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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1096/fj.201802147rr

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1096/fj.201802147RR

Language：English   Publishing type：Research paper (scientific journal)  

In Bacillus subtilis, extracytoplasmic function (ECF) sigma factors are activated by reduction of phosphatidylglycerol (PG) content, absence of glucolipids, or absence of lipoteichoic acid (LTA). LTA is synthesized by polymerization of the glycerophosphate moiety of PG onto diglucosyldiacylglycerol (DGlcDG), a major glucolipid in B. subtilis, in the plasma membrane. Thus, reduction of PG content or absence of glucolipids might cause some changes in LTA, and hence we investigated whether reduction of PG content or absence of glucolipids induces the activation of ECF sigma factors independently from an ensuing change in LTA. Disruption of ugtP, responsible for glucolipid synthesis, in cells lacking LTA caused an additive increase of activation levels of σM, σX, σV and σY (3.1-, 2.2-, 2.1- and 1.4-fold, respectively), relative to their activation levels in cells lacking LTA alone. Reduction of PG content (by repressing Pspac-pgsA) in the cells lacking LTA caused an additive increase of activation levels of σM, σW and σV (2.3-, 1.9- and 2.2-fold, respectively). These results suggested that absence of glucolipids or reduction of PG alone, not the possible secondary alteration in LTA, leads to changes that affect the regulation systems of some ECF sigma factors in the plasma membrane.

DOI： 10.1266/ggs.18-00046

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/jb/mvy083

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Language：English   Publishing type：Research paper (scientific journal)  

Senescent cells accumulate in many tissues as animals age and are considered to underlie several aging-associated pathologies. The tumor suppressors p19ARF and p16INK4a, both of which are encoded in the CDKN2A locus, play critical roles in inducing and maintaining permanent cell cycle arrest during cellular senescence. Although the elimination of p16INK4a-expressing cells extends the life span of the mouse, it is unclear whether tissue function is restored by the elimination of senescent cells in aged animals and whether and how p19ARF contributes to tissue aging. The aging-associated decline in lung function is characterized by an increase in compliance as well as pathogenic susceptibility to pulmonary diseases. We herein demonstrated that pulmonary function in 12-month-old mice was reversibly restored by the elimination of p19ARF-expressing cells. The ablation of p19ARF-expressing cells using a toxin receptor-mediated cell knockout system ameliorated aging-associated lung hypofunction. Furthermore, the aging-associated gene expression profile was reversed after the elimination of p19ARF. Our results indicate that the aging-associated decline in lung function was, at least partly, attributed to p19ARF and was recovered by eliminating p19ARF-expressing cells.

DOI： 10.1172/jci.insight.87732

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC JAPAN  

Extracytoplasmic function (ECF) sigma factors respond to environmental stresses and regulate numerous genes required for adaptation. Under normal growth conditions, the ECF sigma factors are sequestered by transmembrane anti-sigma factor proteins, from which they are released under stress conditions. In Bacillus subtilis ugtP null mutant cells, which lack glucolipids, three of the seven ECF sigma factors, sigma(M), sigma(V) and sigma(X), are activated. The Escherichia coli cell membrane does not contain glucolipids. When the genes for these three ECF sigma and anti-sigma factors were introduced into E. coli cells, expression of lacZ fused to the ECF sigma factor-regulated promoters indicated ECF sigma factor activity. Additional expression of the ugtP gene in these E. coli cells led to the synthesis of small amounts of glucolipids, and the activities of sigma(M) and sigma(V) were repressed, but the activity of sigma(X) was unaffected. It is likely that glucolipids directly influence anti-sigma(M) and anti-sigma(V) factors by stabilizing conformations that sequester the respective ECF sigma factors.

