Kojiro Takahashi

DNA preservation using diamond chips

Abstract The highest density DNA chip reported to date was developed using a DNA solidification technique for vertical binding to the surface of a chemical vapor deposition (CVD) diamond chip. The covalently bound oligonucleotide was approximately 42 pmol (∼2.5×10 13 molecules) per 9 mm 2 of chip surface. Using the oligonucleotide as a linker, large DNA molecules (21 kbp fragment from λ-phage DNA) were covalently bound in the amount of approximately 2.6 fmol (∼1.6×10 9 molecules) per 9 mm 2 of chip surface. To test the potential of the technique for repeated utilization of one piece of the DNA chip, PCR enhancement of a 500 bp region within the 21 kbp λ-DNA fragment was applied over 50 times. The results suggest that a diamond DNA chip is excellent for the preservation of limited and/or valuable gene samples. Furthermore, the technique of high density DNA solidification to a CVD diamond chip will be useful for DNA diagnosis in the future.

Change in cellular localization of a rheumatoid arthritis-related antigen (RA-A47) with downregulation upon stimulation by inflammatory cytokines in chondrocytes.

We previously isolated a rheumatoid arthritis‐related antigen (RA‐A47) protein that had reactivity with RA sera from a human chondrosarcoma‐derived chondrocytic cell line, HCS‐2/8. Sequencing analysis of ra‐a47 cDNA revealed RA‐A47 as a product of the colligin‐2 gene, which is also known as the human heat shock protein (HSP) 47 gene. Expression of hsp47 has been shown to be cooperatively altered with that of collagen genes upon stimulation. In this study, it was confirmed that the mRNA expression of ra‐a47 and COL2A1, a type II collagen gene, was upregulated on stimulation with transforming growth factor (TGF) β in chondrocytes. However, in contrast, inflammatory cytokines such as tumor necrosis factor (TNF) α, interferon (IFN) β, and interleukin (IL)‐6 downregulated the expression of ra‐a47 mRNA, whereas the expression of COL2A1 mRNA was not repressed, or even upregulated, in HCS‐2/8 cells. Of note, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP)‐9 mRNAs were strongly stimulated by TNFα. We also found that cell‐surface type II collagen disappeared upon such a stimulation, suggesting that decrement of RA‐A47 may inhibit the secretion of type II collagen and lead to its accumulation inside the cells. RA‐A47 was detected in the cultured medium of TNFα‐treated HCS‐2/8 cells and of IL‐1‐treated rabbit chondrocytes by Western blot analysis. Under the same conditions, RA‐A47 was detected on the cell surface by immunofluorescence staining. These findings demonstrate that the RA‐A47 chaperone protein is specifically downregulated, causing the intracellular accumulation of unsecretable type II collagen, while the extracellular matrix (ECM) is degraded by MMPs and iNOS through the stimulation of chondrocytes by TNFα. The altered localization of RA‐A47 to the surface or outside of cells may represent the mechanism for the recognition of RA‐A47 as an autoantigen during rheumatoid arthritis. J. Cell. Physiol. 186:268–281, 2001.