CpG methylation is essential for mouse development as well as gene regulation and genome stability. Many features of mammalian DNA methylation are consistent with the action of a de novo methyltransferase that establishes methylation patterns during early development and the post-replicative maintenance of these patterns by a maintenance methyltransferase. The mouse methyltransferase Dnmt1 (encoded by Dnmt) shows a preference for hemimethylated substrates in vitro, making the enzyme a candidate for a maintenance methyltransferase. Dnmt1 also has de novo methylation activity in vitro, but the significance of this finding is unclear, because mouse embryonic stem (ES) cells contain a de novo methylating activity unrelated to Dnmt1 (ref. 10). Recently, the Dnmt3 family of methyltransferases has been identified and shown in vitro to catalyse de novo methylation. To analyse the function of these enzymes, we expressed Dnmt and Dnmt3a in transgenic Drosophila melanogaster. The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When co-expressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development.

Stat3 is an Src homology (SH)2-containing protein constitutively activated in a wide variety of human cancers following its recruitment to YXXQ-containing motifs, which results in resistance to apoptosis. Despite resolution of the crystal structure of Stat3 homodimer bound to DNA, the structural basis for the unique specificity of Stat3 SH2 for YXXQ-containing phosphopeptides remains unresolved. We tested three models of this interaction based on computational analysis of available structures and sequence alignments, two of which assumed an extended peptide configuration and one in which the peptide had a ␤-turn. By using peptide immunoblot affinity assays and mirror resonance affinity analysis, we demonstrated that only phosphotyrosine (Tyr(P)) peptides containing ؉3 Gln (not Leu, Met, Glu, or Arg) bound to wild type Stat3. Examination of a series of wild type and mutant Stat3 proteins demonstrated loss of binding to pYXXQ-containing peptides only in Stat3 mutated at Lys-591 or Arg-609, whose side chains interact with the Tyr(P) residue, and Stat3 mutated at Glu-638, whose amide hydrogen bonds with oxygen within the ؉3 Gln side chain when the peptide ligand assumes a ␤-turn. These findings support a model for Stat3 SH2 interactions that could form the basis for anticancer drugs that specifically target Stat3.

Half of trauma deaths are attributable to hypovolemic circulatory collapse (HCC). We established a model of HCC in rats involving minor trauma plus severe hemorrhagic shock (HS). HCC in this model was accompanied by a 50% reduction in peak acceleration of aortic blood flow and cardiomyocyte apoptosis. HCC and apoptosis increased with increasing duration of hypotension. Apoptosis required resuscitation, which provided an opportunity to intervene therapeutically. Administration of IL-6 completely reversed HCC, prevented cardiac dysfunction and cardiomyocyte apoptosis, reduced mortality 5-fold and activated intracardiac signal transducer and activator of transcription (STAT) 3. Pre-treatment of rats with a selective inhibitor of Stat3, T40214, reduced the IL-6-mediated increase in cardiac Stat3 activity, blocked successful resuscitation by IL-6 and reversed IL-6-mediated protection from cardiac apoptosis. The hearts of mice deficient in the naturally occurring dominant negative isoform of Stat3, Stat3β, were completely resistant to HS-induced apoptosis. Microarray analysis of hearts focusing on apoptosis related genes revealed that expression of 29% of apoptosis related genes was altered in HS vs. sham rats. IL-6 treatment normalized the expression of these genes, while T40214 pretreatment prevented IL-6-mediated normalization. Thus, cardiac dysfunction, cardiomyocyte apoptosis and induction of apoptosis pathway genes are important components of HCC; IL-6 administration prevented HCC by blocking cardiomyocyte apoptosis and induction of apoptosis pathway genes via Stat3 and warrants further study as a resuscitation adjuvant for prevention of HCC and death in trauma patients.

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Platelets are known to contribute to ischemia/reperfusion in several organs, but their role in inflammation and organ injury after hemorrhagic shock (HS) has not been examined. To address this issue, we rendered mice thrombocytopenic (20% of normal platelet count) by treatment with pOp3, a rat monoclonal antibody against platelet glycoprotein Ibalpha, 24 h before subjecting them to either a standard HS or sham protocol. Liver apoptosis increased 3- to 5-fold (P<0.05), and focal liver necrosis increased 11-fold (P<0.01) in placebo-treated shock mice compared with sham; these increased indices of liver injury were completely prevented by pOp3 pretreatment. Neutrophils infiltrating the liver increased nearly 3-fold in placebo-treated shock mice versus sham (P<0.05); this shock-induced increase in neutrophil infiltration was also eliminated by pretreatment with pOp3. Alveolar cross-sectional area, used to histologically assess interstitial lung edema and cellular infiltration, was reduced by 25% in pOp3-treated shock mice versus placebo-treated shock mice (P<0.05). Similar to the results in liver, pOp3 pretreatment decreased neutrophil infiltration in the lung after HS. Thus, platelets contribute to the inflammatory injuries of the liver and lung after HS, in part, perhaps by facilitating neutrophil infiltration into tissues.