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.

Viscoelastic properties of high pressure and heat induced tofu gels

Tofu gels were rheologically examined to determine their storage or elastic (G′) and loss or viscous (G″) moduli as a function of frequency within their linear viscoelastic limits. The tofu gels were made using either glucono-δ-lactone (GDL) or calcium sulphate (CaSO4·2H2O), followed by either heat treatment (heated soymilk at 97 °C prior to coagulation and subsequently held at 70 °C for 60 min, HT) or high pressure treatment (400 MPa at 20 °C for 10 min, HP). The overall moduli values of the GDL gels and CaSO4·2H2O gels of both physical treatments were similar, each gave frequency profiles expected for weak viscoelastic materials. However, although both temperature and high pressure treatments could be used to produce tofu gels, the final products were not the same. Pressure formed gels, despite having a higher overall “consistency” (increasing values of their moduli), had a proportionately higher contribution from the loss modulus (increased tan δ). Differences could also be observed using confocal scanning laser microscopy. While such treatment may give rise to differing systems/structures, with new or modified organoleptic properties, the more “open” structures obtained by pressure treatment may well cause processing difficulties if subsequent reworking or moulding is required.

Viscoelastic properties of high pressure and heat induced tofu gels

Tofu gels were rheologically examined to determine their storage or elastic (G') and loss or viscous (G '') moduli as a function of frequency within their linear viscoelastic limits. The tofu gels were made using either glucono-delta-lactone (GDL) or calcium sulphate (CaSO4 center dot 2H(2)O), followed by either heat treatment (heated soymilk at >= 97 degrees C prior to coagulation and subsequently held at 70 degrees C for 60 min, HT) or high pressure treatment (400 MPa at 20 degrees C for 10 min, HP). The overall moduli values of the GDL gels and CaSO4 center dot 2H(2)O gels of both physical treatments were similar, each gave frequency profiles expected for weak viscoelastic materials. However, although both temperature and high pressure treatments could be used to produce tofu gels, the final products were not the same. Pressure formed gels, despite having a higher overall "consistency" (increasing values of their moduli), had a proportionately higher contribution from the loss modulus (increased tan delta). Differences could also be observed using confocal scanning laser microscopy. While such treatment may give rise to differing systems/structures, with new or modified organoleptic properties, the more "open" structures obtained by pressure treatment may well cause processing difficulties if subsequent reworking or moulding is required. (c) 2007 Elsevier Ltd. All rights reserved.

Evaluation of Serpa cheese proteolysis by nitrogen content and capillary zone electrophoresis

Proteolysis of Serpa cheese produced traditionally (B) and semi-industrially (C) was evaluated for the first time by determination of nitrogen content and capillary zone electrophoresis (CZE). A citrate dispersion of cheese was fractionated to determine the nitrogen in pH 4.4, trichloroacetic and phosphotungstic acid soluble fractions (pH 4.4-SN, TCA-SN and PTA-SN, respectively). The pH 4.4-SN was significantly higher for B ( P < 0.001), while TCA-SN was significantly higher for C ( P < 0.001). PTA-SN was also higher for C but at 60 days ripening no significant difference was found between B and C. Degradation of alpha(s1) - and beta-caseins evaluated by CZE was in good agreement with the maturation index (pH 4.4-SN/TN).

Effects of combined high-pressure and heat treatment on the textural properties of soya gels

sSPI, 7S, and 11S globulin at 12% (w/v) protein concentration, at neutral pH, did not form gels when heat-treated (90 degreesC, 15 min) or when high pressure-treated (300-700 MPa), except for the I IS, which formed a gel when heat-treated. The combination of heat and pressure (that is heating the solutions in a water bath and then pressure-treating at room temperature or the reverse sequence), led to differences: when heat-treatment was before high-pressure treatment, only the I IS fraction formed a self-standing gel; however, when the solutions were pressurised before heat treatment, all the proteins formed self-standing gels. The textural and water-holding properties were measured on the gels formed with the three different soy proteins. (C) 2002 Elsevier Science Ltd. All rights reserved.

