SummaryWe purified a peptidoglycan hydrolase involved in cell separation from a Staphylococcus aureus atl null mutant and identified its gene. Characterization of the gene product shows a 32 kDa N -acetylmuramyl-Lalanine amidase that we designated Sle1. Analysis of peptidoglycan digests showed Sle1 preferentially cleaved N -acetylmuramyl-L -Ala bonds in dimeric cross-bridges that interlink the two murein strands in the peptidoglycan. An insertion mutation of sle1 impaired cell separation and induced S. aureus to form clusters suggesting Sle1 is involved in cell separation of S. aureus . The Sle1 mutant revealed a significant decrease in pathogenesis using an acute infection mouse model. Atl is the major autolysin of S. aureus , which has been implicated in cell separation of S. aureus . Generation of an atl / sle1 double mutant revealed that the mutant cell separation was heavily impaired suggesting that S. aureus uses two peptidoglycan hydrolases, Atl and Sle1, for cell separation. Unlike Atl, Sle1 is not directly involved in autolysis of S. aureus .

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We describe here the purification and quantification of a water-soluble cyclic form of enterobacterial common antigen (ECA CYC ) from Escherichia coli K-12 as well as information regarding its subcellular location and the genetic loci involved in its assembly. Structural characterization of purified ECA CYC molecules obtained from E. coli K-12 revealed that they uniformly contained four trisaccharide repeat units, and they were substituted with from zero to four O-acetyl groups. Cells from overnight cultures contained approximately 2 g ECA CYC per milligram (dry weight), and cell fractionation studies revealed that these molecules were localized exclusively in the periplasm. The synthesis and assembly of ECA CYC were found to require the wzxE and wzyE genes of the wec gene cluster. These genes encode proteins involved in the transmembrane translocation of undecaprenylpyrophosphate-linked ECA trisaccharide repeat units and the polymerization of trisaccharide repeat units, respectively. Surprisingly, synthesis of ECA CYC was dependent on the wzzE gene, which is required for the modulation of the polysaccharide chain lengths of phosphoglyceride-linked ECA (ECA PG ). The presence of ECA CYC in extracts of several other gram-negative enteric organisms was also demonstrated; however, it was not detected in cell extracts of Pseudomonas aeruginosa. These data suggest that in addition to ECA PG , ECA CYC may be synthesized in many, if not all, members of the Enterobacteriaceae.

The transcription factor cAMP response element‐binding protein (CREB) plays important roles in gene expression induced by cAMP signaling and is believed to be activated when its Ser133 is phosphorylated. However, the discovery of Ser133‐independent activation by the activation of transducer of regulated CREB activity coactivators (TORC) and repression by salt inducible kinase cascades suggests that Ser133‐independent regulation of CREB is also important. The activation and repression are mediated by the basic leucine zipper domain of CREB. In this review, we focus on the basic leucine zipper domain in the regulation of transcriptional activity of CREB and describe the functions of TORC and salt inducible kinase.

Age-associated changes of T- and NK-cell (T/NK) potential of human hematopoietic stem cells are unknown. Here, we enumerate and characterize T/NK precursors among CD34-positive/lineage marker-negative (CD34+Lin−) cell populations circulating in normal human adult peripheral blood (PB) by a limiting-dilution assay using co-culture with OP9-DL1 stroma cells expressing Notch 1 ligand, Delta-like 1. The frequency of T-cell precursors in CD34+Lin− cells was found to decrease with donor age, while the ratio of NK- to T-cell precursor frequency (NK/T ratio) increased with age, suggesting that lymphoid differentiation potential of PB progenitors shifts from T- to NK-cell lineage with aging. Clonal analyses of CD34+Lin− cells showed that differences in the NK/T ratio were attributable to different distributions of single- and dual-lineage T/NK precursor clones. Since nearly all of the clones retained monocyte and/or granulocyte differentiation potentials in co-culture with OP9-DL1 cells, T/NK precursors in PB are considered to be contained in the pool of T/NK/myeloid multi-potent progenitors. The age-associated increase in NK- over T-cell commitment might occur in precursor cells with T/NK/myeloid potential.

inducible kinase 2 (SIK2) is expressed abundantly in adipose tissues and represses cAMP-response element-binding protein (CREB)-mediated gene expression by phosphorylating the coactivator transducer of regulated CREB activity (TORC2). Phosphorylation at Ser 587 of SIK2 diminishes its TORC2 phosphorylation activity. In 3T3-L1 white adipocytes, SIK2 downregulates lipogenic gene in response to nutritional stresses. To investigate the impact of SIK2 on the function of brown adipose tissue (BAT), we used T37i brown adipocytes, mice with diet-induced obesity, and SIK2 mutant (S587A) transgenic mice. When T37i adipocytes were treated with insulin, the levels of peroxisome proliferator-activated receptor-coactivator-1␣ (PGC-1␣) and uncoupling protein-1 (UCP-1) mRNA were increased, and the induction was inhibited by overexpression of SIK2 (S587A) mutant or dominant-negative CREB. Insulin enhanced SIK2 phosphorylation at Ser 587 , which was accompanied by decrease in phospho-TORC2. Similarly, the decrease in the level of SIK2 phosphorylation at Ser 587 was observed in the BAT of mice with diet-induced obesity, which was negatively correlated with TORC2 phosphorylation. To confirm the negative correlation between SIK2 phosphorylation at Ser 587 and TORC2 phosphorylation in BAT, SIK2 mutant (S587A) was overexpressed in adipose tissues by using the adipocyte fatty acid-binding protein 2 promoter. The expression of recombinant SIK2 (S587A) was restricted to BAT, and the levels of phospho-TORC2 were elevated in BAT of transgenic mice. Male transgenic mice developed high-fat diet-induced obesity, and their BAT expressed low levels of PGC-1␣

Past exposure to atomic bomb (A-bomb) radiation has exerted various long-lasting deleterious effects on the health of survivors. Some of these effects are seen even after >60 yr. In this study, we evaluated the subclinical inflammatory status of 442 A-bomb survivors, in terms of 8 inflammation-related cytokines or markers, comprised of plasma levels of reactive oxygen species (ROS), interleukin (IL)-6, tumor necrosis factor α (TNF-α), C-reactive protein (CRP), IL-4, IL-10, and immunoglobulins, and erythrocyte sedimentation rate (ESR). The effects of past radiation exposure and natural aging on these markers were individually assessed and compared. Next, to assess the biologically significant relationship between inflammation and radiation exposure or aging, which was masked by the interrelationship of those cytokines/markers, we used multivariate statistical analyses and evaluated the systemic markers of inflammation as scores being calculated by linear combinations of selected cytokines and markers. Our results indicate that a linear combination of ROS, IL-6, CRP, and ESR generated a score that was the most indicative of inflammation and revealed clear dependences on radiation dose and aging that were found to be statistically significant. The results suggest that collectively, radiation exposure, in conjunction with natural aging, may enhance the persistent inflammatory status of A-bomb survivors.

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