Isao Nakanishi

Inactivation of tissue inhibitor of metalloproteinases by neutrophil elastase and other serine proteinases

Tissue inhibitor of metalloproteinases (TIMP) from cultured bovine dental pulp inhibits human rheumatoid synovial matrix metalloproteinase 3 (MMP‐3) with a stoichiometry of 1:1 on a molar basis. Among the serine proteinases examined, human neutrophil elastase, trypsin and α‐chymotrypsin destroyed the inhibitory activity of TIMP against MMP‐3 by degrading the inhibitor molecule into small fragments. In contrast, the inhibitory activity of TIMP was not significantly reduced by the actions of cathepsin G, pancreatic elastase and plasmin. These data indicate that neutrophils which infiltrate tissues in various inflammatory conditions may play an important role in regulating TIMP activity in vivo through the action of neutrophil elastase.

Amplification of c-myc, K-sam, and c-met in gastric cancers : detection by fluorescence in situ hybridization

Gene amplifications of c-myc, K-sam, and c-met were examined in cancer nuclei isolated from 154 primary gastric adenocarcinomas by fluorescence in situ hybridization (FISH) using cosmid probes for 8q24 (c-myc locus) and 7q31 (c-met), as well as a DNA probe for K-sam synthesized by PCR. The results were compared with those of Southern blot analysis. Dual-color FISH using gene locus and chromosome-specific probes detected gene amplifications of c-myc in 24 tumors (15.5%), c-met in 6 tumors (3.9%), and K-sam in 3 tumors (2.9%). The six tumors with c-myc amplification had also been found to have amplified c-erbB-2 in our previous study, and coamplification of c-myc and c-met was found in two other tumors. This technique also differentiated the amplified genes on the homogeneous staining region (HSR) and on double minute chromosomes (DMs) in metaphase spreads and interphase nuclei of cell lines established from poorly differentiated adenocarcinomas, KATO III, SNU 16, and HSC 39. Examination of FISH images of these cell lines suggested that the high-level amplifications of c-myc found in primary tumors occurred mainly on DM in four tumors and on HSR in one, and those of K-sam occured on DM in two tumors and on HSR in one. No high-level amplification of c-met was found. These high-level amplifications were also detected in formalin-fixed, paraffin-embedded tissues from primary gastric tumors and metastatic lymph nodes, in some of which heterogeneity of gene amplification was demonstrated within the same tumor. We conclude that FISH is an important tool for examining the proto-oncogene aberrations in intact cells in solid tumors.

Immunolocalization of matrix metalloproteinase 3 (stromelysin) in rheumatoid synovioblasts (B cells): correlation with rheumatoid arthritis.

Metalloproteinases produced by connective tissue cells may play a key part in the destruction of joints in rheumatoid arthritis. Matrix metalloproteinase 3 (MMP-3; stromelysin) capable of degrading cartilage proteoglycans and type IX collagen and of activating procollagenase was immunolocalised in hyperplastic synovial lining cells in rheumatoid synovium, but not in the cells of normal synovium. Cells responsible for synthesis of MMP-3 have the phenotype of synovioblasts (B cells) by immunoelectron microscopy, but not of phagocytic synovial macrophages (A cells). Cultured monolayer of rheumatoid synovial cells synthesises MMP-3 only under treatment with macrophage conditioned medium. Immunolocalisation of MMP-3 in rheumatoid synovium and cultured synovial cells was possible when the specimens were treated with a monovalent ionophore, monensin. These results suggest that MMP-3 is synthesised and secreted continuously without storage from hyperplastic synovioblasts stimulated by factor(s) derived from activated macrophages present in the synovium.

Collagens in human atherosclerosis. Immunohistochemical analysis using collagen type-specific antibodies.

This study represents a systematic analysis of the distribution of collagen types in human atherosclerotic lesions. Formalin-fixed, paraffin-embedded aortic tissues of 40 lesions from 16 different individuals ranging in age from 1 month to 84 years were examined immunohistochemically using antibodies to type I, III, IV, V, and VI collagens. Preembedding immunoelectron microscopy was used to simultaneously localize type V and VI collagens within the lesions. Localization of type III collagen was very similar to that of type I, and type VI collagen appeared together with these two types of collagen in the thickened intimas of all stages of the lesion. Type V collagen was not detected in either fatty streaks or the mild intimal thickening of the aortas of children. With advancing age and lesion progression, the immunoreactivity with anti-type V collagen antibody became more intense. Type IV collagen was detected in the basement membrane region of intimal cells. In advanced lesions thick deposits of type IV collagen were found around the elongated smooth muscle cells. Using immunoelectron microscopy, type V collagen was found to be localized to cross-banded collagen fibers, and type VI collagen was found to be localized to beaded filaments present throughout the interstitium of the thickened intima. These findings suggest that collagens preserve the pathophysiological and functional integrity of the vascular wall by providing mechanical support as well as assuring the proper interaction of cells during the formation of atherosclerotic lesions.

