Components of the peptidome and transcriptome persist in lin wa pi: the dried skin of the Heilongjiang brown frog Rana amurensis as used in Traditional Chinese Medicine

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

Dermatoxin and phylloxin from the waxy monkey frog, Phyllomedusa sauvagei: cloning of precursor cDNAs and structural characterization from lyophilized skin secretion

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.

Lividins: Novel antimicrobial peptide homologs from the skin secretion of the Chinese Large Odorous frog, Rana (Odorrana) livida: Identification by “shotgun” cDNA cloning and sequence analysis.

Odorous frogs of the sub-genus Odorrana are of oriental distribution, and are so called due to the foul smell of their defensive skin secretions released from specialized skin glands following stress or predator attack. Here we report the application of a “shotgun” skin secretion cDNA library cloning technique which can rapidly expedite identification of secretion bioactive peptides. From a library constructed from the skin secretion of the Large Chinese Odorous frog, Rana (Odorrana) livida, we have identified four novel peptides whose primary structures were deduced initially from cloned precursors. Subsequently, mature peptides were located in and structurally characterized from reverse phase HPLC fractions of skin secretion. Named lividins 1–4, these were found to be structural homologs of known antimicrobial peptide families from Rana frogs. Rapid identification of novel peptides can thus be rapidly achieved using this non-invasive, non-destructive technology and the extensive similarities revealed between antimicrobial peptide precursor organization and nucleic acid sequences would lend support to the hypothesis that they have a common ancestral origin.

Thermogravimetric evaluation of perennial ryegrass (Lolium perenne) for the prediction of in vitro dry matter digestibility

Thermogravimetry (TG) can be used for assessing the compositional differences in grasses that relate to dry matter digestibility (DMD) determined by pepsin-cellulase assay. This investigation developed regression models for predicting DMD of herbage grass during one growing season using TG results. The calibration samples were obtained from a field trial of eight cultivars and two breeding lines. The harvested materials from five cuts were analysed by TG to identify differences in the combustion patterns within the range of 30-600 degrees C. The discrete results including weight loss, peak height, area, temperature, widths and residue of three decomposition peaks were regressed against the measured DMD values of the calibration samples. Similarly, continuous weight loss results of the same samples were also utilised to generate DMD models. The r(2) for validation of the discrete and the best continuous models were 0.90 and 0.95, respectively, and the two calibrations were validated using independent samples from 24 plots from a trial carried out in 2004. The standard error for prediction of the 24 samples by the discrete model (4.14%) was higher than that by the continuous model (2.98%). This study has shown that DMD of grass could be predicted from the TG results. The benefit of thermal analysis is the ability to detect and show changes in composition of cell wall fractions of grasses during different cuts in a year.

Mineralogy of selected geological deposits from the United Kingdom and the Republic of Ireland as possible capping materials for low-level radioactive waste disposal facilities

Solid low-level radioactive waste (LLW) is currently being disposed at a number of facilities in the United Kingdom (UK). The safety of these facilities relies to some extent on the use of engineered barriers, such as a cap, to isolate the waste and protect the environment. Generally, the material used as the barrier layer within such a cap should be of low permeability and it should retain this property over long timescales (beyond a few decades normally required for facilities containing non-radioactive wastes). The objective of this research is to determine the mineralogy of selected geological deposits from the UK and Ireland as part of a larger project to examine their suitability as a capping material, particularly on LLW sites. Mineral transformations, as a result of future climate change, may impact on the long-term performance of the cap and even the disposal facility. X-ray diffraction (XRD) was carried-out on the sand, silt and clay fractions of the London Clay, Belfast Upper Boulder Clay, Irish Glacial Till, Belfast Sleech, and Ampthill Clay geological deposits. Minerals were present that could pose both positive and negative effects on the long-term performance of the cap. Smectite, which has a high shrink swell potential, may produce cracks in London Clay, Belfast Upper Boulder Clay and Ampthill Clay capping material during dry, hotter periods as a possible consequence of future climate change; thus, resulting in higher permeability. Ampthill Clay and Belfast Sleech had elevated amounts of organic matter (OM) at 5.93% and 5.88%, respectively, which may also contribute to cracking. Over time, this OM may decompose and result in increased permeability. Gypsum (CaSO4) in the silt and sand fractions of Ampthill Clay may reduce the impact of erosion during wetter periods if it is incorporated into the upper portion of the cap. There are potential negative effects from the acidity created by the weathering of pyrite (FeS2) present in the silt and sand fractions of Belfast Sleech and Ampthill Clay that could impede the growth of grasses used to stabilize the surface of the capping material if this material is used as part of the vegetative soil layer. Additionally, acidic waters generated from pyrite weathering could negatively impact the lower lying capping layers and the disposal facility in general. However, the calcium carbonate (CaCO3) present in the silt and sand fractions of these deposits, and dolomite (CaMg(CO3)2) in Belfast Sleech, may counter act the acidity.

