Garry J. Rucklidge

The effects of exogenous growth factors on matrix metalloproteinase secretion by human brain tumour cells

Matrix metalloproteinases (MMPs) are a growing family of zinc-dependent endopeptidases that are capable of degrading various components of the extracellular matrix. These enzymes have been implicated in a variety of physiological and pathological conditions including embryogenesis and tumour invasion. The synthesis of many MMPs is thought to be regulated by growth factors, cytokines and hormones. In this study, we investigated the effects of five exogenous growth factors known to be expressed by gliomas [epidermal growth factor (EGF), basic growth factor (bFGF), transforming growth factor beta (TGF-β1,2) and vascular endothelial growth factor (VEGF)] on MMP-2 and MMP-9 expression in an ependymoma, two grade III astrocytomas, a grade III oligoastrocytoma and a benign meningioma. Zymogram analysis revealed that the effects of the growth factors depended upon the cell lines used in the study. Growth factors generally up-regulated MMP-2 and MMP-9 expression in the gliomas but were least effective in the meningioma; the effect being most prominent with TGF-β1 and TGF-β2 in all the cell lines. It is hypothesized that paracrine growth factor interplay may be crucial in the regulation of MMP expression by glioma invasion of the normal brain.

Turnover rates of different collagen types measured by isotope ratio mass spectrometry

The rates of collagen turnover in different tissues have been estimated in growing rats previously exposed to gaseous 18O2. The abundance of the stable isotope was measured using isotope ratio mass spectrometry following combustion of isolated collagen-derived hydroxyproline. Using this method, problems of label reutilization associated with radiolabelling methods are avoided. In general the results confirm the slow turnover rates with half-lives of total collagen in skin, muscle and gut of 74, 45 and 244 d, respectively. The use of cyanogen bromide digests of whole tissues followed by isolation of collagen type-specific peptides has allowed the comparison of turnover rates of collagen types I and III, indicating that collagen type III is turned over more rapidly than type I.

Divergent mechanisms of cis9, trans11-and trans10, cis12-conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach

Conjugated linoleic acids (CLA) affect atherogenesis, but mechanisms are not well understood. We explored how two isomers of CLA, cis9, trans11‐CLA and trans10, cis12‐CLA, affected lipid and glucose metabolism, as well as hepatic protein expression, in apolipoprotein E knockout mice. After 12 wk of intervention, plasma triglyceride, NEFA, and glucose concentrations were significantly higher in the trans10, cis12‐CLA group, whereas plasma triglyceride, NEFA, glucose, and insulin concentrations were significantly lower in the cis9, trans 11‐CLA group, compared with control mice consuming linoleic acid. Proteomics identified significant up‐ or down‐regulation of 113 liver cytosolic proteins by either CLA isomer. Principal component analysis revealed that the treatment effect of cis9, trans11‐CLA was mainly explained by the up‐regulation of different posttranslational forms of heat shock protein 70 kD. In contrast, the treatment effect of trans10, cis12‐CLA was mainly explained by up‐regulation of key enzymes in the gluconeogenic, β‐oxidation, and ketogenesic pathways. Correlation analysis again emphasized the divergent effects of both CLA isomers on different pathways, but also revealed a linkage between insulin resistance and increased levels of hepatic serotransferrin. Thus, our systems biology approach provided novel insights into the mechanisms by which individual CLA isomers differentially affect pathways related to atherogenesis, such as insulin resistance and inflammation. Baukje De Roos, Garry Rucklidge, Martin Reid, Karen Ross, Gary Duncan, Maria A. Navarro, Jose M. Arbones‐Mainar, Mario A. Guzman‐Garcia, Jesus Osada, John Browne, Christine E. Loscher, Helen M. Roche Divergent mechanisms of cis9, trans11‐ and trans10, cis12‐conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach. FASEB J. 19, 1–21 (2005)

Substrate-driven gene expression in Roseburia inulinivorans: Importance of inducible enzymes in the utilization of inulin and starch

Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a β-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the β-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans β-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.

Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells

This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are alpha-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.

Response of apolipoprotein E*3-Leiden transgenic mice to dietary fatty acids: combining liver proteomics with physiological data

Dietary fatty acids have a profound impact on atherosclerosis, but mechanisms are not fully understood. We studied the effects of a saturated fat diet supplemented with fish oil, trans10,cis12 conjugated linoleic acid (CLA), or elaidic acid on lipid and glucose metabolism and liver protein levels of APOE*3 Leiden transgenic mice, a model for lipid metabolism and atherosclerosis. Fish oil lowered plasma and liver cholesterol and triglycerides, plasma free fatty acids, and glucose but increased plasma insulin. CLA lowered plasma cholesterol but increased plasma and liver triglycerides, plasma β‐hydroxybutyrate, and insulin. Elaidic acid lowered plasma and liver cholesterol. Proteomics identified significant regulation of 65 cytosolic and 8‐membrane proteins. Many of these proteins were related to lipid and glucose metabolism, and to oxidative stress. Principal component analysis revealed that fish oil had a major impact on cytosolic proteins, and elaidic acid on membrane proteins. Correlation analysis between physiological and protein data revealed novel clusters of correlated variables, among which a metabolic syndrome cluster. The combination of proteomics and physiology gave new insights in mechanisms by which these dietary fatty acids regulate lipid metabolism and related pathways, for example, by altering protein levels of long‐chain acyl‐CoA thioester hydrolase and adipophilin in the liver.

