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, trans11-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, beta-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.

Leptomeningeal tissue: a barrier against brain tumor cell invasion.

BACKGROUND: Primary brain tumors are characterized by an extensive infiltrative growth into the surrounding brain tissue. This process is confined to the central nervous system, and tumor cell metastasis to other organs is rare. However, other tumors of non-neural origin may frequently metastasize to the central nervous system. PURPOSE: The purpose of the present study was to examine the invasive behavior of different glioma cells into tissues of neural (brain aggregates) as well as non-neural origin (leptomeningeal tissue). Using the same target tissues, the invasive characteristics of two neural metastatic tumors (one malignant melanoma and one small-cell lung carcinoma) were also studied. This direct comparison of the invasive behavior between tumors of neural and non-neural origin provides valuable information regarding the mechanisms of glioma cell dissemination in the central nervous system. METHODS: The in vitro invasive behavior of human tumors of the central nervous system into human leptomeningeal tissue as well as into normal rat brain tissue was studied. For this purpose, a co-culture system consisting of tumor biopsy specimens, human leptomeningeal cell aggregates, and brain cell aggregates was established. Three glioblastomas, one oligodendroglioma, one meningioma, one small-cell lung carcinoma, and one malignant melanoma were studied. RESULTS: In co-cultures of gliomas and leptomeningeal cell aggregates, a well-defined border between the two tissues was observed. The brain cell aggregates, in contrast, were consistently invaded by the glioma cells. The brain metastases showed a different invasion pattern. The metastatic cells invaded and progressively destroyed leptomeningeal cell aggregates, whereas they did not invade the brain cell aggregates. Upon confrontation of the leptomeningeal tissue with the meningioma, a fusion of the two tissues was observed. Immunostaining of the leptomeningeal tissue showed a strong expression of the basement membrane components fibronectin, collagen type IV, and laminin with no expression of glial fibrillary acidic protein, neuron-specific enolase, or S-100 protein. CONCLUSIONS: The present study indicates that there may be important biologic differences between the invasive behavior of gliomas and non-neuroepithelial tumors. Our co-culture experiments suggest that leptomeningeal cells and associated acellular components may constitute a barrier against glioma cell invasion. However, this barrier may not be functional for metastatic tumors to the brain. The presence of glioma cells within the leptomeninges should not necessarily be taken as evidence of aggressive growth or as an indicator of malignancy.

Immunocytochemical characterization of extracellular matrix proteins expressed by cultured glioma cells.

The immunolocalization of type I, III and IV collagens and fibronectin in two rat glioma cell lines in vitro (BT4C and BT4Cn) is described. In addition, antibodies against denatured type I and III collagens were used to study breakdown products of native type I and III collagens. For the BT4C cells, the extracellular matrix expression in monolayer cultures and in multicellular tumor spheroids was compared. Type IV collagen was strongly expressed in BT4C tumor spheroids but was negative in the corresponding monolayer cultures. Denatured type I collagen was found both in monolayers and in spheroids of BT4C, suggesting either a rapid turnover (i.e., synthesis and immediate breakdown) of type I collagen or an altered collagen gene transcription. Both cell lines were negative for native type I and III and denatured type III collagen. Fibronectin was strongly expressed in both cell lines. Supporting the immunofluorescence data, the hydroxyproline content in the tumor spheroids was twice the amount found in monolayer cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunoblotting also verified the immunostaining experiments, showing that glioma spheroids and injected tumor cells have the potential for fibronectin and collagen production, given the appropriate growth conditions.

Immunolocalization of extracellular matrix proteins during brain tumor invasion in BD IX rats.

