Suppression of LPS-induced transcription and cytokine secretion by the dietary isothiocyanate sulforaphane.

PubMed

Folkard, Danielle L; Melchini, Antonietta; Traka, Maria H; Al-Bakheit, Ala'a; Saha, Shikha; Mulholland, Francis; Watson, Andrew; Mithen, Richard F

2014-12-01

Diets rich in cruciferous vegetables are associated with lower levels of pro-inflammatory cytokines, which may contribute to potential health-promoting properties of these vegetables. We investigate whether sulforaphane (SF), an isothiocyanate (ITC) obtained from broccoli, could suppress LPS-induced transcription and subsequent pro-inflammatory cytokine secretion at a physiologically relevant concentration using in vitro models of chronic inflammation. We find that exposure of the LPS receptor Toll-like receptor-4 (TLR4) to physiologically appropriate concentrations of SF under non-reducing conditions results in covalent modification of cysteine residues 246 and 609. We further demonstrate that the changes in expression of 1210 genes (p ≤ 0.01) in THP-1 monocytes and the secretion of pro-inflammatory cytokines in both human peripheral blood mononuclear cells (PBMCs) and THP-1 monocytes induced by LPS exposure can be completely suppressed through exposure with physiologically appropriate concentrations of SF. Finally, we show that in vivo exposure of human PBMCs to ITCs within human circulation reduces secretion of pro-inflammatory cytokines following subsequent ex vivo LPS challenge (p < 0.001). Covalent modification of TLR4 by ITCs and resultant suppression of LPS-induced cell signalling could lead to reductions in levels of pro-inflammatory cytokines in people with chronic diseases who consume diets rich in cruciferous vegetables. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

β-Glucan Reverses the Epigenetic State of LPS-Induced Immunological Tolerance

PubMed Central

Novakovic, Boris; Habibi, Ehsan; Wang, Shuang-Yin; Arts, Rob J.W.; Davar, Robab; Megchelenbrink, Wout; Kim, Bowon; Kuznetsova, Tatyana; Kox, Matthijs; Zwaag, Jelle; Matarese, Filomena; van Heeringen, Simon J.; Janssen-Megens, Eva M.; Sharifi, Nilofar; Wang, Cheng; Keramati, Farid; Schoonenberg, Vivien; Flicek, Paul; Clarke, Laura; Pickkers, Peter; Heath, Simon; Gut, Ivo; Netea, Mihai G.; Martens, Joost H.A.; Logie, Colin; Stunnenberg, Hendrik G.

2018-01-01

Summary Innate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). We apply an integrated epigenomic approach to characterize the molecular events involved in LPS-induced tolerance in a time-dependent manner. Mechanistically, LPS-treated monocytes fail to accumulate active histone marks at promoter and enhancers of genes in the lipid metabolism and phagocytic pathways. Transcriptional inactivity in response to a second LPS exposure in tolerized macrophages is accompanied by failure to deposit active histone marks at promoters of tolerized genes. In contrast, β-glucan partially reverses the LPS-induced tolerance in vitro. Importantly, ex vivo β-glucan treatment of monocytes from volunteers with experimental endotoxemia re-instates their capacity for cytokine production. Tolerance is reversed at the level of distal element histone modification and transcriptional reactivation of otherwise unresponsive genes. PMID:27863248

Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells

PubMed Central

Brancaccio, Mariarita; Coretti, Lorena; Florio, Ermanno; Pezone, Antonio; Calabrò, Viola; Falco, Geppino; Keller, Simona; Lembo, Francesca; Avvedimento, Vittorio Enrico; Chiariotti, Lorenzo

2016-01-01

Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene. PMID:27253528

Selected topics from classical bacterial genetics.

PubMed

Raleigh, Elisabeth A; Elbing, Karen; Brent, Roger

2002-08-01

Current cloning technology exploits many facts learned from classical bacterial genetics. This unit covers those that are critical to understanding the techniques described in this book. Topics include antibiotics, the LAC operon, the F factor, nonsense suppressors, genetic markers, genotype and phenotype, DNA restriction, modification and methylation and recombination.

Stable Chromosomal Expression of Shigella flexneri 2a and 3a O-Antigens in the Live Salmonella Oral Vaccine Vector Ty21a

PubMed Central

Osorio, Manuel; Takeda, Kazuyo; Stibitz, Scott; Kopecko, Dennis J.

2017-01-01

ABSTRACT We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S. Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S. Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever. PMID:29046309

The nucleoid-associated protein Fis directly modulates the synthesis of cellulose, an essential component of pellicle-biofilms in the phytopathogenic bacterium Dickeya dadantii.

PubMed

Prigent-Combaret, Claire; Zghidi-Abouzid, Ouafa; Effantin, Géraldine; Lejeune, Philippe; Reverchon, Sylvie; Nasser, William

2012-10-01

Bacteria use biofilm structures to colonize surfaces and to survive in hostile conditions, and numerous bacteria produce cellulose as a biofilm matrix polymer. Hence, expression of the bcs operon, responsible for cellulose biosynthesis, must be finely regulated in order to allow bacteria to adopt the proper surface-associated behaviours. Here we show that in the phytopathogenic bacterium, Dickeya dadantii, production of cellulose is required for pellicle-biofilm formation and resistance to chlorine treatments. Expression of the bcs operon is growth phase-regulated and is stimulated in biofilms. Furthermore, we unexpectedly found that the nucleoid-associated protein and global regulator of virulence functions, Fis, directly represses bcs operon expression by interacting with an operator that is absent from the bcs operon of animal pathogenic bacteria and the plant pathogenic bacterium Pectobacterium. Moreover, production of cellulose enhances plant surface colonization by D. dadantii. Overall, these data suggest that cellulose production and biofilm formation may be important factors for surface colonization by D. dadantii and its subsequent survival in hostile environments. This report also presents a new example of how bacteria can modulate the action of a global regulator to co-ordinate basic metabolism, virulence and modifications of lifestyle. © 2012 Blackwell Publishing Ltd.

