Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the haematopoietic microenvironment. Recently, several studies have indicated that they are involved in the interaction of haematopoietic stem and stromal cells. However, a detailed characterization of the heparan sulphate proteoglycans synthesized by bone-marrow stromal cells is still lacking. Here we report on the isolation and characterization of proteoglycans from the haematopoietic stromal cell line MS-5, that efficiently supports the growth and differentiation of human and murine haematopoietic progenitor cells. Biochemical characterization of purified proteoglycans revealed that the haematopoietic stromal cell line MS-5 synthesizes, in addition to chondroitin sulphate proteoglycans, several different heparan sulphate proteoglycans. Immunochemical analysis, using specific antibodies against the different members of the syndecan family, glypican, betaglycan and perlecan, showed that MS-5 cells synthesize all these different heparan sulphate proteoglycans. These data were further supported by reverse-transcriptase PCR and confirmed by sequence and Northern blot analysis. The relative abundance of the different heparan sulphate proteoglycans was estimated on the protein and mRNA levels.

Heparan sulphate proteoglycans and the extracellular matrix of bone-marrow-stromal cells are important components of the microenvironment of haematopoietic tissues and are involved in the interaction of haematopoietic stem and stromal cells. Previous studies have emphasized the role of heparan sulphate proteoglycan synthesis by bone-marrow-stromal cells. In the present study we describe the expression of glypican-4 (GPC-4), belonging to the glypican family, in bone-marrow-stromal cells and haematopoietic-progenitor cells of human and murine origin. Expression of GPC-4 was shown on the mRNA-level by reverse transcription-PCR and Northern blot analysis. Amplification products were cloned and sequenced, to confirm these results. To analyze the expression of GPC-4 on the protein level, polyclonal antibodies against selected peptides were raised in rabbits. Western blot analysis showed expression of GPC-4 as a heparan sulphate proteoglycan in the human haematopoietic-progenitor cell line TF-1 and normal human bone marrow. These results were confirmed by FACS analysis of TF-1 cells. Furthermore, GPC-4-positive progenitor cells and stromal cells were enriched from normal human bone marrow by magnetic-cell sorting and analysed by confocal laser-scanning microscopy.

In an effort to gain deeper insight into the molecular processes underlying neurodegeneration in Parkinson's disease, we performed gene expression profiling at several early time points after MPTP-injection into old (1-year) mice. We used a PCR-based gene expression profiling method, digital expression pattern display (DEPD), a method of very high sensitivity and reproducibility, which displays almost all transcripts of a tissue. To identify cell death-associated genes, we defined clusters of differentially expressed transcripts with expression behaviour that correlated with the temporal profile of cell death progression and characterized one of these cell death clusters further. We selected one of the strongest regulated genes, the serum and glucocorticoid-regulated kinase 1 (sgk1), and validated its differential expression by Northern blot analysis, semiquantitative PCR and in situ hybridization. Up-regulation of sgk1 (i) coincides with the onset of dopaminergic cell death in both the 8-week acute and 1-year subacute MPTP models, (ii) spans the entire brain, (iii) is attenuated by the l-deprenyl-mediated inhibition of the MPTP conversion to its active metabolite MPP+ and (iv) is not induced by dehydration. This study demonstrated that the combination of the DEPD technology, clustering analysis and a detailed histopathology is a useful tool for elucidating molecular pathways in neurodegenerative diseases.

The 5' region of the wound-inducible gene wun1, derived from potato, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants. Different 5' deletion fragments were linked to the reporter gene beta-glucuronidase (GUS) as transcriptional fusions, and the expression of these chimeric genes was analyzed in leaf tissue. Sequences 111 base pairs upstream of the transcriptional start site were not able to drive the GUS expression over background levels, whereas sequences between -111 and -571 showed a slightly higher activity with equal levels of transcription in wounded and nonwounded tissue. The addition of further upstream sequences (-571 to -1022) enhanced the level of expression by a factor between 13 and 370. The expression driven by this fragment was inducible by a factor of twofold to ninefold by wounding. Histochemical analysis of different tissue from transgenic plants that contain wun1-GUS fusions demonstrates wound-inducible and cell-specific wun1 promoter activity in plants containing the -1022-base pair fragment. The location of GUS activity appears to be cell-specific, being highest in epidermal cells of leaves and stems and lower in vascular cells. Activity was reduced to levels that could not be detected by histochemical staining in leaves, stems, and roots of plants containing the deleted promoter fragments. Plants that contain the different deletion constructs and plants that carry the -1022-base pair fragment show high expression in anthers and pollen grains that could not be stimulated by wounding.

Objective. We have previously shown that human articular chondrocytes synthesize large amounts of interleukin-6 (IL-6) upon stimulation with proinflam-matory cytokines and that they express the IL-6 receptor. The present study was undertaken to analyze whether different IL-6-type cytokines can induce synthesis of the acute-phase protein 1-antitrypsin in human articular chondrocytes. Methods. Chondrocytes from human articular cartilage, cultured in agarose, were stimulated with IL-6-type cytokines. Total RNA was isolated and analyzed by Northern blotting. Levels of 1-antitrypsin protein were determined by enzyme immunoassay. Results. Stimulation of chondrocytes with on-costatin M (OSM) and IL-6 led to a 5-10-fold increase in 1-antitrypsin synthesis. This increase was dose and time dependent. Furthermore, OSM and IL-6 induced IL-6 synthesis in chondrocytes, resulting in an autocrine amplification loop. Conclusion. Our data strongly suggest the existence of a local acute-phase response in the joint.