Identification of potential serum biomarkers of inflammation and lipid modulation that are altered by fish oil supplementation in healthy volunteers.

Long chain n‐3 polyunsaturated fatty acids (n‐3 LCPUFA) lower risk of coronary heart disease (CHD), but mechanisms are not well understood. We used proteomics to identify human serum proteins that are altered by n‐3 LCPUFA. Such proteins could identify pathways whereby they affect CHD. Eighty‐one healthy volunteers entered a double blind randomised trial to receive 3.5 g of fish oil or 3.5 g of high oleic sunflower oil daily. Serum was collected before and after 6 wk of intervention. Serum was analysed by proteomics using 2‐DE. Proteins that were differentially regulated were identified by MS. We also analysed serum apolipoprotein A1 (apo A1), high‐density lipoprotein (HDL) particle size and haptoglobin. Serum levels of apo A1, apo L1, zinc‐α‐2‐glycoprotein, haptoglobin precursor, α‐1‐antitrypsin precursor, antithrombin III‐like protein, serum amyloid P component and haemopexin were significantly downregulated (all p<0.05) by fish oil compared with high oleic sunflower oil supplementation. Fish oil supplementation caused a significant shift towards the larger, more cholesterol‐rich HDL2 particle. The alterations in serum proteins and HDL size imply that fish oil activates anti‐inflammatory and lipid modulating mechanisms believed to impede the early onset of CHD. These proteins are potential diagnostic biomarkers to assess the mechanisms whereby fish oil protects against CHD in humans.

Evaluation of the effects of swainsonine, captopril, tangeretin and nobiletin on the biological behaviour of brain tumour cells in vitro.

Although intrinsic tumours of the brain seldom metastasize to distant sites, their diffuse, infiltrative–invasive growth within the brain generally precludes successful surgical and adjuvant therapy. Hence, attention has now focused on novel therapeutic approaches to combat brain tumours that include the use of anti‐invasive and anti‐proliferative agents. The effect of four anti‐invasive agents, swainsonine (a locoweed alkaloid), captopril (an anti‐hypertensive drug), tangeretin and nobiletin (both citrus flavonoids), were investigated on various parameters of brain tumour invasion such as matrix metalloproteinase (MMP) secretion, migration, invasion and adhesion. A standard cytotoxicity assay was used to optimize working concentrations of the drugs on seven human brain tumour‐derived cell lines of various histological type and grade of malignancy. A qualitative assessment by gelatin zymography revealed that the effect of these agents varied between the seven cell lines such that the low grade pilocytic astrocytoma was unaffected by three of the agents. In contrast, downregulation of the two gelatinases, MMP‐2 and MMP‐9 was seen in the grade 3 astrocytoma irrespective of which agent was used. Generally, swainsonine was the least effective whereas the citrus flavonoids, particularly nobiletin, showed the greatest downregulation of secretion of the MMPs. Furthermore, captopril and nobiletin were most efficient at inhibiting invasion, migration and adhesion in four representative cell lines (an ependymoma, a grade II oligoastrocytoma, an anaplastic astrocytoma and a glioblastoma multiforme). Yet again, the effects of the four agents varied between the four cell lines. Nobiletin was, nevertheless, the most effective agent used in these assays. In conclusion, the differential effects seen on the various parameters studied by these putative anti‐invasive agents may be the result of interference with MMPs and other mechanisms underlying the invasive phenotype. From these pilot studies, it is possible that these agents, especially the citrus flavonoids, could be of future therapeutic value. However, further work is needed to validate this in a larger study.

ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome

A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.

Invasion of brain tissue by primary glioma : evidence for the involvement of urokinase-type plasminogen activator as an activator of type IV collagenase

The immunocharacterization of a metalloproteinase isolated from rat glioma cell conditioned medium is described and confirms that the enzyme is identical to type IV collagenase. Free, active plasminogen activator (PA) and PA-PAI complexes were identified as being secreted by the same cells. Using affinity-purified metalloproteinase we demonstrate that the enzyme can be partially activated by u-PA but not by plasmin in vitro. On the basis of these findings and previous published work we propose a scheme for the proteolytic degradation of normal brain tissue during tumour invasion.