaviours were not observed in control or pungency control trials. Our re-sults support the idea that the velvet gecko displays generalised anti-predator responses to chemical cues from elapid snakes. Generalised responses to predator chemicals may be common in prey species that co-occur with multiple, ecologically similar, dangerous predators.     

Generalization of predator recognition:Velvet geckos display anti-predator behaviours in response to chemicals from non-dangerous elapid snakes

<正> Many prey species detect chemical cues from predators and modify their behaviours in ways that reduce their risk ofpredation. Theory predicts that prey should modify their anti-predator responses according to the degree of threat posed by thepredator. That is, prey should show the strongest responses to chemicals of highly dangerous prey, but should ignore or respondweakly to chemicals from non-dangerous predators. However, if anti-predator behaviours are not costly, and predators are rarelyencountered, prey may exhibit generalised antipredator behaviours to dangerous and non-dangerous predators. In Australia, mostelapid snakes eat lizards, and are therefore potentially dangerous to lizard prey. Recently, we found that the nocturnal velvetgecko Oedura lesueurii responds to chemicals from dangerous and non-dangerous elapid snakes, suggesting that it displays generalisedanti-predator behaviours to chemicals from elapid snakes. To explore the generality of this result, we videotaped the behaviourof velvet geckos in the presence of chemical cues from two small elapid snakes that rarely consume geckos: the nocturnalgolden-crowned snake Cacophis squamulosus and the diurnal marsh snake Hemiaspis signata. We also videotaped geckos in trialsinvolving unscented cards (controls) and cologne-scented cards (pungency controls). In trials involving Cacophis and Hemiaspischemicals, 50% and 63% of geckos spent long time periods ( 3 min) freezing whilst pressed flat against the substrate, respectively.Over half the geckos tested exhibited anti-predator behaviours (tail waving, tail vibration, running) in response to Cacophis(67%) or Hemiaspis (63%) chemicals. These behaviours were not observed in control or pungency control trials. Our resultssupport the idea that the velvet gecko displays generalised anti-predator responses to chemical cues from elapid snakes.Generalised responses to predator chemicals may be common in prey species that co-occur with multiple, ecologically similar,dangerous predators [Current Zoology 56 (3): 337-342, 2010].     

Monophyly of elapid snakes (Serpentes: Elapidae). An assessment of the evidence

The defining morphological characters of the family Elapidae are analysed in an attempt to evaluate whether the front-fanged, proteroglyphous, snakes constitute a natural (monophyletic) group or whether proteroglyphy is more likely to be a condition achieved independently by a number of higher snake lineages. The evidence relating to presumed elapids whose affinities have been questioned, namely a South African genus Homoroselaps and New World proteroglyphs (Micrurus and Micruroides) , is examined. It concluded that Homoroselaps is a genuinely equivocal case, the evidence for its inclusion in the Elapidae is balanced by features which suggest that it is more closely related to the Aparallactinae. However, Micrurus and Micruroides seem clearly to be more closely related to undisputed elapids than to any other caenophidians. It is suggested that, at least for the present, the family Elapidae be retained in its broad sense to include all proteroglyphous snakes.     

Identification and characterization of a novel toxin-antitoxin module from Bacillus anthracis

Comparative genome analysis of Bacillus anthracis revealed a pair of linked genes encoding pemK (K, killer protein) and pemI (I, inhibitory protein) homologous to pem loci of other organisms. Expression of PemK in Escherichia coli and Bacillus anthracis was bacteriostatic whereas the concomitant expression of PemI reversed the growth arrest. PemK expression effectively inhibited protein synthesis with no significant effect on DNA replication. Coexpression and interaction of these proteins confirmed it to be a Type II addiction module. Thermal denaturation analysis reflected poor conformational stability of PemI as compared to PemK. Circular dichroism analysis indicated that PemI contains twice the amount of beta-sheets as PemK. Gel retardation assays demonstrated that PemI binds to its upstream DNA sequence. This study reports the first evidence of an active chromosome encoded toxin-antitoxin locus in B. anthracis.     

Identification and characterization of a novel arabinoxylanase from wheat flour.

An endogenous wheat (Triticum aestivum) flour endoxylanase was purified to homogeneity from a crude wheat flour extract by ammonium sulfate precipitation and cation-exchange chromatography. The 30-kD protein had an isoelectric point of 9.3 or higher. A sequence of 19 amino acids at the NH2 terminus showed 84.2% identity with an internal sequence of 15-kD grain-softness protein, friabilin. High-performance anion-exchange chromatography and gel-permeation analysis of the hydrolysis products indicated the preferential hydrolysis of highly branched structures by the enzyme; wheat arabinoxylan and rye (Secale cereale) arabinoxylan (high arabinose to xylose ratios) were hydrolyzed more efficiently by this enzyme than oat (Avena sativa) spelt xylan (low arabinose to xylose ratios). The release of the hydrolysis products as a function of time suggested that the endoxylanolytic activity was associated with the release of arabinose units from the polysaccharides, suggesting that the enzyme action is similar to that by endoxylanases from Ceratocystis paradoxa, Aspergillus niger, and Neurospora crassa. Although the enzyme released arabinose from arabinoxylan, it did not hydrolyze p-nitrophenyl-alpha-L-arabinofuranoside. From the above, it follows that the enzyme, called arabinoxylanase, differs from most microbial endoxylanases and from an endoxylanase purified earlier from wheat flour.     

Identification and characterization of novel esterases from a deep-sea sediment metagenome

A deep-sea sediment metagenomic library was constructed and screened for lipolytic enzymes by activity-based approach. Nine novel lipolytic enzymes were identified, and the amino acid sequences shared 56% to 84% identity to other lipolytic enzymes in the database. Phylogenetic analysis showed that these enzymes belonged to family IV lipolytic enzymes. One of the lipolytic enzymes, Est6, was successfully cloned and expressed in Escherichia coli Rosetta in a soluble form. The recombinant protein was purified by Ni-nitrilotriacetic affinity chromatography column and characterized using p-nitrophenyl esters with various chain lengths. The est6 gene consisted of 909 bp that encoded 302 amino acid residues. Est6 was most similar to a lipolytic enzyme from uncultured bacterium (ACL67845, 61% identity) isolated from the South China Sea marine sediment metagenome. The characterization of Est6 revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate (C4) at 20°C and pH 7.5.     

Dentition and diet in snakes: adaptations to oophagy in the Australian elapid genus Simoselaps

Most small fossorial proteroglyphous Australian snakes of the genus Simoselaps feed on adult lizards, but the species of one lineage (the semifasciatus group) feed exclusively on the eggs of squamate reptiles. Examination of cleared, alizarin preparations showed that dentition of the saurophagous species is similar to that of other elapids, but dentition of the oophagous taxa is highly modified. The anterior (palatine and maxillary) teeth other than the fangs are reduced in size and number whereas those of the pterygoid (and in S. 'ropert ', the dentary) are enlarged posteriorly, becoming compressed along a longitudinal plane and angled medially. The shape of the pterygoid and quadrate is also modified.
Two Simoselaps species with broader diets (eating both adult lizards and their eggs) show typical 'saurophagous' dentition in one case, 'oophagous' dentition in the other, showing that either type of dentition can be used to capture and ingest either type of prey. We suggest functional explanations for the dentitional modifications in the egg-eating snakes, primarily in terms of the advantages of applying considerable force to the eggshell. Oophagous modifications within Simoselaps are convergent with those seen in several independently-derived lineages of oophagous colubrid snakes, but (perhaps because of the presence of the fang) differ in having the enlarged blade-like teeth on the pterygoid or dentary rather than the maxilla.