Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the “shotgun” cloning of novel brevinins by means of 3'-RACE, using a “universal” degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.

Isolation and cloning of exendin precursor cDNAs from single samples of venom from the Mexican beaded lizard (Heloderma horridum) and the Gila Monster (Heloderma suspectum)

Reptile venoms are complex cocktails of bioactive molecules, including peptides. While the drug discovery potential of most species remains unrealized, many are endangered and afforded protection under international treaties. In this study, we describe how potential clinically important bioactive peptides and their corresponding mRNAs can be structurally characterized from single, small samples of reptile venom. The potential type-2 diabetes therapeutics, exendin-3 and exendin-4, from the Mexican beaded lizard (Heloderma horridum) and the Gila monster (Heloderma suspectum), respectively, have been characterized at both protein and nucleic acid levels to illustrate the efficacy of the technique and its contribution to biodiversity conservation.

The use of Raman microscopy to determine and localize vitamin E in biological samples

Alpha-tocopherol (aT), the predominant form of vitamin E in mammals, is thought to prevent oxidation of polyunsaturated fatty acids. In the lung, aT is perceived to be accumulated in alveolar type II cells and secreted together with surfactant into the epithelial lining fluid. Conventionally, determination of aT and related compounds requires extraction with organic solvents. This study describes a new method to determine and image the distribution of aT and related compounds within cells and tissue sections using the light-scattering technique of Raman microscopy to enable high spatial as well as spectral resolution. This study compared the nondestructive analysis by Raman microscopy of vitamin E, in particular aT, in biological samples with data obtained using conventional HPLC analysis. Raman spectra were acquired at spatial resolutions of 2-0.8 microm. Multivariate analysis techniques were used for analyses and construction of corresponding maps showing the distribution of aT, alpha-tocopherol quinone (aTQ), and other constituents (hemes, proteins, DNA, and surfactant lipids). A combination of images enabled identification of colocalized constituents (heme/aTQ and aT/surfactant lipids). Our data demonstrate the ability of Raman microscopy to discriminate between different tocopherols and oxidation products in biological specimens without sample destruction. By enabling the visualization of lipid-protein interactions, Raman microscopy offers a novel method of investigating biological characterization of lipid-soluble compounds, including those that may be embedded in biological membranes such as aT.

Treatment of hazardous shipyard wastewater using dolomitic sorbents

Hazardous shipyard wastewater is a worldwide problem, arising from ship repair. In this study an experimental programme was undertaken to establish the suitability of dolomite and dolomitic sorbent materials to remove contaminants from wastewater arising from a commercial shipyard. Experimental data indicate that dolomite and dolomitic sorbents have the ability to significantly reduce the COD concentration of the shipyard effluent (98% reduction). The data gained from trials at a shipyard indicated that the dolomite treatment process could be undertaken in a 8000 L pilot scale reaction vessel. Analysis of the wastewater using ICP-MS during the pilot trial indicated that the dolomite significantly reduced the concentrations of metallic impurities. The concentration of Sn ions, which is indicative of organo-tin complexes commonly found in shipyard wastewater, was reduced by 80% from its initial concentration in the pilot trial. The mechanism for the removal process using dolomite has been ascribed to a metal complexation/sorption process.

Performance of clay samples reinforced with vertical granular columns

This paper reports an experimental study in which samples of soft kaolin clay (100 mm in diameter and 200 mm in height) were reinforced with vertical columns of sand and tested under triaxial conditions. Samples were reinforced with either a single column of sand of 32 mm diameter or three columns of sand, each of 20 mm diameter. The replacement method was used to form the columns. The columns were installed in the clay to depths of 120 and 200 mm. Tests were also carried out on samples that were not reinforced with sand columns. The samples were compressed under both drained and undrained conditions. It was found that the undrained shear strength of samples containing full-depth columns was greatly improved compared with that of the unreinforced samples. In the fully drained tests, the sample installed with a single column of 32 mm diameter exhibited better performance than the sample with three columns of 20 mm diameter, although the area replacement ratio in the case of the three 20 mm diameter columns was higher than that of the single 32 mm diameter column. However, the undrained strength of the composite material was not particularly affected by the number of columns.