Microbial analysis of soil and groundwater from a gasworks site and comparison to a sequenced biological reactive barrier remediation process (SEREBAR)

Aims: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater. Methods and Results: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal. Conclusion: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident. Significance and Impact of the Study: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process.

Proteomic Analysis of Recurrent Joint Inflammation in Juvenile Idiopathic Arthritis

The synovial fluid proteome in juvenile idiopathic arthritis was investigated to isolate joint-specific biomarkers that are expressed in patients displaying recurrent joint inflammation. To identify the synovial specific proteome, matched synovial fluid and plasma samples were subjected to protein separation by 2-dimension electrophoresis (2DE). Forty-three protein spots, overexpressed in the joint, were identified. Synovial fluids from children with single-event knee joint inflammation were then compared with a group with recurrent knee disease. Nine synovial specific proteins were significantly differentially expressed in the recurrent group. Proteolytic fragments of collagen X, fibrin beta-chain, and T-cell receptor alpha-region have been identified among this protein cluster. Putative biomarkers, overexpressed in the joint and differentially expressed in children with recurrent joint inflammation, have been identified. These proteins may play a significant role determining the pathological state within the chronically inflamed joint and influence disease progression in JIA. This is the first study of the synovial proteome in children.

Ring-Down Absorption Spectroscopy for Analytical Microdevices

Ring-down absorption spectroscopy is an emerging ‘‘label-free’’ detection method for analytical microdevices, such as micrototal analysis systems (l-TAS). Developed from the related gas-phase cavity ring-down absorption spectroscopy, fiber-optic-based ring-down techniques for liquid samples offer low detection limits, high sensitivity and fast response. ª 2006 Elsevier Ltd. All rights reserved.

First report of a novel plant lysozyme with both antifungal and antibacterial activities.

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4 kDa in SDS–polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355 U/mg at pH 5.5 and 30 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 °C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.

Azaspiracid: First evidence of protein binding in shellfish

The occurrence of azaspiracid (AZA) toxins in contaminated shellfish has been the focus of much research. The present study investigated the binding properties of these toxins in mussels of the species Mytilus edulis. The work involved extraction of proteins and AZAs from contaminated mussel hepatopancreas and examination of the extracts by isoelectric focusing (IEF), size exclusion chromatography (SEC) and sodium docecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Liquid chromatography coupled with tandem mass spectrometry analysis (LC–MS/MS) was also performed in this study to identify AZAs. Blank mussels were subjected to the same purification and analytical procedures.

AZAs were found to be weakly bound to a protein with a molecular weight of 45 kDa, in samples of contaminated mussels. This protein, which was abundant in contaminated mussels, was also present in blank mussels, albeit at much lower concentrations. It was further noted that a 22 kDa protein was also present only in contaminated mussel samples.

Characterization of melanoidins from a glucose-glycine model system

The properties of melanoidins prepared from glucose and glycine (GG) were investigated by a three step purification protocol consisting of dialysis, gel filtration at high ionic strength and ion metal affinity chromatography. The high molecular weight fraction obtained in the GG system is responsible for 80% of the total brown colour and its antioxidative ability was about 1/4 of that of Trolox measured by the inhibition of linoleic acid oxidation. GG melanoidins have good affinity towards Cu (II) (32% bound to the resin) while it is much lower towards Pb (II) (10%) and Fe (II) (5%). Capillary zone electrophoresis analysis suggests that GG melanoidins are positively charged, although no signal was observed analysing melanoidins by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF/MS).

Effect of roasting on the antioxidant activity of coffee brews

Colombian Arabica coffee beans were roasted to give light, medium, and dark samples. Their aqueous extracts were analyzed by gel filtration chromatography, UV-visible spectrophotometry, capillary electrophoresis, and the ABTS(.+) assay. A progressive decrease in antioxidant activity (associated mainly with chlorogenic acids in the green beans) with degree of roasting was observed with the simultaneous generation of high (HMM) and low molecular mass (LMM) compounds possessing antioxidant activity. Maximum antioxidant activity was observed for the medium-roasted coffee; the dark coffee had a lower antioxidant activity despite the increase in color. Analysis of the gel filtration chromatography fractions showed that the LMM fraction made a greater contribution to total antioxidant activity than the HMM components.

Antioxidant activity of coffee model systems

Coffee model systems prepared from combinations of chlorogenic acid (CGA), N-alpha-acetyl-1-arginine (A), sucrose (S), and cellulose (C) were roasted at 240 degreesC for 4 min prior to analysis by UV-visible spectrophotometry, capillary zone electrophoresis (CZE), and the ABTS radical cation decolorization assay. The A/CGA/S/C and A/S/C systems were also fractionated by gel filtration chromatography. Antioxidant activity of the systems showed a positive, nonlinear relationship with the amount of CGA remaining after roasting. Sucrose degradation was a major source of color in the heated systems. There was no relationship between antioxidant activity and color generation.

Novel approaches to the analysis of the Maillard reaction of proteins

The Maillard reaction comprises a complex network of reactions which has proven to be of great importance in both food science and medicine. The majority of methods developed for studying the Maillard reaction in food have focused on model systems containing amino acids and monosaccharides. In this study, a number of electrophoretic techniques, including two-dimensional gel electrophoresis and capillary electrophoresis, are presented. These have been developed specifically for the analysis of the Maillard reaction of food proteins, and are giving important insights into this complex process.

IQ motif selectivity in human IQGAP1: binding of myosin essential light chain and S100B.

IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition.

