No abstract

Previously, we demonstrated that IL-8 induces rapid mobilization of hematopoietic progenitor cells (HPC) from the bone marrow of rhesus monkeys. Because activation of neutrophils by IL-8 induces the release of gelatinase B (MMP-9), which is involved in the degradation of extracellular matrix molecules, we hypothesized that MMP-9 release might induce stem cell mobilization by cleaving matrix molecules to which stem cells are attached. Rhesus monkeys were treated with a single i.v. injection of 0.1 mg͞kg human IL-8, which resulted in a 10-to 100-fold increase in HPC within 30 min after injection. Zymographic analysis revealed a dramatic instantaneous increase in the plasma levels of MMP-9, followed by the increase in circulating HPC. Enzyme levels decreased at 2 h after injection of IL-8, simultaneously with the decrease in the numbers of circulating HPC. To test the hypothesis that MMP-9 induction was involved in HPC mobilization, rhesus monkeys were treated with a highly specific inhibitory monoclonal anti-gelatinase B antibody. Anti-gelatinase B at a dose of 1-2 mg͞kg completely prevented the IL-8-induced mobilization of HPC, whereas a dose of 0.1 mg͞kg had only a limited effect. Preinjection of inhibitory antibodies did not preclude the IL-8-induced production and secretion of MMP-9. Pretreatment with an irrelevant control antibody did not affect IL-8-induced mobilization, showing that the inhibition by the anti-gelatinase B antibody was specific. In summary, IL-8 induces the rapid systemic release of MMP-9 with concurrent mobilization of HPC that is prevented by pretreatment with an inhibitory anti-gelatinase B antibody, indicating that MMP-9 is involved as a mediator of the IL-8-induced mobilization of HPC.

Human neutrophils were found to release a 91 -kDa gelatinase that is serologically related to tumor-derived gelatinolytic enzymes, as evidenced by immunoprecipitation. In order to identify the neutrophil gelatinase, the activity in conditioned medium from human neutrophil suspensions was purified by affinity chromatography on a gelatin substrate. The 91-kDa active enzyme was further separated from other stainable protein bands by classical SDSjPAGE and blotting to a solid support. Amino-terminal sequence analysis of blotted proteins showed that the 91-kDa enzyme is a truncated form of tumor-derived 92-kDa gelatinase (type IV collagenase), lacking eight residues at the NH2-terminus. Sequence analysis of enzymatically inactive cleavage products of this neutrophil gelatinase demonstrated that the gelatin-binding part of the molecule is restricted to the amino-terminal third.Exocytosis of gelatinase-containing granules from neutrophils occurred spontaneously within 6 h after neutrophil plating. When the cells were triggered with the phorbol ester phorbol 12-myristate 13-acetate, a strong secretagogue, rapid gelatinase release was observed. When granulocytes were stimulated with the neutrophilactivating peptide interleukin-8, maximal exocytosis occurred within 1 h. The almost immediate release of neutrophil gelatinase after stimulation of the cells with a chemotactic factor might play a key role in remodeling of the extracellular matrix during granulocyte movement in response to chemotactic stimuli.Granulocytes, in particular neutrophils, play an important role in destroying pathogenic microorganisms as part of the aspecific immune response during acute inflammation. They are also involved in a variety of chronic inflammatory processes occurring in, for example, autoimmune diseases. Destruction and remodeling of connective tissues in acute and chronic inflammatory disorders is regulated by cytokines and mediated through the combined effect of molecules such as reactive oxidants and various proteolytic enzymes. Some cytokines, e. g. interleukin-I (IL-I), have been reported to induce proteinase secretion in the inflamed tissue directly, whereas others such as locally produced interleukin-8 (IL-8) attract leukocytes which can then release an array of proteolytic enzymes [l]. Among these are leukocytic serine proteinases, e.g. elastase and plasminogen activators and the metalloproteinases collagenase and gelatinase. These enzymes are actively synthesized and secreted or released from preformed granules by the infiltrating leukocytes [2 -41.Human polymorphonuclear leukocytes have been shown to secrete a specific type of collagenase not found in other cell types. This fully characterized protein has a molecular mass of 75 kDa and degrades native collagen types I, I1 and 111 [5 -71. A less well characterized gelatinase of molecular mass 92 kDa has also been detected in polymorphonuclear leukocytes [8, 91. This enzyme degrades denatured collagens (gelatins) and types IV and V collagen. Both enzymes have been shown to be stor...

