Prolonged activation of protein kinase Cs (PKCs) by long-term treatment of cells with phorbol ester tumor promoters down-regulates the expression of many PKCs. To investigate the molecular mechanisms involved in the down-regulation of PKCZ, we expressed the novel PKCs Z and y and various mutant forms in baby hamster kidney cells. Upon overexpression, constitutively active PKCZ, but not wild type or kinase-dead PKCZ, underwent rapid degradation to generate several lower molecular weight polypeptides. When co-expressed with active kinases, kinase-dead PKCZ with a pseudosubstrate site mutation designed to give an active conformation was down-regulated while the wild type PKCZ was not. These results suggest requirements for kinase activity and an active conformation for down-regulation of PKCZ. Treatment with the proteasome inhibitors N-AcLeu-Leu-norleucinal and lactacystin led to accumulation of PKCZ proteolytic products and potentially ubiquitinated forms. While wild type PKCZ localizes mostly to the detergent-soluble fraction of the cell, a signi®cant portion of full-length constitutively active PKCZ and of kinase-dead, active conformation PKCZ were found in the detergent-insoluble fraction. Several proteolytic fragments of constitutively active PKCZ also were found in the detergent insoluble fraction. These full-length and proteolytic fragments of PKCZ in the detergent-insoluble fraction accumulated further in the presence of proteasome inhibitors. These data suggest that active conformation PKCZ accumulates in the detergent-insoluble compartment, is degraded by proteolysis in the presence of kinase activity, and that the cleavage products undergo further degradation via ubiquitin-mediated degradation in the proteasome.

Dengue viruses (DEN), causative agents of dengue fever (DF) and more severe dengue hemorrhagic fever (DHF)/dengue shock syndrome, infect over 100 million people every year. Among those infected, up to one-half million people develop DHF, which requires an extensive hospital stay. Recent reports indicate that there is a significant correlation between virus titer in the bloodstream of infected individuals and the severity of the disease, especially the development of DHF. This suggests that if there is a procedure to reduce viremia in infected subjects, then the severity of the disease may be controlled during the critical early stages of the disease before it progresses to DHF. We have generated bispecific mAb complexes (heteropolymer(s), HP), which contain a mAb specific for the DEN envelope glycoprotein cross-linked with a second mAb specific for the primate E complement receptor 1. These HP facilitate rapid binding of DEN to human and monkey E in vitro, with ∼90% bound within 5 min. Furthermore, in a passive viremia monkey model established by continuous steady state infusion of DEN, injection of HP during the steady state promoted rapid binding of DEN to the E, followed by subsequent clearance from the vascular system. Moreover, HP previously infused into the circulation is capable of efficiently capturing a subsequent challenge dose of DEN and binding it to E. These data suggest that HP potentially can be useful for alleviating DEN infection-associated symptoms by reducing titers of free virus in the vascular system.