DOI： 10.1266/ggs.90.109

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background: HuR (human antigen R) is a ubiquitously expressed member of the Hu/ELAV family of proteins that is involved in diverse biological processes. HuR has also been shown to play an important role in cell cycle arrest during replicative senescence in both human and mouse cells. Senescent cells not only halt their proliferation, but also activate the secretion of proinflammatory cytokines. A persistent DNA damage response is essential for the senescence-associated secretory phenotype (SASP), and increasing evidence has suggested that the SASP is associated with malignancy.
Methods: Senescence-associated phenotypes were analyzed in MEFs and other cell line in which HuR expression is inhibited by sh-RNA-mediated knockdown.
Results: RNAi-mediated HuR inhibition resulted in an increase in SASP-related cytokines. The induction of SASP factors did not depend on ARF-p53 pathway-mediated cell cycle arrest, but required NF-kappa B activity. In the absence of HuR, cells were defective in the DNA-damage response, and single strand DNA breaks accumulated, which may have caused the activation of NF-kappa B and subsequent cytokine induction.
Conclusions: In the absence of HuR, cells exhibit multiple senescence-associated phenotypes. Our findings suggest that HuR regulates not only the replicative lifespan, but also the expression of SASP-related cytokines in mouse fibroblasts.
General significance: RNA-binding protein HuR protects cells from undergoing senescence. Senescence-associated phenotypes are accelerated in HuR-deficient cells. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2014.07.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

p19(ARF) plays an essential role in the senescence of mouse cells, and its expression is lost by methylation or deletion of the ARF locus; otherwise, p53 is inactivated to bypass senescence. ARF expression is tightly regulated, but little is known about its posttranscriptional regulation. Here, we show that an RNA-binding protein, HuR (human antigen R), represses ARF mRNA translation, thereby maintaining the replicative life span of mouse embryonic fibroblasts (MEFs). Loss of HuR results in premature senescence, with concomitant increases in p19(ARF) but not p16(Ink4a) levels, and this senescence is not observed in ARF-null MEFs that retain an intact Ink4a locus. HuR depletion does not alter ARF transcription or stability but enhances ribosome association with ARF mRNA. Under these conditions, ARF mRNA accumulates in nucleoli, where it associates with nucleolin. Furthermore, adipose-specific deletion of the HuR gene results in increased p19(ARF) expression in aged animals, which is accompanied by decreased insulin sensitivity. Together, our findings demonstrate that p19(ARF) is also regulated at the translational level, and this translational regulation restrains the cellular life span and tissue functions in vivo.

DOI： 10.1128/MCB.01277-12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Lipoteichoic acid (LTA) is an important cell envelope component of Gram-positive bacteria. Bacillus subtilis has four homologous genes for LTA synthesis: ltaS (yflE), yfnI, yqgS and yvgJ. The products LtaS (YflE), YfnI and YqgS are bona fide LTA synthetases, whereas YvgJ functions only as an LTA primase. To clarify whether defects in LTA on the cell envelope trigger extracytoplasmic function (ECF) sigma factors, mRNA levels of the autoregulated ECF sigma factors in cells with singly and multiply deleted alleles of the ltaS homologues were examined by real-time RT-PCR. This revealed that sigM and sigX were induced in cells with a null allele of Delta ltaS and Delta yfnI, respectively, and that no ECF sigma factor was induced in cells with a single null allele of Delta yqgS or Delta yvgJ. In cells with double null alleles (Delta ltaS and Delta yfnI), sigW and ylaC were induced in addition to sigM and sigX. Cells with triple null alleles (Delta ltaS Delta yfnI and Delta yqgS) showed a pattern of induction similar to that of the double null. In cells with quadruple null alleles, sigV and sigY were newly induced. Cells with Delta ltaS had approximatley 1/4 the diglucosyldiacylglycerol and over 10 times the CDP-diacylglycerol of wild-type cells. Compensatory elevation of the mRNA level of other homologues was observed (in Delta ltaS cells the level of yfnI was elevated; in Delta yfnI cells that of yqgS and yvgJ was elevated; both were even higher in Delta ltaS Delta yfnI cella In Delta ltaS cells, the mRNA level of yfnI was corroborated to be regulated by sigma(M), which is activated in the null mutant cells. In Delta yfnI cells, the mRNA levels of yqgS and yvgJ reverted to less than those of wild-type when a defective sigX allele was introduced. Since sigX was activated in cells with Delta yfnI, this suggests that the induction of yqgS and yvgJ is dependent on sigma(X). The LTAs produced by the four ltaS homologues seem to play distinct physiological roles to maintain the full function of LTA on the B. subtilis cell envelope.