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A new aspartic protease inhibitory chemotype bearing a 2-amino-3,4-dihydroquinazoline ring was identified by high-throughput screening for the inhibition of BACE-1. X-ray crystallography revealed that the exocyclic amino group participated in a hydrogen bonding array with the two catalytic aspartic acids of BACE-1 (Asp(32), Asp(228)). BACE-1 inhibitory potency was increased (0.9 microM to 11 nM K(i)) by substitution into the unoccupied S(1)' pocket.

Gelatinases in inflammatory demyelinating diseases of the central nervous system (CNS) were studied using actively induced experimental autoimmune encephalomyelitis (EAE) in mice as a model system. Clinical disease scores correlated in time and in intensity with pathology parameters such as cytosis in the cerebrospinal fluid (CSF), inflammatory infiltrates, and demyelination in the CNS. Zymographic analysis was employed to measure gelatinases A and B in the CSF from individual animals. According to their apparent molecular weight (MW), gelatinases A and B appeared with a MW of 65 and 95 kDa, respectively. The 65 kDa form was present in all samples, even in those derived from non-induced animals, whereas the 95 kDa form was present only in samples from animals developing EAE. The levels of 95 and 65 kDa gelatinase correlated with the CSF cytosis. In vitro digestion of myelin basic protein (MBP) with gelatinase B and analysis of the cleavage products by protein sequence analysis pinpointed two cleavage sites in conserved regions of MBP. Gelatinase production within the CNS may constitute an important pathogenic mechanism for both the disruption of the blood-brain barrier and the destruction of myelin, as observed in several neuroinflammatory disorders.

Akt (also known as PKB or RAC-PK) is an intracellular serine/threonine kinase involved in regulating cell survival. Although this makes it a promising target for the discovery of drugs to treat human cancer, a complicating factor may be the role played by Akt in insulin signalling. Two human isoforms, Akt-1 and Akt-2, have been described previously and a third isoform has been identified in rats (here termed Akt-3, but also called RAC-PK-g or PKB-g). We describe the identification of the corresponding human isoform of Akt-3. The gene encoding human Akt-3 was localized to chromosome 1q43±44. The predicted protein sequence is 83% identical to human Akt-1 and 78% identical to human Akt-2, and contains a pleckstrin homology domain and a kinase domain. In contrast to the published rat Akt-3 isoform, human and mouse Akt-3 also possess a C-terminal`tail' that contains a phosphorylation site (Ser472) thought to be involved in the activation of Akt kinases. In addition to phosphorylation of Ser472, phosphorylation of Thr305 also appears to contribute to the activation of Akt-3 because mutation of both these residues to aspartate increased the catalytic activity of Akt-3, whereas mutation to alanine inhibited activation. Akt-3 activity could be inhibited by the broad spectrum kinase inhibitor staurosporine and by the PKC inhibitor Ro 31-8220, but not by other PKC or PKA inhibitors tested. Although Akt-3 is expressed widely, it is not highly expressed in liver or skeletal muscle, suggesting that its principle function may not be in regulating insulin signalling. These observations suggest that Akt-3 is a promising target for the discovery of novel chemotherapeutic agents which do not interfere with insulin signalling.

Gelatinase B is a matrix metalloproteinase (MMP-9) involved in tissue remodeling, development, cancer, and inflammation. Neutrophils produce three major forms of (pro)gelatinase B: 92 kDa monomers, homodimers, and complexes of gelatinase B covalently bound to neutrophil gelatinase B-associated lipocalin (NGAL). In contrast to the case for other proteinases, little information about the glycosylation of any natural human MMP is available. Here, both gelatinase B and NGAL were purified from human peripheral blood neutrophils, and the entire contents of the released N- and O-glycan pools were analyzed simultaneously using recently developed high-performance liquid chromatography-based technology. The results are discussed within the context of the domain structure of gelatinase B and a molecular model of NGAL based on data from this study and the three-dimensional nuclear magnetic resonance (NMR) structure of the protein. More than 95% of the N-linked glycans attached to both gelatinase B and NGAL were partially sialylated, core-fucosylated biantennary structures with and without outer arm fucose. The O-linked glycans, which were estimated to comprise approximately 85% of the total sugars on gelatinase B, mainly consisted of type 2 cores with Galbeta1,4GlcNAc (lactosamine) extensions, with or without sialic acid or outer arm fucose. This paper also contains the first report of O-linked glycans attached to NGAL. Although both proteins were isolated from neutrophils and contained O-linked glycans mainly with type 2 cores, the glycans attached to individual serine/threonine residue(s) in NGAL were significantly smaller than those on gelatinase B. In contrast to NGAL, gelatinase B contains a region rich in Ser, Thr, and Pro typical of O-glycosylated mucin-like domains.