The distribution of several native extracellular matrix proteins (type I, III, and IV collagens and fibronectin) using immunofluorescent localization is described for in two different malignant gliomas (BT4A and BT4An). In addition, antibodies against denatured forms of type I and III collagens were used to localize areas of active degradation within the tumors. We have shown that both tumors express the native connective tissue components studied, although the distribution of these components within and between the tumors was different. In addition, native type I and III collagens and fibronectin were overexpressed in the tumors compared to the normal brain. Morphometry on immunostained type IV collagen sections showed an increase in vascular elements in both tumors compared to normal brain tissue. The BT4A tumor, which by light microscopy showed a degradative mode of invasion, expressed denatured type I and III collagens at the tumor-brain border zone, suggesting that this tumor has collagenolytic activity. The present article suggests that the distribution and changes in extracellular matrix protein synthesis and degradation may play an important role in the progressive growth of brain tumors in vivo.

Preparation and analysis of the products of non-enzymatic protein glycosylation and their relationship to cross-linking of proteins.

The presence of glycosylated protein-bound lysine residues has led to much speculation regarding changes in structure and function of the modified protein. The synthesis of hexose-lysine adducts and their separation using an amino acid analyser is described. These compounds are also produced during borohydride reduction and subsequent hydrolysis of modified proteins, and misidentification of these may occur depending upon precise chromatographic procedures. The possibility that glucose might participate in a cross-linking reaction between two protein-bound lysine residues was tested but no evidence for such a mechanism was found. The presence of 14C-labelled urinary hexosyllysine indicated that body protein breakdown in addition to ingested dietary hexosyllysine contributes to the excretion of this component.

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.

Identification of lysine-derived crosslinks in porcine collagen type X from growth plate and newly mineralized bone.

Intact collagen type X cannot readily be extracted from the growth plate. Both the use of pepsin to release this molecule from tissue and the relative solubility of collagen type X following treatment of chick embryos with beta-aminopropionitrile (Chen et al., 1992) suggest that the insolubility may by brought about by the formation of lysine-derived crosslinks. By immunocytochemical labelling using antibodies specific for collagen type X, we have shown that this collagen type persists in the cartilaginous spicules present in metaphyseal bone and appears to be colocalized with collagen type II. The combined concentration of the reducible bifunctional crosslinks, dihydroxylysinonorleucine and monohydroxylysinonorleucine, in collagen type X isolated from the premineralized and newly mineralized growth plate was about 0.6 residues/ molecule, a level which might explain the relative intractability of collagen type X. Pyridinoline and deoxypyridinoline were present in very small amounts in collagen type X; this suggests that, unlike the situation in other types of collagen, few of the bifunctional crosslinks undergo maturation to pyridinium compounds. Although it is clear that collagen type X contains lysinederived crosslinks, work is in progress to establish which molecule also participates in the formation of these crosslinks.

Primary structure of the helical domain of porcine collagen X.

The entire primary structure of the collagen X helical region is presented, including identification of the extensive post-ribosomal modifications by amino acid sequencing and mass spectrometry. As in collagen I, a single residue of 3-hydroxyproline was identified, but for collagen X this was located near the N-terminal end of the helix. Lysine residues in collagen X are extensively hydroxylated/glycosylated: at least 11 sites were localized and shown to be fully glycosylated, exclusively as glucosyl-galactosyl derivatives. The lysine-derived crosslinks, dihydroxylysinonorleucine and hydroxylysinonorleucine, were shown to be present in a 3:2 molar ratio primarily within the C-terminal portion of the helix.

Immunohistochemical localization of native and denatured collagen types I and II in fetal and adult rat long bones.

Collagen turnover during rat long bone development and growth was investigated using immunofluorescence methods with specific polyclonal antibodies against native (triple helix) and denatured (breakdown products) forms of type I and II collagen. Labeling of cryostat sections with anti-native and denatured collagen type II antibodies resulted in a positive staining throughout the cartilage matrix of fetal and adult long bones. Likewise, native and denatured type I collagen could be detected in mineralized and non-mineralized bone matrix. Moreover, labeling with anti-denatured type I antibody evoked a strong intracellular staining of osteoblasts, but not of osteocytes. Denatured type I was also localized intra-pericellularly in the small chondrocytes comprising the primitive cartilage cores and the epiphyses of older long bones. On the other hand, apart from its localization in the cartilage matrix, denatured type II collagen was found specifically within the chondrocytes. These observations indicate that a continuous turnover of the major collagen types takes place in fetal and adult rat long bone tissue. Degradation of collagen apparently occurs intra- and extracellularly, and is mainly independent of the presence and activity of osteoclasts. The presence of denatured type I collagen in cartilage suggests that chondrocytes synthesize small amounts of type I collagen, which is immediately degraded to a denatured form.