Recent advances in the application of capillary electrophoresis for food analysis

This article reviews recent developments in the application of capillary electrophoresis (CE) for the analysis of foods and food components. CE has been applied to a number of important areas of food analysis and is fast becoming an established technique within food analytical and research laboratories. Papers are reviewed that were published during the two years to date following the previous review.

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.

Plastic compression of a collagen gel forms a much improved scaffold for ocular surface tissue engineering over conventional collagen gels

We compare the use of plastically compressed collagen gels to conventional collagen gels as scaffolds onto which corneal limbal epithelial cells (LECs) are seeded to construct an artificial corneal epithelium. LECs were isolated from bovine corneas (limbus) and seeded onto either conventional uncompressed or novel compressed collagen gels and grown in culture. Scanning electron microscopy (SEM) results showed that fibers within the uncompressed gel were loose and irregularly ordered, whereas the fibers within the compressed gel were densely packed and more evenly arranged. Quantitative analysis of LECs expansion across the surface of the two gels showed similar growth rates (p > 0.05). Under SEM, the LECs, expanded on uncompressed gels, showed a rough and heterogeneous morphology, whereas on the compressed gel, the cells displayed a smooth and homogeneous morphology. Transmission electron microscopy (TEM) results showed the compressed scaffold to contain collagen fibers of regular diameter and similar orientation resembling collagen fibers within the normal cornea. TEM and light microscopy also showed that cell–cell and cell–matrix attachment, stratification, and cell density were superior in LECs expanded upon compressed collagen gels. This study demonstrated that the compressed collagen gel was an excellent biomaterial scaffold highly suited to the construction of an artificial corneal epithelium and a significant improvement upon conventional collagen gels.

Structural and morphological studies of the dipeptide based L-Pro-L-Val organocatalytic gels and their rheological behaviour

Organocatalytic gels based on the dipeptide sequence L-Pro-L-Val have been studied by two different FTIR techniques. This suggests a different arrangement of the gelator molecules in the self-assembled fibers depending on the organic solvent employed. In acetonitrile and nitromethane the structure of the supramolecular aggregates is similar and provides similar catalytic properties (supramolecularenhancement of basicity). In contrast, the self-assembled fibers obtained in toluene clearly presented a different molecular arrangement consistent with its different catalytic behaviour (enamine-based catalysis). In addition these gels have been studied by microscopy and rheology.

Combined use of two genetic fingerprinting methods, pulsed-field gel electrophoresis and ribotyping, for characterization of Escherichia coli O157 isolates from food animals, retail meats, and cases of human disease

Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207 Escherichia coli O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom). In addition, 164 of these isolates were also phage typed. The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (n = 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (n = 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs. PFGE was conducted with the restriction endonuclease XbaI. while for ribotyping, two restriction endonucleases (PstI and SphI) were combined to digest genomic DNAs simultaneously. The 207 E. coli O157 isolates produced 97 PFGE profiles and 51 ribotypes. The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated E. coli O157 isolates in the study (Simpson's index of diversity [D] = 0.98 and 0.94 for PFGE typing and ribotyping, respectively). There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters. Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually. Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the E. coli O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of E. coli O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise.

Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota

With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intenstity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans.

Natural caprine whey oligosaccharides separated by membrane technology and profile evaluation by capillary electrophoresis

The functional food market is growing rapidly and membrane processing offers several advantages over conventional methods for separation, fractionation and recovery of bioactive components. The aim of the present study was to select a process that could be implemented easily on an industrial scale for the isolation of natural lactose-derived oligosaccharides (OS) from caprine whey, enabling the development of functional foods for clinical and infant nutrition. The most efficient process was the combination of a pre-treatment to eliminate proteins and fat, using an ultrafiltration (UF) membrane of 25 kDa molecular weight cut off (MWCO), followed by a tighter UF membrane with 1 kDa MWCO. Circa 90% of the carbohydrates recovered in the final retentate were OS. Capillary electrophoresis was used to evaluate the OS profile in this retentate. The combined membrane-processing system is thus a promising technique for obtaining natural concentrated OS from whey. Powered

The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation

Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (Pgels may regulate corneal epithelialisation through secretion of inhibitory proteins.