Neuropeptide YY1 receptor in human dental pulp cells of noncarious and carious teeth

Aim To determine the distribution of the NPY Y1 receptor in carious and noncarious human dental pulp tissue using immunohistochemistry. A subsidiary aim was to confirm the presence of the NPY Y1 protein product in membrane fractions of dental pulp tissue from carious and noncarious teeth using western blotting. Methodology Twenty two dental pulp samples were collected from carious and noncarious extracted teeth. Ten samples were processed for immunohistochemistry using a specific antibody to the NPY Y1 receptor. Twelve samples were used to obtain membrane extracts which were electrophoresed, blotted onto nitrocellulose and probed with NPY Y1 receptor antibody. Kruskal-Wallis one-way analysis of variance was employed to test for overall statistical differences between NPY Y1 levels in noncarious, moderately carious and grossly carious teeth. Results Neuropeptide Y Y1 receptor immunoreactivity was detected on the walls of blood vessels in pulp tissue from noncarious teeth. In carious teeth NPY Y1 immunoreactvity was observed on nerve fibres, blood vessels and inflammatory cells. Western blotting indicated the presence and confirmed the variability of NPY Y1 receptor protein expression in solubilised membrane preparations of human dental pulp tissue from carious and noncarious teeth. Conclusions Neuropeptide Y Y1 is expressed in human dental pulp tissue with evidence of increased expression in carious compared with noncarious teeth, suggesting a role for NPY Y1 in modulation of caries induced pulpal inflammation. © 2008 International Endodontic Journal.

Dissociative Recombination of Protonated Formic Acid: Implications for Molecular Cloud and Cometary Chemistry

At the heavy ion storage ring CRYRING in Stockholm, Sweden, we have investigated the dissociative recombination of DCOOD2+ at low relative kinetic energies, from ~1 meV to 1 eV. The thermal rate coefficient has been found to follow the expression k(T) = 8.43 × 10-7 (T/300)^-0.78 cm3 s-1 for electron temperatures, T, ranging from ~10 to ~1000 K. The branching fractions of the reaction have been studied at ~2 meV relative kinetic energy. It has been found that ~87% of the reactions involve breaking a bond between heavy atoms. In only 13% of the reactions do the heavy atoms remain in the same product fragment. This puts limits on the gas-phase production of formic acid, observed in both molecular clouds and cometary comae. Using the experimental results in chemical models of the dark cloud, TMC-1, and using the latest release of the UMIST Database for Astrochemistry improves the agreement with observations for the abundance of formic acid. Our results also strengthen the assumption that formic acid is a component of cometary ices.

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2

Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5 Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys18 and Cys24 and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133–160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr6)-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.

A novel and potent trypsin inhibitor peptide, AC-17, from the skin secretion of the edible frog, Rana esculenta

Protease inhibitors are found in many venoms and evidence suggests that they occur widely in amphibian skin secretions. Kunitz inhibitors have been found in the skin secretions of bombinid toads and ranid frogs, Kazal inhibitors in phyllomedusine frogs and Bowman–Birk inhibitors in ranid frogs. Selective protease inhibitors could have important applications as therapeutics in the treatment of diseases in which discrete proteases play an aetiologcal role. Here we have examined the skin secretion of the edible frog, Rana esculenta, for protease inhibitors using trypsin as a model. HPLC fractions of secretions were screened for inhibitory activity using a chromogenic substrate as reporter. Three major peptides were resolved with trypsin inhibitory activity in HPLC fractions — one was a Kunitz-type inhibitor, a second was a Bowman–Birk inhibitor but the third represented a novel class of trypsin inhibitor in European frog skin. Analysis of the peptide established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17. Peptide AC-17 resembled a typical “Rana box” antimicrobial peptide but while it was active against Escherichia coli (MIC 30 µM) it was devoid of activity against Staphylococcus aureus and of haemolytic activity. In contrast, the peptide was a potent inhibitor of trypsin with a Ki of 5.56 µM. AC-17 represents the prototype of a novel trypsin inhibitor from the skin secretion of a European ranid frog that may target a trypsin-like protease present on the surface of Gram-negative bacteria.