The effect of exogenous gangliosides on matrix metalloproteinase secretion by human glioma cells in vitro.

Matrix metalloproteinases (MMPs) are zinc-dependent peptidases and are amongst those enzymes responsible for extracellular matrix (ECM) degradation during tumour-cell migration. Gangliosides are a family of acidic membrane glycolipids thought to play a role during cell development, differentiation and oncogenic transformation. In this descriptive study, we investigated the effects of six exogenous gangliosides (GM1, GM3, GD1a, GD1b, GD3 and GT1b) on the secretion of MMP-2 (72 kDa gelatinase or gelatinase-A) and MMP-9 (92 kDa gelatinase or gelatinase-B). Cell-conditioned media from eight human glioma-derived cell-lines served as the source of MMPs and were investigated using SDS-PAGE zymography. Six of the cell lines showed upregulation of secretion of both enzymes by all six gangliosides. Of the remaining two cell lines, one showed inhibition of MMP secretion by all gangliosides and the other had a small but differential response to the range of gangliosides investigated. These results suggest that gangliosides may stimulate glioma cell invasiveness by promoting MMP expression.

An inverse correlation between expression of NCAM-A and the matrix-metalloproteinases gelatinase-A and gelatinase-B in human glioma cells in vitro.

Matrix metalloproteinases (MMPs) are an homologous family of proteolytic enzymes capable of degrading components of the extracellular matrix (ECM) and thereby facilitating the invasion of tumour cells into normal tissues. The neural cell adhesion molecules (NCAMs) of neuronal and glial cells provide a Ca2+-independent mechanism for cell-cell and cell-ECM adhesion. NCAMs are downregulated to promote cell disaggregation during cell migration in the developing nervous system whereas MMPs facilitate migration. Recent studies have shown downregulation of MMP secretion in rat glioma cells transfected with an NCAM cDNA, implying an inverse correlation between NCAM and MMP expression. The purpose of this study was to establish whether such a correlation could be demonstrated in a panel of nine human glioma cell-lines, one metastatic carcinoma and one foetal astrocyte derived cell line. The secretion of two MMPs, 72 kDa gelatinase (MMP-2 or gelatinase-A) and 92 kDa gelatinase (MMP-9 or gelatinase-B), was investigated using SDS-PAGE zymography; NCAM-A was assayed by an immunochemiluminescent assay following SDS-PAGE of whole-cell extracts. An inverse correlation was found between the expression of NCAM-A and that of both MMPs studied although the patterns of expression showed no obvious correlation with histological type or grade of the parent tumours. Our results suggest that downregulation of NCAM-A may contribute to tumour invasiveness by promoting both cell disaggregation and protease secretion.

Comparative analysis of matrix metalloproteinases by immunocytochemistry, immunohistochemistry and zymography in human primary brain tumours.

Matrix metalloproteinases (MMPs) are a growing family of zinc-dependent endopeptidases which are characterised by their ability to degrade various extracellular matrix (ECM) components. The family includes collagenases, gelatinases, stromelysins, metalloelastase and membrane type metalloproteinases. Consistent with their proteolytic activities, MMPs have been implicated in a variety of physiological and pathological conditions, such as normal embryogenesis, tissue morphogenesis and are thought to play a role in facilitating tumour cell invasion of the normal brain. In this comparative study, we have used zymography, immunohistochemical and immunocytochemical techniques to demonstrate the expression of gelatinase-A and B (MMP-2 and 9, respectively) and membrane type metalloproteinase (MMP-14) in 8 intrinsic human primary brain tumours of various histological type and grade. Zymography results showed that MMP-2 was the most prominent proteolytic enzyme in all the cell lines studied (with one exception), while MMP-9 was only faintly expressed. However, the corresponding paraffin sections showed no expression of either MMP-2, 9 or 14 within the tumour cells, positivity being confined to haematogenous cells and the vascular endothelium. Fluorescence immunocytochemical studies, using monoclonal antibodies to MMP-2, 9 and 14, showed granular cytoplasmic reactivity in vitro. In addition, there was strong focal positivity at the cell membrane with MMP-14 in some high grade tumours suggesting that MMPs are produced at the leading edge of the cell by individual subpopulations of invading glia, in small quantities and on demand in vivo. It can be concluded that local microenvironmental conditions in vitro appear to stimulate such MMP activity.

