Involvement of Ca2+/calmodulin kinase II (CaMK II) in genistein-induced potentiation of leucine/glutamine-stimulated insulin secretion.

Genistein has been reported to potentiate glucose-stimulated insulin secretion (GSIS). Inhibitory activity on tyrosine kinase or activation of protein kinase A (PKA) was shown to play a role in the genistein-induced potentiation effect on GSIS. The aim of the present study was to elucidate the mechanism of genistein-induced potentiation of insulin secretion. Genistein augmented insulin secretion in INS-1 cells stimulated by various energy-generating nutrients such as glucose, pyruvate, or leucine/glutamine (Leu/Gln), but not the secretion stimulated by depolarizing agents such as KCl and tolbutamide, or Ca(2+) channel opener Bay K8644. Genistein at a concentration of 50 µM showed a maximum potentiation effect on Leu/Gln-stimulated insulin secretion, but this was not sufficient to inhibit the activity of tyrosine kinase. Inhibitor studies as well as immunoblotting analysis demonstrated that activation of PKA was little involved in genistein-induced potentiation of Leu/Gln-stimulated insulin secretion. On the other hand, all the inhibitors of Ca(2+)/calmodulin kinase II tested, significantly diminished genistein-induced potentiation. Genistein also elevated the levels of [Ca(2+)]i and phospho-CaMK II. Furthermore, genistein augmented Leu/Gln-stimulated insulin secretion in CaMK II-overexpressing INS-1 cells. These data suggest that the activation of CaMK II played a role in genistein-induced potentiation of insulin secretion.

A novel agonistic antibody to human death receptor 4 induces apoptotic cell death in various tumor cells without cytotoxicity in hepatocytes.

The proapoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors death receptor (DR) 4 and DR5 are attractive targets to develop the receptor-specific agonistic monoclonal antibodies (mAb) as anticancer agents because of their tumor-selective cell death-inducing activity. Here, we report a novel agonistic mAb, AY4, raised against human DR4 in mice. ELISA analysis revealed that AY4 specifically bound to DR4 without competition with TRAIL for the binding. Despite distinct binding regions of AY4 on DR4 from those of TRAIL, AY4 as a single agent induced caspase-dependent apoptotic cell death of several tumor types through the extrinsic and/or intrinsic pathways without substantial cytotoxicity to normal human hepatocytes. Further, the AY4-sensitive cells followed the same cell death characteristics classified as type I and type II cells by the response to TRAIL, suggesting that the cell death profiles in responses to DR4 and/or DR5 stimulation are determined by the downstream signaling of the receptor rather than the kind of receptor. Noticeably, AY4 efficiently induced cell death of Jurkat cells, which have been reported to be resistant to other anti-DR4 agonistic mAbs, most likely due to the unique epitope property of AY4. In vivo administration of AY4 significantly inhibited tumor growth of human non-small cell lung carcinoma preestablished in athymic nude mice. Conclusively, our results provide further insight into the DR4-mediated cell death signaling and potential use of AY4 mAb as an anticancer therapeutic agent, particularly for DR4-responsive tumor types.

Characterization of a novel anti-human TNF-alpha murine monoclonal antibody with high binding affinity and neutralizing activity.

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta. TSK114 was found to be of IgG1 isotype with kappa light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-alpha by surface plasmon resonance technique revealed a binding affinity (K(D)) of approximately 5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, respectively. TSK114 neutralized human TNF-alpha-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-alpha-neutralizing antibody with picomolar affinity.