Activation of matrix metalloproteinase 3 (stromelysin) and matrix metalloproteinase 2 (‘gelatinase’) by human neutrophil elastase and cathepsin G

The ability of human neutrophil elastase and cathepsin G to activate matrix metalloproteinase 3 (MMP‐3 = stromelysin) and MMP‐2 (‘gelatinase’) purified from human rheumatoid synovial fibroblasts in culture was examined. The zymogen of MMP‐3 (proMMP‐3) was activated to full activity with elastase and cathepsin G by limited proteolysis of the molecule into two active forms of M r ∼ 45000 and M r ∼ 25000. In contrast, proMMP‐2 was not activated at all by these neutrophil serine proteinases, although it was degraded into small fragments. These data suggest that neutrophil elastase and cathepsin G may play an important role in the activation of proMMP‐3 in vivo in various inflammatory conditions, but proMMP‐2 may be activated in different ways.

Microvascular angiogenesis and apoptosis in the survival of free fat grafts.

Objectives/Hypothesis Autologous fat is an ideal material for augmentation in plastic surgery because of its minimal tissue reaction and easy availability, but its long‐term graft survival is somewhat unpredictable. This study was conducted to determine how fat grafts get their vascular supply from the recipient bed and why they keep reducing in volume and weight.

Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice.

Chronic intermittent injection of carbon tetrachloride (CCI4) for more than 10 weeks induced liver fibrosis in mice, as evidenced by positive Azan staining and increased intrahepatic collagen content. Preceding the onset of liver fibrosis, interleukin‐6 (IL‐6) gene expression was enhanced in liver and immunoreactive IL‐6 was detected in infiltrating inflammatory cells. To delineate the role of IL‐6 in this process, we treated IL‐6‐deficient mice with CCl4 in a similar manner for 12 weeks, after which fibrotic changes were less evident and serum albumin levels were lower in IL‐6‐deficient than wild‐type mice. Moreover, CCl4‐induced expression of transforming growth factor β1 and hepatocyte growth factor genes in liver was significantly reduced in IL‐6‐deficient mice. Thus, IL‐6 may be vitally involved in fibrotic changes and maintenance of serum albumin levels, partly by modulating intrahepatic expression of these cytokines. J. Leukoc. Biol. 66: 601–608; 1999.

Induction and stimulation of 92-kDa gelatinase / type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumor necrosis factor α

Production of a 92-kDa gelatinase/type IV collagenase and tissue inhibitor of metalloproteinases (TIMP) was investigated with human sarcoma cell lines. Among the cytokines and growth factors examined, only human recombinant tumor necrosis factor alpha (TNF alpha) induced and stimulated the proteinase with concomitant increase in TIMP expression, but matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) expression was unchanged. These data suggest that gene expression of the two metalloproteinases is regulated in a different fashion and TNF alpha may be important to allow cancer cells to be more invasive and metastatic.

Expression of glial fibrillary acidic protein gfap in peripheral nerve sheath tumors a comparative study of immunoreactivity of gfap vimentin s 100 protein and neurofilament in 38 schwannomas and 18 neurofibromas

Immunoreactivity of glial fibrillary acidic protein (GFAP) in 38 schwannomas and 18 neurofibromas was evaluated and compared with the reactivity of vimentin, S-100 protein, and neurofilament protein. All cases were positive for vimentin and S-100 protein. GFAP was positively stained in the neoplastic cells of 15 of 38 schwannomas (38%) and in two of 18 neurofibromas (11%). The extensively stained GFAP-positive tumors tended to be deeply situated in the body. The GFAP-positive cells were usually spindle-shaped and appeared preferentially in the perivascular region of hyalinized, thick blood vessels.

A complementary DNA for human choline acetyltransferase induces two forms of enzyme with different molecular weights in cultured cells

Complementary DNA (cDNA) clones containing the entire coding region of human choline acetyltransferase (ChAT) were isolated from cDNA libraries prepared from the autopsied spinal cord. In the human cDNA, the ATG codon assigned to the putative initiation codon for pig, rat and mouse ChAT cDNAs was replaced by ACG. The human cDNA contained an in-frame ATG codon 324 nucleotides upstream of the ACG codon. Therefore, human ChAT cDNA should code for a 748 amino acid polypeptide of 82.6 kDa. This deduced molecular weight was larger than that of ChAT protein purified from the human brain and placenta (64-70 kDa). The human ChAT cDNA containing the entire coding region was ligated to an expression vector and introduced into African green monkey kidney (COS) cells and Chinese hamster ovary (CHO) cells. The cells expressed high ChAT activity and produced two protein bands immunostained with an antibody to monkey ChAT. The molecular weight of the proteins was estimated to be approximately 70 and 80 kDa by polyacrylamide-SDS gel electrophoresis. When partial cDNAs that lacked the first ATG but contained the replaced ACG codon were introduced into COS cells, the cells expressed moderate ChAT activity and an immunoreactive protein band of 70 kDa. These results indicate that translation of human ChAT mRNA starts at two sites and produces two enzyme proteins with different molecular weights. It might be that the larger form of ChAT molecule is an enzyme precursor for processing or that the N-terminal extrapeptide is needed for subcellular localization of the enzyme.