Laminin: a potential inhibitor of rat glioma cell invasion in vitro.

The lack of metastatic behaviour of primary glioma is poorly understood. A possible natural barrier accounting for this phenomenon may be the proteins of the extracellular matrix which are found in the basement membranes of the blood vascular system. This hypothesis is reinforced by the finding that glioma invasion in vitro using a syngeneic model system results in a lack of invasion of areas of target tissue which contain extracellular matrix proteins. The study was extended by examining the effect of the incorporation of these proteins during the formation of fetal rat brain cell aggregates and glioma spheroids and on the invasion of aggregates by tumour spheroids. Laminin was shown to reduce the size of the aggregates and spheroids during their formation while fibronectin and type IV collagen had no effect. Laminin also prevented the invasion of the tumour spheroid into the target aggregate and appeared to inhibit migration of glioma cells on laminin coated tissue culture plastic.

A metalloproteinase, capable of destroying cultured brain tissue isolated from rat glioma cells.

A continuous rat glioma cell line, BT5C, which causes severe invasion and tissue destruction when cocultured with fetal rat brain aggregates, secreted into the culture medium a metalloproteinase in a hMr latent (86000) and an lMr active (76000) isoform. Purified preparations of the enzyme were added to cultures of fetal rat brain aggregates, which were then examined by light and by scanning electron microscopy. After 48h of enzyme exposure, destruction of the neural tissue was observed. This was morphologically similar to the tissue destruction seen when BT5C spheroids were cocultured with brain aggregates. The protease had no effect on the morphology of BT 5C tumour spheroids. The report suggests a possible role of metalloproteinases in tissue degradation during brain tumour invasion.

The influence of sequential, in vitro passage on secretion of matrix metalloproteinases by human brain tumour cells.

Matrix metalloproteinases (MMP) are a family of zinc-dependent enzymes which degrade various components of the extracellular matrix (ECM) and play an important role in facilitating tumour cell invasion of the normal brain. The family includes the gelatinases, stromelysins and collagenases. Preliminary studies have shown that there is a differential expression four metalloproteinases in human brain tumour cell lines derived from neoplasms of various histological types and grades of malignancy. Morphological and antigenic changes in human glioma-derived cell lines over many serial in vitro passages have been reported in earlier studies. When established cell lines are maintained in culture over a long period, it is possible that the secretion of enzymes such as metalloproteinases may differ according to the passage level examined. This report presents a study on the secretion of four matrix metalloproteinases - interstitial collagenase (MMP-), 72-kDa and 92-kDa gelatinases (MMP-2 and MMP-9 respectively), and stromelysin (MMP-3) - in three human brain tumour-derived cell lines at sequentially increasing passage numbers, ranging from passage 2 to passage 50; foetal astrocytes were used as a positive control. Reverse zymography and substrate degradation analysis were employed to demonstrate the presence of these enzymes in cell- conditioned culture medium. Aminophenyl mercuric acetate (APMA) was used to activate latent zymogen. Results demonstrate that there is no definite pattern of change in the levels of enzyme secretion common to all cell lines studied. Instead, the fluctuations in APMA- activated metalloproteinase activity in serial passage seems to vary considerably depending on the cell line and the type of enzyme studied. The variation in metalloproteinase expression observed on serial passage may be due to in vitro selection processes or karyotype evolution where the transcription of either the enzyme and/or its inhibitor may be affected. Thus an imbalance of the two products could be occurring in serial passage. Ideally, experiments requiring the measurement of relative enzyme activities should use cultures as near to the biopsy stage as possible, i.e. very low passages, to avoid artifacts that may arise on prolonged culturing.

