Analysis of pSC138, the multidrug resistance plasmid of Salmonella enterica serotype Choleraesuis SC-B67

Salmonella enterica serotype Choleraesuis (S. Choleraesuis) usually causes systemic infections in man and needs antimicrobial treatment. Multidrug resistance (MDR) in S. Choleraesuis is thus a great concern in the treatment of systemic non-typhoid salmonellosis. A large plasmid, pSC138, was identified in 2002 from a S. Choleraesuis strain SC-B67 that was resistant to all antimicrobial agents commonly used to treat salmonellosis, including ciprofloxacin and ceftriaxone. Complete DNA sequence of the plasmid had been determined previously (Chiu et al., 2005). In the present study, the sequence of pSC138 was reannotated in detail and compared with several newly sequenced plasmids. Some transposable elements and drug resistance genes were further delineated. Plasmid pSC138 was 138,742 bp in length and consisted of 177 open reading frames (ORFs). While 134 of the ORFs displayed significant identity levels to other plasmid and prokaryotic sequences, the remaining 43 ORFs have not been previously reported. Mobile elements, including two integrons, seven insertion sequences and eight transposons, and a truncated prophage together encompass at least 66,781 bp (48.1%) of the plasmid genome. The sequence of pSC138 consists of three major regions: a large composite transposable region Tn6088 with a Tn21-like backbone inserted by a variety of integrons or transposable elements; a transfer/maintenance region that contains a conserved ISEcp1-mediated transposon-like element Tn6092, carrying an AmpC gene, bla(CMY-2), that confers the ceftriaxone resistance; and a Rep_3 type of replication region. Another seven bacteremic strains of S. Choleraesuis that expressed the same MDR phenotype were identified during 2003-2008. The same Rep_3 type replicase and the bla(CMY-2)-containing, ISEcp1-mediated transposon-like element were found in the MDR isolates, suggesting a successful preservation and dissemination of the MDR plasmid. Comparison of pSC138 with other recently published plasmids revealed a high identity level between partial sequences of pSC138 and plasmids of the same or different incompatibility groups. The large MDR region found in pSC138 may provide a niche for the future evolution of the plasmid by acquisition of relevant resistance genes through the panoply of mobile elements and illegitimate recombination events.     

苏云金芽孢杆菌gerA操纵子在芽孢萌发中的作用

芽孢萌发的营养诱导剂通过与特异的萌发受体结合激活下游的萌发过程,从而使芽孢经过一系列的遗传变化及生化反应恢复营养生长.从苏云金芽孢杆菌(Bacillus thuringiensis)中克隆到一个与枯草芽孢杆菌(Bacillus subtilis)gerA操纵子和蜡状芽孢杆菌(Bacillus cereus)gerR操纵子同源的gerA操纵子.苏云金芽孢杆菌gerA操纵子含有3个开放读码框:gerAA、gerAC和gerAB,该操纵子在产孢起始3个小时后开始转录.gerA的破坏阻断了L-丙氨酸诱导的芽孢萌发并且延迟了肌苷诱导的萌发.在L-丙氨酸诱导芽孢萌发的过程中D-环丝氨酸能够提高芽孢的萌发率.     

Adenosine kinase regulation of cardiomyocyte hypertrophy

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKT(Ser??3) phosphorylation but did attenuate sustained phosphorylation of Raf(Ser33?) (24-48 h), mTOR(Ser2???) (24-48 h), p70S6k(Thr3??) (2.5-48 h), and ERK(Thr2?2/Tyr2??) (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k(Thr3??) phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK(Thr202/Tyr204) and AKT(Ser??3). Reduction of Raf-induced p70S6k(Thr3??) phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of Raf signaling to mTOR/p70S6k.     

Protective effects of activated vitamin D receptor on radiation‐induced intestinal injury

Radiation‐induced intestinal injury (RIII) is a common complication after radiation therapy in patients with pelvic, abdominal, or retroperitoneal tumours. Recently, in the model of DSS (Dextran Sulfate Sodium Salt) ‐induced intestinal inflammatory injury, it has been found in the study that transgenic mice expressing hVDR in IEC (Intestinal Epithelial Cell) manifest highly anti‐injury properties in colitis, suggesting that activated VDR in the epithelial cells of intestine may inhibit colitis by protecting the mucosal epithelial barrier. In this study, we investigated the effect of the expression and regulation of VDR on the protection of RIII, and the radiosensitivity in vitro experiments, and explored the initial mechanism of VDR in regulating radiosensitivity of IEC. As a result, we found that the expression of VDR in intestinal tissues and cells in mice can be induced by ionizing radiation. VDR agonists are able to prolong the average survival time of mice after radiation and reduce the radiation‐induced intestinal injury. For lack of vitamin D, the radiosensitivity of intestinal epithelial cells in mice increased, which can be reduced by VDR activation. Ensuing VDR activation, the radiation‐induced intestinal stem cells damage is decreased, and the regeneration and differentiation of intestinal stem cells is promoted as well. Finally, on the basis of sequencing analysis, we validated and found that VDR may target the HIF/PDK1 pathway to mitigate RIII. We concluded that agonism or upregulation of VDR expression attenuates radiation‐induced intestinal damage in mice and promotes the repair of epithelial damage in intestinal stem cells.     

Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.     

Spironolactone Attenuates Bleomycin-Induced Pulmonary Injury Partially via Modulating Mononuclear Phagocyte Phenotype Switching in Circulating and Alveolar Compartments

Background

Recent experimental studies provide evidence indicating that manipulation of the mononuclear phagocyte phenotype could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis. Mineralocorticoid receptor (MR) has been reported as a target to regulate macrophage polarization. The present work was designed to investigate the therapeutic potential of MR antagonism in bleomycin-induced acute lung injury and fibrosis.

Methodology/Principal Findings

We first demonstrated the expression of MR in magnetic bead-purified Ly6G-/CD11b+ circulating monocytes and in alveolar macrophages harvested in bronchoalveolar lavage fluid (BALF) from C57BL/6 mice. Then, a pharmacological intervention study using spironolactone (20mg/kg/day by oral gavage) revealed that MR antagonism led to decreased inflammatory cell infiltration, cytokine production (downregulated monocyte chemoattractant protein-1, transforming growth factor β1, and interleukin-1β at mRNA and protein levels) and collagen deposition (decreased lung total hydroxyproline content and collagen positive area by Masson’ trichrome staining) in bleomycin treated (2.5mg/kg, via oropharyngeal instillation) male C57BL/6 mice. Moreover, serial flow cytometry analysis in blood, BALF and enzymatically digested lung tissue, revealed that spironolactone could partially inhibit bleomycin-induced circulating Ly6C^hi monocyte expansion, and reduce alternative activation (F4/80+CD11c+CD206+) of mononuclear phagocyte in alveoli, whereas the phenotype of interstitial macrophage (F4/80+CD11c-) remained unaffected by spironolactone during investigation.

Conclusions/Significance

The present work provides the experimental evidence that spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially via inhibition of MR-mediated circulating monocyte and alveolar macrophage phenotype switching.