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The chemical compounds synthesised and secreted from the dermal glands of amphibian have diverse bioactivities that play key roles in the hosts' innate immune system and in causing diverse pharmacological effects in predators that may ingest the defensive skin secretions. As new biotechnological methods have developed, increasing numbers of novel peptides with novel activities have been discovered from this source of natural compounds. In this study, a number of defensive skin secretion peptide sequences were obtained from the European edible frog, P. kl. esculentus, using a 'shotgun' cloning technique developed previously within our laboratory. Some of these sequences have been previously reported but had either obtained from other species or were isolated using different methods. Two new skin peptides are described here for the first time. Esculentin-2c and Brevinin-2Tbe belong to the Esculentin-2 and Brevinin-2 families, respectively, and both are very similar to their respective analogues but with a few amino acid differences. Further, [Asn-3, Lys-6, Phe-13] 3-14-bombesin isolated previously from the skin of the marsh frog, Rana ridibunda, was identified here in the skin of P. kl. esculentus. Studies such as this can provide a rapid elucidation of peptide and corresponding DNA sequences from unstudied species of frogs and can rapidly provide a basis for related scientific studies such as those involved in systematic or the evolution of a large diverse gene family and usage by biomedical researchers as a source of potential novel drug leads or pharmacological agents.

Objective: To investigate the changes in the functional activity of glycogen synthase kinase-3 (GSK-3) in the hepatic tissue after endotoxin (lipopolysaccharide, LPS) tolerance and explore the effects of LPS-induced GSK-3 inhibition on glycogen metabolism in the liver., Methods: Male SD rats were randomly divided into normal control, endotoxin pretreatment and GSK-3 inhibitor (lithium chloride) groups with corresponding pretreatments prior to a large dose of LPS challenge (10 mg/kg) to induce liver injury. Glycogen deposition and content in the hepatic tissue was detected using periodic acid-Schiff (PAS) staining and a glycogen quantification kit, respectively. Western blotting was performed for semi-quantitative analysis of protein level and inhibitory phosphorylation of GSK-3, and a Coomassie brilliant blue G-250-based colorimetric assay was used to detect calpain activity in the liver., Results: Glycogen content in the liver decreased significantly after LPS challenge in all the 3 groups (P<0.05) but showed no significant difference among the groups (P>0.05). Both LPS and lithium chloride pretreatments caused a significant increase of liver glycogen content (P<0.05). LPS pretreatment induced inhibitory phosphorylation of GSK-3β (P<0.05) and partial cleavage of GSK-3α but did not affect the expression of GSK-3 protein (P>0.05). Large-dose LPS challenge significantly increased the activity of calpain in the liver tissue (P<0.05) to a comparable level in the 3 groups (P>0.05)., Conclusion: Endotoxin pretreatment induces inhibitory phosphorylation of GSK-3β and partial cleavage of GSK-3α and promotes the deposition of liver glycogen but does not affect the activity of calpain, which may contribute to an increased glycogen reserve for energy supply in the event of large-dose LPS challenge.

The skin secretions of Neotropical phyllomedusine leaf frogs have proven to be a rich source of biologically active peptides, including antimicrobials. The major families of antimicrobial peptides (AMPs) reported are the dermaseptins and phylloseptins and the minor families are the dermatoxins, phylloxins, plasticins, distinctins, and medusins. Here, we report a novel AMP of 10 amino acid residues (LRPAILVRIKamide), named balteatide, from the skin secretion of wild Peruvian purple-sided leaf frogs, Phyllomedusa baltea. Balteatide was found to exhibit a 90% sequence identity with sauvatide, a potent myotropic peptide from the skin secretion of Phyllomedusa sauvagei. However, despite both peptides exhibiting only a single amino acid difference (I/T at position 9), sauvatide is devoid of antimicrobial activity and balteatide is devoid of myotropic activity. Balteatide was found to have differential activity against the Gram-positive bacterium, Staphylococcus aureus; the Gram-negative bacterium, Escherichia coli; and the yeast, Candida albicans, and unusual for phyllomedusine frog skin AMPs, was most potent (MIC 32 mg/L) against the yeast. Balteatide was also devoid of haemolytic activity up to concentrations of 512 mg/L. Phyllomedusine frog skin secretions thus continue to provide novel AMPs, some of which may provide templates for the rational design of new classes of anti-infective therapeutics.

Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity. Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A highly selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantitating dextromethorphan (DXM) and its metabolite dextrophan (DXO) in rat plasma using pirfenidone as an internal standard. Protein precipitation with acetonitrile was employed for the sample preparation. Chromatographic separation was achieved on a SB-C18 column at 25 degrees C, with a gradient elution programme of which acetonitrile-0.1% formic acid in water as mobile phase. The flow rate was 0.4 mL/min. Detection is carried out by multiple reaction monitoring (MRM) on a ion-trap LC-MS/MS system with an electrospray ionization interface. The assay is linear over the range 1-500 ng/mL for DXM and 1-250 ng/mL for DXO, with a lower limit of quantitation of 1 ng/mL for both. Intra- and inter-day precision of the assay were less than 9.80% and the accuracy were in the range 96.35-106.39%. The developed method was successfully applied to analyze the drug in samples of rat plasma for pharmacokinetic study.

Evidence supporting the functionality of Smoothened (SMO), an essential transducer in most pathways engaged by Hedgehog (Hh), as a G(i)-coupled receptor contrasts with the lack of an apparently consistent requirement for G(i) in Hh signal transduction. In the present study, we sought to evaluate the role of SMO-G(i) coupling in fibroblast migration induced by Sonic Hedgehog (Shh). Our results demonstrate an absolute requirement for G(i) in Shh-induced fibroblast migration. We found that Shh acutely stimulates the small Rho GTPases Rac1 and RhoA via SMO through a G(i) protein- and PI3K-dependent mechanism, and that these are required for cell migration. These responses were independent of transcription by Gli and of the C-terminal domain of SMO, as we show using a combination of molecular and genetic tools. Our findings provide a mechanistic model for fibroblast migration in response to Shh and underscore the role of SMO-G(i) coupling in non-canonical Hh signaling.

Quantitative detection of hydrogen peroxide in solution above a Streptococcus gordonii (Sg) bacterial biofilm was studied in real time by scanning electrochemical microscopy (SECM). The concentration of hydrogen peroxide was determined to be 0.7 mM to 1.6 mM in the presence of 10 mM glucose over a period of 2 to 8 h. The hydrogen peroxide production measured was higher near the biofilm surface in comparison to Sg grown planktonically. Differential hydrogen peroxide production was observed both by fluorometric as well as by SECM measurements. The interaction between two different species in a bacterial biofilm of Sg and Aggregatibacter actinomycetemcomitans (Aa) in terms of hydrogen peroxide production was also studied by SECM. One-directional y-scan SECM measurements showed the unique spatial mapping of hydrogen peroxide concentration across a mixed species biofilm and revealed that hydrogen peroxide concentration varies greatly dependent upon local species composition.

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 microm circle plasmid (2 microm). Here we show that accumulation of 2 microm in the SUMO pathway mutants siz1Delta siz2Delta, slx5Delta, and slx8Delta is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 microm. Characterization of this species from siz1Delta siz2Delta showed that it contains tandem copies of the 2 mum sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 microm-encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 microm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir(o) strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 microm amplification.

Attachment of the ubiquitin-like protein SUMO to other proteins is an essential process in Saccharomyces cerevisiae. However, yeast mutants lacking the SUMO ligases Siz1 and Siz2/Nfi1 are viable, even though they show dramatically reduced levels of SUMO conjugation. This siz1Delta siz2Delta double mutant is cold sensitive and has an unusual phenotype in that it forms irregularly shaped colonies that contain sectors of wild-type-appearing cells as well as sectors of enlarged cells that are arrested in G(2)/M. We have found that these phenotypes result from misregulation of the copy number of the endogenous yeast plasmid, the 2 microm circle. siz1Delta siz2Delta mutants have up to 40-fold-higher levels of 2 microm than do wild-type strains. Furthermore, 2 microm is responsible for the siz1Delta siz2Delta mutant's obvious growth defects, as siz1Delta siz2Delta [cir(0)] strains, which lack 2 microm, are no longer heterogeneous and show growth characteristics similar to those of the wild type. Possible mechanisms for SUMO's effect on 2 microm are suggested by the finding that both Flp1 recombinase and Rep2, two of the four proteins encoded by 2 microm, are covalently modified by SUMO. Our data suggest that SUMO attachment negatively regulates Flp1 levels, which may partially account for the increased 2 microm copy number in the siz1Delta siz2Delta strain.