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,

Re-establishing blood flow to ischaemic tissues causes greater injury than that induced during the ischaemic period. This type of tissue injury, reperfusion injury, is involved in frostbite, multiple organ failure after hypovolaemia and in myocardial infarction. Depletion of neutrophils alleviates reperfusion injury, implying a causal role of neutrophil infiltration. Among members of the recently discovered family of chemotactic cytokines (chemokines), interleukin-8 (IL-8) is a major neutrophil chemotactic and activating factor produced by various types of human cells. We investigated its pathophysiological role in a rabbit model of a lung reperfusion injury. Reperfusion of ischaemic lung caused neutrophil infiltration and destruction of pulmonary structure, as well as local production of IL-8. Furthermore, the administration of a neutralizing monoclonal antibody against IL-8 prevented neutrophil infiltration and tissue injury, proving a causal role of locally produced IL-8 in this model.

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During systematic analysis of nonbonded contacts in protein-ligand complexes derived from crystal structures in the Protein Data Bank, Cl-p interactions have been found, not only in the well-documented serine proteases but also, to a lesser extent, in other proteins. From geometric analysis of such Cl-p interactions in the crystal structures, two distinct geometries were found: the ''edge-on'' approach of a Cl atom to a ring atom or C-C bond and the ''face-on'' approach toward the ring centroid with an average interatomic distance of 3.6 Å . High-level ab initio calculations using benzene-chlorohydrocarbon model systems elucidated that the calculated Cl-p interaction energy is À2.01 kcal/mol, and the dispersion force is the major source of attraction. We also discussed the geometric flexibility in Cl-p interactions and a relationship between the intensity of the p density in an aromatic ring and the interaction position of the Cl atom.

Aims: To investigate collagen remodelling in the interstitium of the heart in patients with diabetes. Methods: Immunohistochemical study of the biopsied myocardium using type specific anticollagen antibodies (I, III, IV, V, VI) was performed in 12 patients with non-insulin dependent diabetes mellitus and six non-diabetic patients. There was no history of hypertension or coronary artery stenosis in any of the patients. Results: Noticeable accumulations of collagen types I, III, andVI in the myocardial interstitium were recognised in both groups, but little accumulation of types IV or V was found. Types I and III mainly stained in the perimysium and perivascular region, while type VI predominantly stained in the endomysium. There was no disease specific accumulation of collagen in diabetes mellitus. The percentage of total interstitial fibrosis in the myocardium was significantly higher in the diabetic group than in the control group (p < 0.05). Although the percentages of coliagen types I and VI did not differ between the two groups, the percentage type of III was significantly higher in the diabetic group than in the controls (p < 0-01). Conclusions: Collagen remodelling mainly as a result of an increase in collagen type III in the perimysium and perivascular region, occurs in the hearts of patients with diabetes.