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Spleen tyrosine kinase (Syk) tyrosine kinase plays essential roles in receptors for Fc portion of immunoglobulins and B cell receptor complex signaling in various inflammatory cells; therefore, inhibitors of Syk kinase may show potential as antiasthmatic/allergic therapeutics. We identified 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride (BAY 61-3606), a potent (K i ϭ 7.5 nM) and selective inhibitor of Syk kinase. BAY 61-3606 inhibited not only degranulation (IC 50 values between 5 and 46 nM) but also lipid mediator and cytokine synthesis in mast cells. BAY 61-3606 was highly efficacious in basophils obtained from healthy human subjects (IC 50 ϭ 10 nM) and seems to be at least as potent in basophils obtained from atopic (high serum IgE) subjects (IC 50 ϭ 8.1 nM). B cell receptor activation and receptors for Fc portion of IgG signaling in eosinophils and monocytes were also potently suppressed by BAY 61-3606. Oral administration of BAY 61-3606 to rats significantly suppressed antigen-induced passive cutaneous anaphylactic reaction, bronchoconstriction, and bronchial edema at 3 mg/kg. Furthermore, BAY 61-3606 attenuated antigen-induced airway inflammation in rats. Based on these anti-inflammatory effects of BAY 61-3606 both in vitro and in vivo, it was demonstrated that Syk may play a very critical role in the pathogenesis of allergic reactions.

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Cathepsin K is a recently identified cysteine protease which is abundantly and selectively expressed in osteoclasts. To evaluate the contribution of cathepsin K to bone resorption processes, we investigated the effect of cathepsin K antisense phosphothiorate oligodeoxynucleotide (S-ODN) on the bone-resorbing activity of osteoclasts. Rabbit osteoclasts were cultured on dentine slices for 24 h in the presence or absence of antisense S-ODN in a medium containing 100 nM Tfx TM -50, polycationic liposome, as a carrier of the S-ODN. Uptake of the S-ODN by osteoclasts was confirmed microscopically using fluorescein-labeled S-ODN. The treatment with antisense significantly decreased the amount of cathepsin K protein in osteoclasts. The antisense inhibited the osteoclastic pit formation in a concentration-dependent fashion. At 10 M the antisense reduced the total pit number and area and average pit depth by 46, 52, and 30%, respectively. The sense and mismatch SODNs, which were used as negative controls, had no effect on either the cathepsin K protein level or the pit formation. A nonspecific cysteine protease inhibitor, E-64, also reduced pit formation in a concentration-dependent manner with maximum reductions at 1 M of 46, 48, and 35% in the above pit parameters. The inhibitory effect of the antisense almost equal to that of E-64 demonstrates that cathepsin K is a cysteine protease playing a crucial role in osteoclastic bone resorption.Bone tissue is a composite matrix comprising of hydroxyapatite and fibrous proteins (mainly Type I collagen) and is constantly subjected to a cycle of bone resorption and bone formation (1). Bone resorption is mainly carried out by osteoclasts which are multinucleate giant cells. In osteoclastic bone resorption, demineralization, in which osteoclasts release protons to solubilize the inorganic salt (2), is followed by the degradation of the protein fibers with cysteine proteases (1, 3). The involvement of the cysteine proteases has been verified in both in vitro and in vivo studies showing that various types of cysteine protease inhibitors reduce bone resorption (1,(3)(4)(5)(6)(7)(8)(9)(10). From studies based on substrate preference, inhibitor preference, and immunoreactivity, the cathepsins L and B were suggested to be responsible for osteoclastic bone resorption processes (8 -14).Recently, several research laboratories (including our own) have successfully cloned cDNAs for a novel cysteine protease, namely cathepsin K, from rabbit and human cDNA libraries (15)(16)(17)(18)(19), and its role in bone resorption has been the focus of recent attention. Human cathepsin K is highly and selectively expressed in osteoclasts (16 -21); in fact its expression level is much greater than those of cathepsins B, L, and S (20, 21). Brömme et al. (22) and Bossard et al. (23) showed that cathepsin K expresses a potent proteolytic activity against Type I collagen. Saneshige et al. (24) demonstrated that retinoic acid, a vitamin A metabolite, both up-regulates the gene expression of cathepsin K in ost...

