<Emphasis Type="Italic">In silico</Emphasis> identification and experimental validation of PmrAB targets in <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>by regulatory motif detection

Background  

The PmrAB (BasSR) two-component regulatory system is required for Salmonella typhimurium virulence. PmrAB-controlled modifications of the lipopolysaccharide (LPS) layer confer resistance to cationic antibiotic polypeptides, which may allow bacteria to survive within macrophages. The PmrAB system also confers resistance to Fe³⁺-mediated killing. New targets of the system have recently been discovered that seem not to have a role in the well-described functions of PmrAB, suggesting that the PmrAB-dependent regulon might contain additional, unidentified targets.     

International Society for Wildlife Endocrinology: the future of endocrine measures for reproductive science, animal welfare and conservation biology

Hormone analysis is a precise and widely accepted tool for monitoring reproductive function and responses to stressors. Although hormones are present and can be measured in various biological matrices, non-invasive methods have gained popularity over the past 30 years as a more practical approach for assessing ovarian, testicular and, more recently, adrenocortical activity in intractable wildlife species. Non-invasive hormone monitoring also has been key to understanding biological mechanisms related to observed behaviours of captive and free-ranging animals. Despite the increasing popularity of this research field, wildlife endocrinologists have not had a specific forum for sharing and discussing their latest findings, technical developments and common challenges. To provide such a communication platform, the International Society for Wildlife Endocrinology (ISWE) was established in 2010, followed by an international meeting held on 3-4 November 2011 at the Toronto Zoo, Canada. Over several sessions, keynote speakers and participants discussed recent developments of new and innovative methods for hormone monitoring, as well as the latest advances in basic endocrinology as applied to adrenal function, reproductive physiology, animal health, ecology and evolution. Here, we introduce ISWE to the scientific community and discuss how this new society will serve as a resource for wildlife endocrinologists worldwide.     

Blockage of heme oxygenase-1 abrogates the protective effect of regulatory T cells on murine pregnancy and promotes the maturation of dendritic cells

Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect fetal antigens from maternal effector cells. Their effect is associated with the up-regulation of tolerance-associated molecules at the fetal-maternal interface. Among these, Heme Oxygenase-1 (HO-1, coded by Hmox1) is of special importance as its blockage correlates with increased abortion rates and its up-regulation positively affects pregnancy outcome. Here, we aimed to investigate whether the protective effect of Treg is mediated by HO-1 in a mouse model. HO-1 blockage by Zinc Protoporhyrin (ZnPPIX) abrogated the protective effect of Treg transfer. We found that HO-1 is important in maintaining maternal dendritic cells (DCs) in an immature state, which contributes to the expansion of the peripheral Treg population. This brings to light one essential pathway through which Treg mediates the semi-allogeneic fetus tolerance.     

Metabolic Profiling Reveals Sphingosine-1-Phosphate Kinase 2 and Lyase as Key Targets of (Phyto-) Estrogen Action in the Breast Cancer Cell Line MCF-7 and Not in MCF-12A

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.     

Lipid profiling of the Arabidopsis hypersensitive response reveals specific lipid peroxidation and fragmentation processes: biogenesis of pimelic and azelaic Acid

Lipid peroxidation (LPO) is induced by a variety of abiotic and biotic stresses. Although LPO is involved in diverse signaling processes, little is known about the oxidation mechanisms and major lipid targets. A systematic lipidomics analysis of LPO in the interaction of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae revealed that LPO is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species and precedes programmed cell death. Singlet oxygen was identified as the major cause of lipid oxidation under basal conditions, while a 13-lipoxygenase (LOX2) and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection. Analysis of lox2 mutants revealed that LOX2 is essential for enzymatic membrane peroxidation but not for the pathogen-induced free jasmonate production. Despite massive oxidative modification of plastid lipids, levels of nonoxidized lipids dramatically increased after infection. Pathogen infection also induced an accumulation of fragmented lipids. Analysis of mutants defective in 9-lipoxygenases and LOX2 showed that galactolipid fragmentation is independent of LOXs. We provide strong in vivo evidence for a free radical-catalyzed galactolipid fragmentation mechanism responsible for the formation of the essential biotin precursor pimelic acid as well as of azelaic acid, which was previously postulated to prime the immune response of Arabidopsis. Our results suggest that azelaic acid is a general marker for LPO rather than a general immune signal. The proposed fragmentation mechanism rationalizes the pathogen-induced radical amplification and formation of electrophile signals such as phytoprostanes, malondialdehyde, and hexenal in plastids.     

Mass spectrometry for the molecular imaging of angiotensin metabolism in kidney

To better understand the tissue distribution and activity of enzymes involved in angiotensin II (Ang II) processing, we developed a novel molecular imaging method using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Mouse kidney sections (12 μm) were incubated with 10-1,000 μmol/l Ang II for 5-15 min at 37°C. The formed peptides Ang III and Ang-(1-7) were identified by MALDI-TOF/TOF. A third metabolite, Ang-(1-4), was generated from further degradation of Ang-(1-7). Enzymatic processing of Ang II was dose and time dependent and absent in heat-treated kidney sections. Distinct spatial distribution patterns (pseudocolor images) were observed for the peptides. Ang III was localized in renal medulla, whereas Ang-(1-7)/Ang-(1-4) was present in cortex. Regional specific peptide formation was confirmed using microdissected cortical and medullary biopsies. In vitro studies with recombinant enzymes confirmed activity of peptidases known to generate Ang III or Ang-(1-7) from Ang II: aminopeptidase A (APA), Ang-converting enzyme 2 (ACE2), prolyl carboxypeptidase (PCP), and prolyl endopeptidase (PEP). Renal medullary Ang III generation was blocked by APA inhibitor glutamate phosphonate. The ACE2 inhibitor MLN-4760 and PCP/PEP inhibitor Z-pro-prolinal reduced cortical Ang-(1-7) formation. Our results establish the power of MALDI imaging as a highly specific and information-rich analytical technique that will further aid our understanding of the role and site of Ang II processing in cardiovascular and renal pathologies.     

Triazoloamides as potent γ-secretase modulators with reduced hERG liability

Synthesis and SAR studies of novel aryl triazoles as gamma secretase modulators (GSMs) are presented in this communication. Starting from our aryl triazole leads, optimization studies were continued and the series progressed towards novel amides and lactams. Triazole 57 was identified as the most potent analog in this series, displaying single-digit nanomolar Aβ42 IC(50) in cell-based assays and reduced affinity for the hERG channel.     

Possible Origins of CTnBST, a Conjugative Transposon Found Recently in a Human Colonic Bacteroides Strain

A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species.