Tribbles homolog 2 (Trib2) and HoxA9 cooperate to accelerate acute myelogenous leukemia.

Trib2 is a member of the Trib family of serine/threonine kinase-like proteins (Trib1, Trib2, Trib3). Mice reconstituted with hematopoietic stem cells (HSC) retrovirally expressing Trib2 uniformly developed fatal transplantable acute myelogenous leukemia (AML). Trib2-induced AML was clonal and we sought to identify cooperating genes in Trib2-induced AML. Using Splinkerette PCR techniques, we identified proviral insertion near HoxA9 in a Trib2 monoclonal tumor, which resulted in greatly elevated HoxA9 expression. Mice reconstituted with HSC cotransduced with HoxA9 and Trib2 had accelerated onset of AML compared to either gene alone. These data identify Trib2 and HoxA9 as cooperating genes in AML.

Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a.

Chemical rescue is an established approach that offers a directed strategy for designing mutant enzymes in which activity can be restored by supplying an appropriate exogenous compound. This method has been used successfully to study a broad range of enzymes in vitro, but its application to living systems has received less attention. We have investigated the feasibility of using chemical rescue to make a conditional-lethal mutant of the cytomegalovirus (CMV) maturational protease. The 28-kDa CMV serine protease, assemblin, has a Ser-His-His catalytic triad and an internal (I) cleavage site near its midpoint. We found that imidazole can restore I-site cleavage to mutants inactivated by replacing the critical active site His with Ala or with Gly, which rescued better. Comparable rescue was observed for counterpart mutants of the human and simian CMV assemblin homologs and occurred in both living cells and in vitro. Cleavage was established to be at the correct site by amino acid sequencing and proceeded at approximately 11%/h in bacteria and approximately 30%/h in vitro. The same mutations were unresponsive to chemical rescue in the context of the assemblin precursor, pUL80a. This catalytic difference distinguishes the two forms of the CMV protease.

TNFalpha inhibition in MRL/lpr mice ameliorates pulmonary but not renal disease.

TNFalpha inhibition has a clearly beneficial effect in a number of arthritides and in Crohn's disease. The exact mechanism of action is uncertain with studies showing inhibition of chemokines, inhibition of adhesion molecule expression, and improved T-cell function. Unlike most therapeutic interventions for autoimmune disease, TNFalpha inhibition appears to act on specific pathologic processes. It is not known how wide-spread these TNFalpha-mediated pathologic processes are. Efforts to expand the use of TNFalpha inhibition have had notable successes but have been disappointing in other disorders. We hypothesized that TNFalpha-mediated pathologic processes might play a significant role in the end-organ effects seen in SLE. We modeled SLE by using MRL/lpr mice and treated with two types of TNFalpha inhibitor. Pulmonary disease was significantly improved in the treated groups compared to controls. In contrast, renal disease was unaffected suggesting that in lupus, where multiple organs are affected, different pathologic processes may be mediating the end-organ damage. This has important implications for designing therapeutics for SLE.