Antimicrobial peptides from the skin secretion of the Wuyi Mountain torrent frog, Amolops wuyiensis: An inhabitant of a pristine aquatic environment

The antimicrobial peptides of amphibian skin secretions are proposed to aid survival in microbe-rich environments. While many amphibians inhabit such environments, other such as the Wuyi Mountain torrent frog, Amolops wuyiensis, live in pristine waters flowing from underground mountain springs. This species thus represents an interesting model in which to study antimicrobial peptides. “Shotgun” cloning of a skin-derived cDNA library from this species identified transcripts encoding a brevinin-1 and a ranatuerin-2. Peptides with coincident molecular masses to both predicted mature peptides were identified in HPLC fractions of skin secretion. Synthetic replicates of both peptides were generated by solid-phase peptide synthesis and tested for activity using Staphylococcus aureus, Escherichia coli and Candida albicans. The brevinin was found to be broad-spectrum and potent with minimum inhibitory concentrations (MICs) of 24 µM (Sa), 5 µM (Ec) and 20 µM (Ca). In contrast, the ranatuerin was less effective and of narrower spectrum with an MIC > 200 µM for Sa, 40 µM (Ec) and 120 µM (Ca). Thus this species of amphibian that lives in a pristine environment does indeed possess at least one potent and broad-spectrum antimicrobial peptide in its skin secretion arsenal. This phenomenon could be explained in several ways. Firstly, it may represent an ancestral peptide required when the stem species inhabited microbe-rich environments. However, there is mounting evidence for the second reason, that suggests the function of such peptides is not primarily in antimicrobial defence.

Genomic instability after targeted irradiation of human lymphocytes: Evidence for inter-individual differences under bystander conditions

Environmental (222)radon exposure is a human health concern, and many studies demonstrate that very low doses of high LET alpha-particle irradiation initiate deleterious genetic consequences in both radiated and non-irradiated bystander cells. One consequence, radiation-induced genomic instability (RIGI), is a hallmark of tumorigenesis and is often assessed by measuring delayed chromosomal aberrations We utilised a technique that facilitates transient immobilization of primary lymphocytes for targeted microbeam irradiation and have reported that environmentally relevant doses, e.g. a single He-3(2+) particle traversal to a single cell, are sufficient to Induce RIGI Herein we sought to determine differences in radiation response in lymphocytes isolated from five healthy male donors Primary lymphocytes were irradiated with a single particle per cell nucleus. We found evidence for inter-individual variation in radiation response (Rid, measured as delayed chromosome aberrations) Although this was not highly significant, it was possibly masked by high levels of intra-individual variation While there are many studies showing a link between genetic predisposition and RIGI, there are few studies linking genetic background with bystander effects in normal human lymphocytes In an attempt to investigate inter-individual variation in the induction of bystander effects, primary lymphocytes were irradiated with a single particle under conditions where fractions of the population were traversed We showed a marked genotype-dependent bystander response in one donor after exposure to 15% of the population The findings may also be regarded as a radiation-induced genotype-dependent bystander effect triggering an instability phenotype (C) 2010 Elsevier B.V. All rights reserved.

Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical -KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

Design, Manufacture and Performance of Germanium Bipolar Transistors

Germanium NPN bipolar transistors have been manufactured using phosphorus and boron ion implantation processes. Implantation and subsequent activation processes have been investigated for both dopants. Full activation of phosphorus implants has been achieved with RTA schedules at 535?C without significant junction diffusion. However, boron implant activation was limited and diffusion from a polysilicon source was not practical for base contact formation. Transistors with good output characteristics were achieved with an Early voltage of 55V and common emitter current gain of 30. Both Silvaco process and device simulation tools have been successfully adapted to model the Ge BJT(bipolar junction transistor) performance.

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard (Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)(5)-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides. (C) 2011 Published by Elsevier Inc.

Phosphonoacetate biosynthesis: In vitro detection of a novel NADP(+)-dependent phosphonoacetaldehyde-oxidizing activity in cell-extracts of a marine Roseobacter

A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.