Elements of the granular gland peptidome and transcriptome persist in air-dried skin of the South American orange-legged leaf frog, Phyllomedusa hypocondrialis.

The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin—a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and North American Rana frogs share the same families of skin antimicrobial peptides.

The Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis) and the North American pickerel frog (Rana palustris) occupy different ecological niches on two different continents with no overlap in geographical distribution. R. palustris skin secretions contain a formidable array of antimicrobial peptides including homologs of brevinin-1, esculentin-1, esculentin-2, ranatuerin-2, a temporin and a family of peptides considered of unique structural attributes when isolated, palustrins 1–3. Here we describe the structures of mature peptides and precursors of eight putative antimicrobial peptides from the skin secretion of the Chinese bamboo leaf odorous frog (Rana (Odorrana) versabilis). Each peptide represents a structural homolog of respective peptide families isolated from R. palustris, including two peptides identical in primary structure to palustrin 1c and palustrin 3b. Additionally, two peptides were found to be structural homologs of ranatuerin 2B and ranatuerin 2P from the closely-related North American species, Rana berlandieri (the Rio Grande leopard frog) and Rana pipiens (the Northern leopard frog), respectively. Both palustrins and ranatuerins have hitherto been considered unique to North American ranid frogs. The use of primary structures of amphibian skin antimicrobial peptides is thus questionable as a taxonomic device or alternatively, the micro-evolution and/or ancestry of ranid frogs is more highly complex than previously thought.

Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine 0-sulfation motif.

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position -1 and the basic amino acid residue (Arg) at position -2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.

Bradykinin-related peptides, including a novel structural variant, (Val1)-bradykinin, from the skin secretion of Guenther's frog, Hylarana guentheri and their molecular precursors.

Multiple bradykinin-related peptides including a novel bradykinin structural variant, (Val1)-bradykinin, have been identified from the defensive skin secretion of Guenther's frog, Hylarana guentheri by a tandem mass spectrometry method. Subsequently, four different preprobradykinin cDNAs, which encoded multiple bradykinin copies and its structural variants, were consistently cloned from a skin derived cDNA library. These preprobradykinin cDNAs showed little structural similarity with mammalian kininogens and the kininogens from the skin of toads, but have regions that are highly conserved in the kininogens from another ranid frog, Odorrana schmackeri. Alignment of these preprobradykinins revealed that preprobradykinin 1, 2 and 3 may derive from a single gene by alternative exon splicing.

The effect of tungsten trioxide thin films at ferroelectric-electrode boundaries on fatigue behaviour

A conventional thin film capacitor heterostructure, consisting of sol-gel deposited lead zirconium titanate (PZT) layers with sputtered platinum top and bottom electrodes, was subjected to fatiguing pulses at a variety of frequencies. The fatigue characteristics were compared to those of a similarly processed capacitor in which a ~20nm tungsten trioxide layer had been deposited, using pulsed laser deposition, between the ferroelectric and upper electrode. The expectation was that, because of its ability to accommodate considerable oxygen non-stoichiometry, tungsten trioxide (WO3) might act as an efficient sink for any oxygen vacancies flushed to the electrode-ferroelectric boundary layer during repetitive switching, and hence would improve the fatigue characteristics of the thin film capacitor. However, it was found that, in general, the addition of tungsten trioxide actually increases the rate of fatigue. It appears that any potential benefit from the WO3, in terms of absorbing oxygen vacancies, is far outweighed by it causing dramatically increased charge injection in the system.

The structural organization of aurein precursor cDNAs from the skin secretion of the Australian green and golden bell frog, Litoria aurea.

Aureins are a family of peptides (13-25 residues), some of which possess potent antimicrobial and anti-cancer properties, which have been classified into 5 subgroups based upon primary structural similarities. They were originally isolated from the defensive skin secretions of the closely related Australian bell frogs, Litoria aurea and Litoria raniformis, and of the 23 aurein peptides identified, 10 are common to both species. Using a recently developed technique, we have constructed a cDNA library from the defensive secretion of the green and golden bell frog, L. aurea, and successfully cloned a range of aurein precursor transcripts containing entire open-reading frames. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of aurein. The deduced precursor structures for the most active aureins (2.2 and 3.1) confirmed the presence of a C-terminal amidation motif whereas that of aurein 5.3 did not. Processed peptides corresponding in molecular mass to aureins 2.2, 2.3, 2.5, 3.1 and 5.3 were identified in the same secretion sample using LC/MS. The application of this technique thus permits parallel peptidomic and transcriptomic analyses on the same lyophilized skin secretion sample circumventing sacrifice of specimens of endangered herpetofauna.

Novel brevinins from Chinese piebald odorous frog (Huia schmackeri) skin deduced from cloned biosynthetic precursors

Antimicrobial peptides represent the most characterized and diverse class of peptides within the defensive skin secretions of anuran amphibians. With an ever expanding database of primary structures, the current accepted rules for nomenclature have become increasingly difficult to apply to peptides whose primary structural attributes are either unique or that fall between those that define existing groups. An additional factor that adds to the confusion is the regular re-classification or revision of existing taxa. In the present study, we have identified five new antimicrobial peptide homologs in the defensive skin secretion of the Chinese piebald odorous frog, Huia schmackeri (formerly Rana (Odorrana) schmackeri), by cloning of their respective biosynthetic precursors. As these peptides are obvious homologs of the brevinin-1 and brevinin-2 families we have named these in accordance: (1) brevinin-1HS1, (2) brevinin-2HS1, (3) brevinin-2HS2, (4) brevinin-2HS3 and (5) brevinin-1HS2. The reasons for adopting these names are discussed. It is clear that with an ever-increasing number of amphibian skin antimicrobial peptides appearing in the literature that a consistent nomenclature scheme needs to be established.

The rotational molding characteristics of metallocene polyethylene skin/foam structures

This article reports on the results of ongoing work in which the foaming characteristics of metallocene-catalyzed linear low density polyethylenes for rotational molding are investigated. Earlier publications related rheological and thermal parameters to the polymer structure and mechanical properties and found that metallocene polyethylene can be used in rotational foam molding to produce a foam that will perform as well as a Ziegler-Natta catalyzed foam. Through adjustments to molding conditions, the significant processing and physical material parameters, which optimize metallocene catalyzed linear low-density polyethylene foam structure, have been identified. This article details the optimum processing route for the production of two layer skin/foam parts using the drop box method. © SAGE Publications 2007.

Skin-to-Skin contact

Skin to skin contact facilitates the initiation of breast feeding, neonatal thermoregulation and the promotion of maternal-infant attachment. However, there are cultural influences in society and in the labour ward that may make implemention of skin to skin contact difficult.

Electrode kinetics and mechanism of iodine reduction in the room-temperature ionic liquid [C(4)mim][NTf2]

The fast electrochemical reduction of iodine in the RTIL 1-butyl-3-methylimidazolium bis(trifluoromethyl-sulfonyl)imide, [C(4)mim][NTf2], is reported and the kinetics and mechanism of the process elucidated. Two reduction peaks were observed. The first reduction peak is assigned to the process

Extraction of electrode kinetic parameters from microdisc voltammetric data measured under transport conditions intermediate between steady-state convergent and transient linear diffusion as typically applies to room temperature ionic liquids

The extraction of electrode kinetic parameters for electrochemical couples in room-temperature ionic liquids (RTILs) is currently an area of considerable interest. Electrochemists typically measure electrode kinetics in the limits of either transient planar or steady-state convergent diffusion for which the voltammetic response is well understood. In this paper we develop a general method allowing the extraction of this kinetic data in the region where the diffusion is intermediate between the planar and convergent limits, such as is often encountered in RTILs using microelectrode voltammetry. A general working surface is derived, allowing the inference of Butler-Volmer standard electrochemical rate constants for the peak-to-peak potential separation in a cyclic voltammogram as a function of voltage scan rate. The method is applied to the case of the ferrocene/ferrocenium couple in [C(2)mim][N(Tf)(2)] and [C(4)mim][N(Tf)(2)].