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BackgroundNectin-2 is a Ca2+-independent cell-cell adhesion molecule that is one of the plasma membrane components of adherens junctions. However, little has been reported about the involvement of Nectin-2 in cancer.MethodsTo determine the expression of Nectin-2 in cancer tissues and cancer cell lines, we performed gene expression profile analysis, immunohistochemistry studies, and flow cytometry analysis. We also investigated the potential of this molecule as a target for antibody therapeutics to treat cancers by generating and characterizing an anti-Nectin-2 rabbit polyclonal antibody (poAb) and 256 fully human anti-Nectin-2 monoclonal antibodies (mAbs). In addition, we tested anti-Nectin-2 mAbs in several in vivo tumor growth inhibition models to investigate the primary mechanisms of action of the mAbs.ResultsIn the present study, we found that Nectin-2 was over-expressed in clinical breast and ovarian cancer tissues by using gene expression profile analysis and immunohistochemistry studies. Nectin-2 was over-expressed in various cancer cell lines as well. Furthermore, the polyclonal antibody specific to Nectin-2 suppressed the in vitro proliferation of OV-90 ovarian cancer cells, which express endogenous Nectin-2 on the cell surface. The anti-Nectin-2 mAbs we generated were classified into 7 epitope bins. The anti-Nectin-2 mAbs demonstrated antibody-dependent cellular cytotoxicity (ADCC) and epitope bin-dependent features such as the inhibition of Nectin-2-Nectin-2 interaction, Nectin-2-Nectin-3 interaction, and in vitro cancer cell proliferation. A representative anti-Nectin-2 mAb in epitope bin VII, Y-443, showed anti-tumor effects against OV-90 cells and MDA-MB-231 breast cancer cells in mouse therapeutic models, and its main mechanism of action appeared to be ADCC.ConclusionsWe observed the over-expression of Nectin-2 in breast and ovarian cancers and anti-tumor activity of anti-Nectin-2 mAbs via strong ADCC. These findings suggest that Nectin-2 is a potential target for antibody therapy against breast and ovarian cancers.

A mutant form of L-lactate oxidase (LOX) from Aerococcus viridans in which alanine 95 was replaced by glycine was constructed as a mimic of L-lactate monooxygenase but proved instead to be a mimic of the long chain ␣-hydroxyacid oxidase from rat kidney. A95G-LOX keeps oxidase activity with L-lactate at the same level as wild type LOX but has much enhanced oxidase activity with longer chain L-␣-hydroxyacids, ␣-hydroxy-n-butyric acid, ␣-hydroxy-n-valeric acid, etc., and also the aromatic ␣-hydroxyacid, L-mandelic acid. Kinetic analysis of the activity with these substrates indicates that the reduction of the enzyme bound flavin by substrates is the rate-limiting step in A95G-LOX. The affinity of pyruvate for the reduced enzyme is increased, and sulfite binding to the oxidized enzyme is weaker in A95G-LOX than in native enzyme. Wild type LOX stabilizes both the neutral and anionic flavin semiquinones with a pK a of 6.1, but A95G LOX stabilizes only the anionic semiquinone form. These results strongly suggest that the environment around the N5-C4a region of the flavin isoalloxazine ring is changed by this mutation.␣-Hydroxyacid oxidizing enzymes, a family of flavoprotein enzymes, share a series of common characteristics. Within the family, the crystal structures of glycolate oxidase and flavocytochrome b 2 have been solved by x-ray diffraction studies (1-4). The reaction mechanisms of L-lactate monooxygenase from Mycobacterium smegmatis and flavocytochrome b 2 from Saccharomyces cerevisiae have been studied extensively (5, 6). L-Lactate monooxygenase utilizes L-lactate and other L-␣-hydroxyacids but is unique in accelerating the oxidative decarboxylation of the products, pyruvate (or corresponding keto acid) and hydrogen peroxide, to acetic acid (or corresponding carboxylic acid), carbon dioxide, and water. New members of this flavoenzyme family have been described including those utilizing aromatic and bulky substrates like mandelate and long chain ␣-hydroxyacids (7-9).We are studying one of the new enzymes of this family, L-lactate oxidase from Aerococcus viridans (10). This enzyme utilizes L-lactate and oxygen as L-lactate monooxygenase does but forms pyruvate and hydrogen peroxide as the final products instead of catalyzing the oxidative decarboxylation reaction. We believe that lactate oxidase is an ideal enzyme for studying the reaction mechanism of this enzyme family because of its considerable stability, and it is also a useful enzyme for the construction of a lactate sensor for biological applications.By analogy with the crystal structures of glycolate oxidase and flavocytochrome b 2 , it has been pointed out that glycine 99 in L-lactate monooxygenase is unique in the family of enzymes (except now mandelate dehydrogenase (11) also with glycine), because the other family members have an alanine residue at the homologous position (6). This residue, in glycolate oxidase and flavocytochrome b 2 , is in close contact with the flavin N-5 position on the re-face of the flavin. It was considered possible that the s...

The 1 :I complex between an osmate ester and bovine serum albumin was found to be effective as a n enantioselective catalyst in the cis-hydroxylation of alkenes, affording diols in up t o 68% e.e. and turnover of the catalyst with t-butyl hydroperoxide.