Investigation of DNA polymorphisms in SMAD genes for genetic predisposition to diabetic nephropathy in patients with type 1 diabetes mellitus

Aims/hypothesis: SMAD proteins are involved in multiple signalling pathways and are key modulators of gene expression. We hypothesised that genetic variation in selected SMAD genes contributes to susceptibility to diabetic nephropathy. Methods: We selected 13 haplotype tag (ht) single nucleotide polymorphisms (SNPs) from 67 variants identified by resequencing the SMAD2 and SMAD3 genes. For SMAD1, SMAD4 and SMAD5 genes, genotype data were downloaded for 217 SNPs from Phase II of the International HapMap project. Of these, 85 SNPs met our inclusion criteria, resulting in the selection of 13 tag SNPs for further investigation. A case-control approach was employed, using 267 nephropathic patients and 442 controls with type 1 diabetes from Ireland. Two further populations (totalling 1,407 patients, 2,238 controls) were genotyped to validate initial findings. Genotyping was conducted using iPLEX, TaqMan and gel electrophoresis.
Results: The distribution of genotypes was in Hardy-Weinberg equilibrium. Analysis by the ? 2 test of genotype and allele frequencies in patients versus controls in the Irish population (n?=?709) revealed evidence for the association of one allele at 5% level of significance (rs10515478, p uncorrected?=?0.006; p corrected?=?0.04). This finding represents a relatively small difference in allele frequency of 6.4% in the patient group compared with 10.7% in the control group; this difference was not supported in subsequent investigations using DNA from European individuals with similar phenotypic characteristics.
Conclusions/interpretation: We selected an appropriate subset of variants for the investigation of common genetic risk factors and assessed SMAD1 to SMAD5 genes for association with diabetic nephropathy. We conclude that common polymorphisms in these genes do not strongly influence genetic susceptibility to diabetic nephropathy in white individuals with type 1 diabetes mellitus.

LDL particle size in habitual exercisers, lean sedentary men and abdominally obese sedentary men

Habitual exercisers enjoy considerable protection from coronary heart disease (CHD). Often, however, only modest differences in traditional CHD risk factors are apparent between habitual exercisers and their sedentary counterparts. For this reason, there is increasing interest in novel predictors of CHD, such as a preponderance of small, dense low-density lipoprotein (LDL) particles. Polyacrylamide gel electrophoresis was used to separate lipoprotein subfractions in 32 lean exercisers, 36 lean sedentary men and 21 obese sedentary men aged 30-45 years. Well-validated equations were used to determine LDL concentration and peak particle diameter. Waist girth was used to identify lean (<100 cm) and obese ( >= 100cm) individuals. LDL concentration was lower in lean exercisers than in lean sedentary men (2.64 +/- 0.44 vs. 3.76 +/- 0.79 mmol.l(-1), p <0.001), suggesting that habitual exercise influences this risk factor. In contrast, there were no significant differences in LDL peak particle diameter between lean exercisers, lean sedentary men and obese sedentary men (27.92 +/- 0.67, 28.09 +/- 0.62 and 27.77 +/- 0.77 nm, respectively). In multiple linear regression analysis, triglyceride concentration was the only significant predictor of LDL PPD. These data suggest that habitual exercise influences LDL concentration but does not influence LDL particle size in men aged 30-45 years.

Comparative analysis of synovial fluid and plasma proteomes in juvenile arthritis - Proteomic patterns of joint inflammation in early stage disease

Synovial fluid is a potential source of novel biomarkers for many arthritic disorders involving joint inflammation, including juvenile idiopathic arthritis. We first compared the distinctive protein ‘fingerprints’ of local inflammation in synovial fluid with systemic profiles within matched plasma samples. The synovial fluid proteome at the time of joint inflammation was then evaluated across clinical subgroups to identify early disease associated proteins. We measured the synovial fluid and plasma proteomes using the two-dimensional fluorescence difference gel electrophoresis approach. Image analysis software was used to highlight the expression levels of joint and subgroup associated proteins across the study cohort (n = 32). A defined subset of 30 proteins had statistically significant differences (p < 0.05) between sample types such that synovial fluid could be differentiated from plasma. Furthermore distinctive synovial proteome expression patterns segregate patient subgroups. Protein expression patterns localized in the chronically inflamed joint therefore have the potential to identify patients more likely to suffer disease which will spread from a single joint to multiple joints. The proteins identified could act as criteria to prevent disease extension by more aggressive therapeutic intervention directed at an earlier stage than is currently possible.

Fragmentation and plasmid strand breaks in pure and gold-doped DNA irradiated by beams of fast hydrogen atoms

The results of an investigation into the damage caused to dry plasmid DNA after irradiation by fast (keV) hydrogen atoms are presented. Agarose gel electrophoresis was used to assess single and double strand break yields as a function of dose in dry DNA samples deposited on a mica substrate. Damage levels were observed to increase with beam energy. Strand break yields demonstrated a considerable dependence on sample structure and the method of sample preparation. Additionally, the effect of high-Z nanoparticles on damage levels was investigated by irradiating DNA samples containing controlled amounts of gold nanoparticles. In contrast to previous (photonic) studies, no enhancement of strand break yields was observed with the particles showing a slight radioprotective effect. A model of DNA damage as a function of dose has been constructed in terms of the probability for the creation of single and double strand breaks, per unit ion flux. This model provides quantitative conclusions about the effects of both gold nanoparticles and the different buffers used in performing the assays and, in addition, infers the proportion of multiply damaged fragments.

Modelling the comet assay

The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters.