Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Techniques for analysis of proteins by SDS-polyacrylamide gel electrophoresis and Western blotting

The separation of mixtures of proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a technique that is widely used—and, indeed, this technique underlies many of the assays and analyses that are described in this book. While SDS-PAGE is routine in many labs, a number of issues require consideration before embarking on it for the first time. We felt, therefore, that in the interest of completeness of this volume, a brief chapter describing the basics of SDS-PAGE would be helpful. Also included in this chapter are protocols for the staining of SDS-PAGE gels to visualize separated proteins, and for the electrotransfer of proteins to a membrane support (Western blotting) to enable immunoblotting, for example. This chapter is intended to complement the chapters in this book that require these techniques to be performed. Therefore, detailed examples of why and when these techniques could be used will not be discussed here.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes, Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Difference gel electrophoresis

DIGE is a protein labelling and separation technique allowing quantitative proteomics of two or more samples by optical fluorescence detection of differentially labelled proteins that are electrophoretically separated on the same gel. DIGE is an alternative to quantitation by MS-based methodologies and can circumvent their analytical limitations in areas such as intact protein analysis, (linear) detection over a wide range of protein abundances and, theoretically, applications where extreme sensitivity is needed. Thus, in quantitative proteomics DIGE is usually complementary to MS-based quantitation and has some distinct advantages. This review describes the basics of DIGE and its unique properties and compares it to MS-based methods in quantitative protein expression analysis.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye (TM) and analysed by two-dimensional difference gel. electrophoresis. Gel images analysed off-line, using the DeCyder (TM) image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying. A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased. A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process. The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.

Effects of drying methods and conditions on antimicrobial activity of edible chitosan films enriched with galangal extract

The aim of this work was to study the effects of drying methods and conditions (i.e., ambient drying, hot air drying at 40 degrees C, vacuum drying and low-pressure superheated steam drying within the temperature range of 70-90 degrees C at an absolute pressure of 10 kPa) as well as the concentration of galangal extract on the antimicrobial activity of edible chitosan films against Staphylococcus aureus. Galangal extract was added to the film forming solution as a natural antimicrobial agent in the concentration range of 0.3-0.9 g/100 g. Fourier transform infrared (FTIR) spectra and swelling of the films were also evaluated to investigate interaction between chitosan and the galangal extract. The antimicrobial activity of the films was evaluated by the disc diffusion and viable cell count method, while the morphology of bacteria treated with the antimicrobial films was observed via transmission electron microscopy (TEM). The antimicrobial activity, swelling and functional group interaction of the antimicrobial films were found to be affected by the drying methods and conditions as well as the concentration of the galangal extract. The electron microscopic observations revealed that cell wall and cell membrane of S. aureus treated by the antimicrobial films were significantly damaged. (C) 2009 Elsevier Ltd. All rights reserved.

Effects of restricted water availability and increased temperature on the grain filling, drying and quality of winter wheat

Experiments in controlled environments examined the effects of the timing and severity of drought, and increased temperature, on grain development of Hereward winter wheat. Environmental effects on grain specific weight, protein content, Hagberg Falling Number, SDS-sedimentation volume, and sulphur content were also studied. Drought and increased temperature applied before the end of grain filling shortened the grain filling period and reduced grain yield, mean grain weight and specific weight. Grain filling was most severely affected by drought between days 1-14 after anthesis. Protein content was increased by stresses before the end of grain growth, because nitrogen harvest index was less severely affected than was dry matter harvest index. Hagberg Falling Number was increased to the greatest extent by stresses applied 15-28 days after anthesis. Treatment effects on grain sulphur content were similar to those on protein content, such that N:S ratio was not significantly affected by drought nor temperature stresses. The effects of restricted water on grain yield and quality were linearly related to soil moisture between 44 and about 73% field capacity (FC) from days 15-28. Drought stress (but not temperature stress) before the end of grain filling decreased SDS-sedimentation volume relative to drought applied later. (C) 2003 Elsevier Science Ltd. All rights reserved.

A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g gluconodelta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.

Insights into redox cycling of sulfur and iron in peatlands using high-resolution diffusive equilibrium thin film (DET) gel probe sampling

High spatial resolution vertical profiles of pore-water chemistry have been obtained for a peatland using diffusive equilibrium in thin films (DET) gel probes. Comparison of DET pore-water data with more traditional depth-specific sampling shows good agreement and the DET profiling method is less invasive and less likely to induce mixing of pore-waters. Chloride mass balances as water tables fell in the early summer indicate that evaporative concentration dominates and there is negligible lateral flow in the peat. Lack of lateral flow allows element budgets for the same site at different times to be compared. The high spatial resolution of sampling also enables gradients to be observed that permit calculations of vertical fluxes. Sulfate concentrations fall at two sites with net rates of 1.5 and 5.0nmol cm− 3 day− 1, likely due to a dominance of bacterial sulfate reduction, while a third site showed a net gain in sulfate due to oxidation of sulfur over the study period at an average rate of 3.4nmol cm− 3 day− 1. Behaviour of iron is closely coupled to that of sulfur; there is net removal of iron at the two sites where sulfate reduction dominates and addition of iron where oxidation dominates. The profiles demonstrate that, in addition to strong vertical redox related chemical changes, there is significant spatial heterogeneity. Whilst overall there is evidence for net reduction of sulfate within the peatland pore-waters, this can be reversed, at least temporarily, during periods of drought when sulfide oxidation with resulting acid production predominates.

Self-assembly and hydrogelation of an amyloid peptide fragment

The self-assembly of a fragment of the amyloid beta peptide that has been shown to be critical in amyloid fibrillization has been studied in aqueous solution. There are conflicting reports in the literature on the fibrillization of A beta (16-20), i.e., KLVFF, and our results shed light on this. In dilute solution, self-assembly of NH2-KLVFF-COOH is strongly influenced by aromatic interactions between phenylalanine units, as revealed by UV spectroscopy and circular dichroism. Fourier transform infrared (FTIR) spectroscopy reveals beta-sheet features in spectra taken for more concentrated solutions and also dried films. X-ray diffraction and cryo-transmission electron microscopy (cryo-TEM) provide further support for beta-sheet amyloid fibril formation. A comparison of cryo-TEM images with those from conventional dried and negatively stained TEM specimens highlights the pronounced effects of sample preparation on the morphology. A comparison of FTIR data for samples in solution and dried samples also highlights the strong effect of drying on the self-assembled structure. In more concentrated phosphate-buffered saline (PBS) solution, gelation of NH2-KLVFF-COOH is observed. This is believed to be caused by screening of the electrostatic charge on the peptide, which enables beta sheets to aggregate into a fibrillar gel network. The rheology of the hydrogel is probed, and the structure is investigated by light scattering and small-angle X-ray scattering.

Oxidatively drying coating composition

coating composition comprising an oxidatively drying coating binder and a chelate comprising at least one group according to the following formula (I): forming a complex with a metal ion, A1 and A2 both being an aromatic residue, R1 and R3 being covalently bonded groups, and R2 being a divalent organic radical, wherein at least one solubilizing group is coivalently bonded to the chelating compound. The solubilizing group is a non-polar group, preferable an aliphatic group having at least four carbon atoms, covalently bonded to A1 and/or A2. The metal ion is a divalent ion of a metal selected from the group of manganese, cobalt, copper, lead, zirconium, iron, lanthanium, cerium, vanadium, and clacium or a trivalent ion of a metal selected from the group of manganese, cobalt, lead, zirconium, iron, lanthanium, cerium, and vanadium, combined with a monovalent counterion.

Longitudinal investigation of the faecal microbiota of healthy full-term infants using fluorescence in situ hybridization and denaturing gradient gel electrophoresis.

From birth onwards, the gastrointestinal (GI) tract of infants progressively acquires a complex range of micro-organisms. It is thought that by 2 years of age the GI microbial population has stabilized. Within the developmental period of the infant GI microbiota, weaning is considered to be most critical, as the infant switches from a milk-based diet (breast and/or formula) to a variety of food components. Longitudinal analysis of the biological succession of the infant GI/faecal microbiota is lacking. In this study, faecal samples were obtained regularly from 14 infants from 1 month to 18 months of age. Seven of the infants (including a set of twins) were exclusively breast-fed and seven were exclusively formula-fed prior to weaning, with 175 and 154 faecal samples, respectively, obtained from each group. Diversity and dynamics of the infant faecal microbiota were analysed by using fluorescence in situ hybridization and denaturing gradient gel electrophoresis. Overall, the data demonstrated large inter- and intra-individual differences in the faecal microbiological profiles during the study period. However, the infant faecal microbiota merged with time towards a climax community within and between feeding groups. Data from the twins showed the highest degree of similarity both quantitatively and qualitatively. Inter-individual variation was evident within the infant faecal microbiota and its development, even within exclusively formula-fed infants receiving the same diet. These data can be of help to future clinical trials (e.g. targeted weaning products) to organize protocols and obtain a more accurate outline of the changes and dynamics of the infant GI microbiota.

Multiple sol-gel transitions of PEG-PCL-PEG triblock copolymer aqueous solution

An aqueous solution of a poly(ethylene glycol)-polycaprolactone-poly(ethylene glycol) (PEG-PCL-PEG) with a composition of EG13CL23EG13 undergoes multiple transitions, from sol-to-gel (hard gel)-to-sol-to-gel (soft gel)-to-sol, in the concentration range 20.0∼35.0 wt.-%. Through dynamic mechanical analysis, UV-vis spectrophotometry, small angle X-ray scattering, differential scanning calorimetry, microcalorimetry and 13C NMR spectroscopy, the mechanism of these transitions was investigated. The hard gel and soft gel are distinguished by the crystalline and amorphous state of the PCL. The extent of PEG dehydration and the molecular motion of each block also played a critical role in the multiple transitions. This paper suggests a new mechanism for these multiple transitions driven by temperature changes.