Protein arginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that catalyzes the posttranslational conversion of arginine to citrulline (Arg --> Cit) in a number of proteins, including histones. While the gene encoding this enzyme has been implicated in the pathophysiology of rheumatoid arthritis (RA), little is known about its mechanism of catalysis, its in vivo role, or its role in the pathophysiology of RA; however, recent reports suggest that this enzyme can act as a transcriptional corepressor for the estrogen receptor. Herein, we report our initial kinetic and mechanistic characterization of human PAD4. Specifically, these studies confirm that PAD4 catalyzes the hydrolytic deimination of Arg residues to produce Cit and ammonia. The metal dependence of PAD4 has also been evaluated, and the results indicate that PAD4 activity is highly specific for calcium. Calcium activation of PAD4 catalysis exhibits positive cooperativity with K(0.5) values in the mid to high micromolar range. Evidence indicating that calcium binding causes a conformational change is also presented. Additionally, the steady-state kinetic parameters for a number of histone H4-based peptide substrates and benzoylated Arg derivatives have been determined. K(m) values for these compounds are in the high micromolar to the low millimolar range with k(cat) values ranging from 2.8 to 6.6 s(-)(1). The ability of PAD4 to catalyze the deimination of methylated Arg residues has also been evaluated, and the results indicate that these compounds are poor PAD4 substrates (V/K

Macrophages are an important source of inflammatory cytokines generated during the innate immune response,but in the microenvironment of certain tumors,macrophages promote tumor progression through their preferential secretion of cytokines that support tumor cell growth and suppress antitumoral immune responses. KSHV is the causative agent of KS and lymphomas preferentially arising in immuno compromised patients, and specific cytokines, including IL-6 and IL-10, have been implicated in KSHV-associated cancer pathogenesis. However, the contribution of KSHV-infected macrophages to the cytokine milieu within KSHV-related tumors is unclear. We found that individual KSHV-encoded miRNA induce IL-6 and IL-10 secretion independently and additively by murine macrophages and human myelomonocytic cells. Bioinformatics analysis identified KSHV miRNA binding sites formiR-K12-3 and miR-K12-7 within the 3'UTR of the basic region/leucine zipper motif transcription factor C/EBPbeta, a known regulator of IL-6 and IL-10 transcriptional activation.Subsequent immunoblot analyses revealed that miR-K12-3 and miR-K12-7 preferentially reduce expression of C/EBPbeta p20 (LIP), an isoform of C/EBPbeta known to function as a negative transcription regulator. In addition,RNA interference specifically targeting LIP induced basal secretion of IL-6 and IL-10 by macrophages.Taken together, these data support a role for KSHV miRNA in the programming of macrophage cytokine responses in favor of KSHV-related tumor progression.

Objectives was FIGO IB1 (n = 10), IB2 (n = 13), IIA (n = 4), IIB (n = 19), IIIA (n = 2), IIIB (n = 16), IVA (n = 5) and IVB (n = 2). The median gravidity was 3 (range 1-10) and median parity was 3 (range 0-10). Seven patients had a preimplant history of pelvic infection, four had a history of intrauter-

Cisplatin/irinotecan chemoradiotherapy is tolerable, demonstrating similar efficacy for squamous cell and adenocarcinoma esophageal cancers.