DOI： 10.1099/mic.0.063420-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC JAPAN  

The dgkB gene is essential for the growth of Bacillus subtilis. It encodes a diacylglycerol (DG) kinase that converts DG to phosphatidic acid to reintroduce it into the phospholipid synthesis pathway. Repression of the dgkB gene placed under a regulatable promoter causes accumulation of DG and leads to lethality. DG is formed as a byproduct of the synthesis of lipoteichoic acid (LTA), a polyanionic component of the cell envelope. B. subtilis synthesizes LTA by polymerizing the glycerophosphate moiety of phosphatidylglycerol (PG) onto a glucolipid membrane anchor, and releasing the DG moiety of PG. B. subtilis has four genes homologous to Staphylococcus aureus ltaS, which encodes LTA synthase. Disruption of either or both of two genes, yflE and yfnI, whose products show higher homology with S. aureus LtaS among the four homologues, suppressed the lethality caused by dgkB repression. In cells with dgkB repression, DG was accumulated to 43 +/- 3% of total lipids, about three times the content of wild type cells (13 +/- 1%). Disruption of yfnI in the dgkB-repressed cells reduced the DG content to 15 +/- 2%, but yflE-disruption did not (42 +/- 1%); this was probably due to efficient LTA synthesis by YfnI in the yflE-disrupted cells. Further introduction of a disrupted allele of ugtP, encoding glucolipid synthase that consumes DG as a substrate, partially lowered the colony forming capacity in strains with yflE-disruption. A disrupted dgkB allele was successfully introduced into strains disrupted for either or both of yflE and yfnI, indicating that the essential gene dgkB is dispensable in mutants defective in LTA synthesis.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC JAPAN  

Bacillus subtilis Marburg 168 cells with disrupted ugtP, which encodes UDP-glucosyltransferase involved in glucolipid synthesis, were bent and distended. In the ugtP mutant cells, the extracytoplasmic function sigmas SigM, SigV and SigX, were found to be activated. Introduction of a disrupted allele of sigM into the ugtP strain caused even more abnormal morphology, with cells taking on a balloon-like shape; growth of these cells in LB medium was hampered by addition of 1.5% NaCl. Addition of MgSO4 or MnCl2 suppressed the abnormal morphology. In ugtP mutant cells the transcription of the mreB operon from an upstream promoter in maf (designated Pupstream mreB) and PmreBH was 4.3- and 2.3-fold higher, respectively, and localization of GFP-MreB was not in discrete dots (in an apparently helical pattern), but faint and in irregular clusters. GFP-MreB protein was reduced in the ugtP mutant cells. We suggest that glucolipids are important for MreB isoforms to take on the configuration that appears as discrete dots and plays a role in shaping cells into straight rods.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC JAPAN  

The Bacillus subtilts gene pgsA, which codes for the phosphatidylglycerophosphate synthase that catalyzes the committed step for the synthesis of phosphatidylglycerol (PG), is essential since Pspac-pgsA cells require IPTG for growth. Removal of the inducer caused a dramatic decrease of PG content in the membranes of cells and retarded growth. At 60 min and 120 min after removal, it was reduced to 14.1% and 8.9% of total lipid, respectively, from an initial content of 28.1%. We conjectured that the activity of some extracytoplasmic function (ECF) sigma factors, most of which are caught and regulated directly by cognate transmembrane anti-sigma factors, are affected by altered lipid composition of the membranes. Induction of the activities of ECF sigma factors (sigma(M) and sigma(V)) was observed after removal of IPTG, though that of sigma(V) was small. But other ECF sigma factors (sigma(W), sigma(X), sigma(Y), sigma(YlaC) and sigma(Z)) and the general stress sigmas sigma(B) and sigma(I) were not induced. Especially sigma(M) was activated strongly with the reduction of PG content and sustained a high level of activity, in contrast to the transient activation in PG normal cells after exposure to high salinity. This study demonstrates a new relationship between the alterations of lipid composition in the membranes and the activation of ECF sigma factors.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：GENETICS SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：GENETICS SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：GENETICS SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：GENETICS SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：GENETICS SOC JAPAN  

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