We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.The mammalian DNA methyltransferases (DNA methyltransferase 1 [Dnmt1], Dnmt3a, and Dnmt3b) establish and maintain genomic methylation patterns which are of critical importance in various biological processes, including development, genomic imprinting, silencing of parasitic sequence elements, and tumorigenesis (3,14,17,31). The individual role of each of the DNA methyltransferases in establishing and maintaining these patterns is still unclear and has been confounded by their overlapping activities with respect to their abilities to methylate unmethylated and hemimethylated DNA in the test tube (21, 30). Embryonic stem (ES) cells deficient in one or more of these enzymes can be used in one of several approaches to elucidate the roles of the individual enzymes in living cells. Earlier studies using cells deficient in the Dnmt1 enzyme showed considerable decreases in the level of genomic DNA methylation at CpG-rich repetitive elements and imprinted genes (17,25,27). Recent studies using cells deficient in both the Dnmt3a and -3b enzymes showed that CpG-rich retroviral and intracisternal A particle (IAP) elements became slightly demethylated, and Igf-2 and Xist became extensively demethylated, in the absence of these enzymes, implying that Dnmt1 by itself had sequence specificity in maintaining the methylation of these sequences (20).These previous studies all focused on the methylation of CpG-rich sequences in knockout cells. However, most methylation in mammalian cells is found in non-CpG-rich regions of DNA (5), and the roles of the various enzymes in establishing and maintaining these methylation patterns have not been investigated. We have therefore used a genome-scanning approach to investigate the patterns of methylation in the various knockout cells in CpG-poor and CpG-rich regions to determine the roles of the enzymes in carrying out the bulk of methylation in mouse ES cells.We found that methylatio...

For RGCs at ambient pressure, IL-6 secreted from astrocytes and microglia under pressure is adequate to abate other proapoptotic signals from these glia. For RGCs challenged by elevated pressure, decreased IL-6 in astrocyte medium is insufficient to counteract these signals. Increased IL-6 in microglia medium counters not only proapoptotic signals from these cells but also the pressure-induced apoptotic cascade intrinsic to RGCs.

Cornea specialists correctly differentiated bacterial from fungal keratitis more often than chance, but in fewer than 70% of cases. More specific categorization led to less successful clinical distinction. Although certain clinical signs of infectious keratitis may be associated with a bacterial or fungal etiology, this study highlights the importance of obtaining appropriate microbiological testing during the initial clinical encounter.

SummaryCorneal scarring due to injury is a leading cause of blindness worldwide and results from dysregulated inflammation and angiogenesis during wound healing. Here we demonstrate that the extracellular matrix metalloproteinase MMP12 (macrophage metalloelastase) is an important regulator of these repair processes. Chemical injury resulted in higher expression of the fibrotic markers a-smooth muscle actin and type I collagen, and increased levels of angiogenesis in corneas of Mmp12 2/2 mice compared with corneas of wild-type mice. In vivo, we observed altered immune cell dynamics in Mmp12 2/2 corneas by confocal imaging. We determined that the altered dynamics were the result of an altered inflammatory response, with delayed neutrophil infiltration during the first day and excessive macrophage infiltration 6 days later, mediated by altered expression levels of chemokines CXCL1 and CCL2, respectively. Corneal repair returned to normal upon inhibition of these chemokines. Taken together, these data show that MMP12 has a protective effect on corneal fibrosis during wound repair through regulation of immune cell infiltration and angiogenesis.