Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDSjPAGE with M , = 72000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-AspVal-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with M , = 67 000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-GlnIle. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,lO-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.Connective tissue cells in culture synthesize and secrete tissue collagenase (matrix matalloproteinase 1 ; EC 3.4.24.7) and at least two other matrix metalloproteinases (MMP-2 and MMP-3) that digest various components of the extracellular matrix. The deduced primary sequences of these three MMPs from their cDNA sequencing have indicated that they are structurally related [l -61, but the substrate specificities of these enzymes are distinct. MMP-1 digests collagen types I, I1 and I11 at specific sites and generates characteristic threequarter and one-quarter fragments of the native collagen molecules [7 -91. It has also been shown to digest collagen typesCorrespondence to H. Nagase,

BackgroundTo compare the frequency of Y-chromosome microdeletions in Japanese and African azoospermic and oligozoospermic men and describe embryo characteristics and reproductive outcome following in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI).MethodsOur study was performed prospectively at two centers, a private IVF clinic and a university hospital. Japanese and African (Tanzanian) men with nonobstructive azoospermia (NOA) and oligozoospermia (concentration < 5 × 106 /ml) were evaluated for Y-chromosome microdeletions (n = 162). Of the 47 men with NOA, 26 were Japanese and 21 were Africans. Of the 115 men with oligozoospermia, 87 were Japanese and 28 were Africans. Reproductive outcomes of patients with Y-chromosome microdeletions were then compared with those of 19 IVF+ICSI cycles performed on couples with Y-chromosome intact males/tubal factor infertility which served as a control group.ResultsSeven azoospermic and oligozoospermic patients had Y-chromosome deletions; the total number of deletions in the AZFc region was five. There was only one deletion in the AZFa region and one complete deletion involving all three regions (AZFa, b, and c) within AZF. In our study population, microdeletion frequency among Japanese men was 6.2% (95% CI, 4.25% – 14.45%), whereas no deletions were identified in the African group (95% CI, 0.0% – 7.27%). The difference between the two groups was not statistically significant, however. Embryos derived from ICSI utilizing sperm with Y-chromosome microdeletion showed reduced rates of fertilization, blastocyst development, implantation, and pregnancy compared to the Y-chromosome intact group, although these observed differences were not statistically significant.ConclusionThe observed frequency of Y-chromosome microdeletion was 6.2% among Japanese azoospermic and oligozoospermic males; no microdeletions were identified among our African study patients. In this population of couples undergoing IVF+ICSI, there was no statistically significant difference in embryo characteristics or pregnancy outcome between patients with Y-chromosome microdeletion and those with an intact Y-chromosome.

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Objective: The aim of the present study was to show variant species of ADAM15 and unique Src homology 3 (SH3)-binding motifs, which strongly bound Src family proteins compared with ADAM15. Methods and Results: RT-PCR using primers for the cytoplasmic domain revealed the presence of different species, designated ADAM15v1 and ADAM15v2, which had characteristic SH3-binding class I and class II motifs. The mRNA of ADAM15v1 and ADAM15v2 was mainly found in peripheral blood mononuclear cells, T lymphocytes and monocytic cell lines. ADAM15v2 protein interacted more strongly with the Src family proteins Lck and Hck than did ADAM15 protein, as examined by pull-down analysis and immunoprecipitation followed by immunoblot analysis. The binding with Lck and Hck was enhanced by the phosphorylation of ADAM15v2 protein. Conclusions: These results suggest that the cytoplasmic domain of ADAM15v2 strongly interacts with Lck and Hck and regulates leukocyte function.

CD156 (ADAM8) is part of the ADAM family of proteins with the catalytic site consensus sequence of metalloprotease and disintegrins. To examine the role of CD156 in vivo, we generated mutant CD156 (eCD156) transgenic mice expressing the ectodomain of CD156 under the control of the α1-antitrypsin (AT) promoter. One of the transgenic mice designated ATMS2-TG18 expressed a 1.84 kb mRNA which was predicted to be a truncated CD156. The expression of the transgenic CD156 mRNA in ATMS2-TG18 mice was abundant in the liver and slight in kidney. Turpentine oil (TO) and lipopolysaccharide (LPS) markedly upregulated the expression. Soluble CD156 (sCD156) was produced constitutively, and increased after the treatment with TO. Casein-induced peritoneal leukocyte infiltration was significantly less extensive in ATMS2-TG18 than non-transgenic mice. The expression of L-selectin in neutrophils (PMN) from peripheral blood leukocytes (PBL) was more strongly downregulated in ATMS2-TG18 than non-transgenic mice, suggesting that L-selectin in PMN from ATMS2-TG18 mice was shed by sCD156. In contrast, oxazolone (Ox)-induced contact hypersensitivity reactions (CHR) were more marked in ATMS2-TG18 than non-transgenic mice. The expression of E-selectin mRNA was detected in inflammatory skin sites from ATMS2-TG18, but not non-transgenic mice, suggesting that sCD156 may activate the endothelial cells and lead to the upregulation of E-selectin. These results suggest that CD156 regulates leukocyte infiltration directly or indirectly.