Deer antler does not represent a typical endochondral growth system: immunoidentification of collagen type X but little collagen type II in growing antler tissue.

The collagen isotypes present at early (6 week) and late (5 month) stages of growing deer antler were isolated and identified. Pepsin-digested collagens were separated by differential salt fractionation, SDS-PAGE and Western blotting and subsequently identified by immunostaining. Cyanogen bromide digestion of antler tissue was used to establish a collagen type-specific pattern of peptides, and these were also identified by immunoblotting. Collagen type I was found to be the major collagen in both early- and late-stage antler. Collagen type II was present in the young antler in small amounts but was not confined to the soft "cartilaginous" tip of the antler. Collagen type XI was found in the pepsin digest of the young antler, but collagen type IX was not present at either stage of antler growth. Collagen type X was found in the young antler in all fractions studied. Microscopic study showed that the deer antler did not possess a discrete growth plate as found in endochondral bone growth. Unequivocal immunolocalization of the different collagen types in the antler were unsuccessful. These results show that, despite the presence in the antler of many cartilage collagens, growth does not occur through a simple endochondral process.

Enzyme-linked immunosorbent assay for the measurement of immunoglobulin G concentrations in porcine plasma and colostrum.

An enzyme-linked immunosorbent assay is described for the estimation of porcine immunoglobulin G in either colostrum or plasma samples. The inter-assay coefficient of variation was 9.1 per cent and the intraassay coefficient of variation was 7.2 per cent. The repeatability or intra-class correlation coefficient of the assay was 0.9. The assay proved to be a sensitive, inexpensive and rapid method for assessing the immune status of pigs.

Concentration of collagen, aggrecan and cartilage oligomeric matrix protein (COMP) in synovial fluid from equine middle carpal joints.

The aim of the present investigation was to study the metabolic activity of the third carpal bone and the release of COMP, aggrecan and collagen type II molecules in the synovial fluid as a result of injury. Cartilage oligomeric matrix protein (COMP), aggrecan and collagen type II or fragments of these molecules released to the synovial fluid and serum (COMP) were quantified in samples from 73 left equine middle carpal joints from 2 breeds with different activity profiles (52 Standardbred trotters [STB] and 21 Swedish Warmblood riding horses [SWH]) and different articular cartilage lesions. Synovial and serum samples were analysed using inhibition ELISA for COMP and aggrecan. An ELISA that combines features of both the competitive and capture ELISAs was used for collagen type II. COMP and aggrecan concentrations decreased in synovial fluid from the joints with moderate lesions of STB compared with the normal joints; COMP from 16.6 to 12.0 microg/ml and aggrecan from 93.0 to 68.1 microg/ml. In serum, COMP concentrations were also lowered in the STB with moderate lesions compared with the normal joints, while in the SWH, the COMP concentration in synovial fluids from joints with moderate lesions was somewhat increased at 19.6 microg/ml compared with the normal joints (17.6 microg/ml). The ratio between aggrecan/COMP in the synovial fluid from joints with moderate lesions was higher in the STB (6.2) than in the SWH (3.4). The level of collagen type II in synovial fluid was higher in the SWH (8.8 microg/ml) than the STB (1.6 microg/ml), but there was no correlation between joint damage and collagen concentrations in synovial fluids (10.0 and 1.8 microg/ml in joints with moderate lesions from SWH and STB, respectively). A marked difference in COMP synthesised upon metabolic labelling between the normal and osteoarthritic cartilage was seen and the synthesis of COMP in the articular cartilage of the third carpal bone with moderate articular lesions (from an STB) was lower than in the joint with mild lesions. This difference between breeds may reflect different load characters, in release of macromolecules in osteoarthritic and normal joints. This a novel finding that should be considered in studies of equine traumatic arthritis.

A new metabolism cage suitable for the study of mice.

Nitrogen recoveries of 98% have been obtained, significantly better than those obtained using a commercial metabolism cage.