Isolation of RNA from small human articular cartilage specimens allows quantification of mRNA expression levels in local articular cartilage defects.

Human adult cartilage is an inherently difficult tissue from which to isolate RNA. The RNA isolation techniques described so far have generally only been successfully applied to the isolation of RNA from larger amounts of cartilage. However, it is important to be able to analyse focal cartilage lesions in order to understand the local processes in the cartilage degeneration process. Therefore, we have developed a protocol for isolating RNA directly from as little as 10 mg wet weight of cartilage followed by quantitative PCR analysis. We were able to analyse the expression levels of several genes in parallel including aggrecan and type II collagen.

The black-pearl gene of Drosophila defines a novel conserved protein family and is required for larval growth and survival.

Using a transposon insertion line of the Drosophila Genome Project we have cloned the black-pearl gene (blp), analyzed cDNA clones, generated various mutants, and characterized their phenotypes. The blp gene codes for a protein of 15.7 kDa calculated molecular weight that has been conserved from yeast to plants and mammals with high homology. A domain of these new proteins shows distant similarity to DnaJ domains indicating a functionally relevant interaction with other proteins. The P element insertion in line P1539 lies within the 5' untranslated leader of the black-pearl gene. Flies homozygous for this insertion are semi-lethal, escapers produce very few offspring and show melanotic inclusions in the hemocoel ('black pearls') similar to various melanotic 'tumor' mutants. Two small deletions confined to the blp gene and two EMS-induced mutations are homozygous lethal. These null mutants appear normal up to a prolonged first instar larval stage but fail to grow and die. Thus in Drosophila the blp gene is specifically required for larval growth. The evolutionary conservation in both unicellular and multicellular organisms suggests for the new protein family described here a fundamental role in cell growth.

Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology.

OBJECTIVE: To understand changes in gene expression levels that occur during osteoarthritic (OA) cartilage degeneration, using complementary DNA (cDNA)-array technology. METHODS: Nine normal, 6 early degenerated, and 6 late-stage OA cartilage samples of human knee joints were analyzed using the Human Cancer 1.2 cDNA array and TaqMan analysis. RESULTS: In addition to a large variability of expression levels between different patients, significant expression patterns were detectable for many genes. Cartilage types II and VI collagen were strongly expressed in late-stage specimens, reflecting the high matrix-remodeling activity of advanced OA cartilage. The increase in fibronectin expression in early degeneration suggests that fibronectin is a crucial regulator of matrix turnover activity of chondrocytes during early disease development. Of the matrix metalloproteinases (MMPs), MMP-3 appeared to be strongly expressed in normal and early degenerative cartilage and down-regulated in the late stages of disease. This indicates that other degradation pathways might be more important in late stages of cartilage degeneration, involving other enzymes, such as MMP-2 and MMP-11, both of which were up-regulated in late-stage disease. MMP-11 was up-regulated in OA chondrocytes and, interestingly, also in the early-stage samples. Neither MMP-1 nor MMP-8 was detectable, and MMP-13 and MMP-2 were significantly detectable only in late-stage specimens, suggesting that early stages are characterized more by degradation of other matrix components, such as aggrecan and other noncollagenous molecules, than by degradation of type II collagen fibers. CONCLUSION: This investigation allowed us to identify gene expression profiles of the disease process and to get new insights into disease mechanisms, for example, to develop a picture of matrix proteinases that are differentially involved in different phases of the disease process.

Effective isolation of high-quality total RNA from human adult articular cartilage.

The isolation of large quantities of good-quality RNA from human articular cartilage has been a long-standing problem for researchers working with human articular cartilage. In this paper we report a protocol which we have developed based on the Qiagen RNeasy procedure to produce high yields of purified, DNA-free RNA from normal and osteosteoarthritic human articular cartilage. The average yield of RNA was 8.39 microg/g (n = 59) for normal and 6.69 microg/g (n = 58) for osteoarthritic cartilage (average ratio OD 260/280 = 1.8-1.9). Quantitative PCR, cDNA array technology, and Northern blot analysis were used to verify the quality of the RNA.

Up-regulation of MDC15 (metargidin) messenger RNA in human osteoarthritic cartilage.

OBJECTIVE: The aim of the study was to investigate the messenger RNA (mRNA) expression of the disintegrin metalloproteinase MDC15 (metargidin, or ADAM-15) in normal and osteoarthritic (OA) articular cartilage. METHODS: In situ hybridization experiments and reverse transcription-polymerase chain reaction (RT-PCR) were performed on tissue samples of adult normal and OA articular cartilage. RESULTS: MDC15 mRNA could be detected in normal articular cartilage by RT-PCR using tissue-extracted total RNA as a template. However, the mRNA level remained below the sensitivity of in situ hybridization. In contrast, in situ hybridizations of OA cartilage revealed an intense staining with the MDC15-specific riboprobes. The extension of the analysis to chondrosarcomas showed a strong up-regulation of MDC15 mRNA in these malignant transformed cells. CONCLUSION: Our results demonstrate a markedly strong up-regulation of MDC15 in adult OA and neoplastic cartilage compared with adult normal articular cartilage, indicating a potential role of the disintegrin metalloproteinase in cartilage remodeling.