[Effect of Yersinia pestis EV 76 lypopolysaccharides with different levels of toxicity on dynamics of TNF-alpha and INF-gamma synthesis by human monocytes].

PubMed

Sokolova, E P; Demidova, G V; Ziuzina, V P; Alekseeva, L P; Bespalova, I A; Tynianova, V I

2010-01-01

AIM. To study dynamics of synthesis of TNF-alpha and INF-gamma by cell line U-937 human monocytes under the effect of Yersinia pestis EV 76 lypopolysaccharides (LPS) with different levels of toxicity: original LPS28 and LPS37 as well as their conformationally--changed variants with enhanced toxicity--complex of LPS with murine toxin (MT) of Y. pestis, and LPS modified by biologicall active compound (BAC) obtained from human erythrocytes. Using phenol method, LPS were obtained from Y. pestis EV 76 cells grown at 28 and 37 degrees C. Production of cytokines was measured by ELISA. It was shown that original and modified forms of LPS28 and LPS37 induce synthesis of both TNF-alpha and INF-gamma by human monocytes. Expression of genes for two ways of synthesis of these cytokines points to activation and transmission of signal induced by all studied forms of Y. pestis EV 76 LPS through TLR4. Levels of activity of MyD88-dependent and MyD88-independent signaling pathways are different and depend from chemical structure of LPS28 and LPS37, conformation of their modified forms and duration of their exposition with monocytes. Dynamics ofcytokine synthesis corresponds to response of synergized TLR on activation with profound agonistic/antagonistic effect. It was determined that conformational modifications of Y. pestis EV76 LPS occurring due to effect of MT and BAC accompanied by quantitative, qualitative and temporal changes of TNF-alpha and INF-gamma synthesis by human monocytes and correlate with increase of their toxic properties.

LPS causes pericyte loss and microvascular dysfunction via disruption of Sirt3/angiopoietins/Tie-2 and HIF-2α/Notch3 pathways.

PubMed

Zeng, Heng; He, Xiaochen; Tuo, Qin-Hui; Liao, Duan-Fang; Zhang, Guo-Qiang; Chen, Jian-Xiong

2016-02-12

Recent studies reveal a crucial role of pericyte loss in sepsis-associated microvascular dysfunction. Sirtuin 3 (SIRT3) mediates histone protein post-translational modification related to aging and ischemic disease. This study investigated the involvement of SIRT3 in LPS-induced pericyte loss and microvascular dysfunction. Mice were exposed to LPS, expression of Sirt3, HIF-2α, Notch3 and angiopoietins/Tie-2, pericyte/endothelial (EC) coverage and vascular permeability were assessed. Mice treated with LPS significantly reduced the expression of SIRT3, HIF-2α and Notch3 in the lung. Furthermore, exposure to LPS increased Ang-2 while inhibited Ang-1/Tie-2 expression with a reduced pericyte/EC coverage. Intriguingly, knockout of Sirt3 upregulated Ang-2, but downregulated Tie-2 and HIF-2α/Notch3 expression which resulted in a dramatic reduction of pericyte/EC coverage and exacerbation of LPS-induced vascular leakage. Conversely, overexpression of Sirt3 reduced Ang-2 expression and increased Ang-1/Tie-2 and HIF-2α/Notch3 expression in the LPS treated mice. Overexpression of Sirt3 further prevented LPS-induced pericyte loss and vascular leakage. This was accompanied by a significant reduction of the mortality rate. Specific knockout of prolyl hydroxylase-2 (PHD2) increased HIF-2α/Notch3 expression, improved pericyte/EC coverage and reduced the mortality rate in the LPS-treated mice. Our study demonstrates the importance of SIRT3 in preserving vascular integrity by targeting pericytes in the setting of LPS-induced sepsis.

Modulation of Caenorhabditis elegans immune response and modification of Shigella endotoxin upon interaction.

PubMed

Kesika, Periyanaina; Prasanth, Mani Iyer; Balamurugan, Krishnaswamy

2015-04-01

To analyze the pathogenesis at both physiological and molecular level using the model organism, Caenorhabditis elegans at different developmental stages in response to Shigella spp. and its pathogen associated molecular patterns such as lipopolysaccharide. The solid plate and liquid culture-based infection assays revealed that Shigella spp. infects C. elegans and had an impact on the brood size and pharyngeal pumping rate. LPS of Shigella spp. was toxic to C. elegans. qPCR analysis revealed that host innate immune genes have been modulated upon Shigella spp. infections and its LPS challenges. Non-destructive analysis was performed to kinetically assess the alterations in LPS during interaction of Shigella spp. with C. elegans. The modulation of innate immune genes attributed the surrendering of host immune system to Shigella spp. by favoring the infection. LPS appeared to have a major role in Shigella-mediated pathogenesis and Shigella employs a tactic behavior of modifying its LPS content to escape from the recognition of host immune system. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chronic DON exposure and acute LPS challenge: effects on porcine liver morphology and function.

PubMed

Renner, Lydia; Kahlert, Stefan; Tesch, Tanja; Bannert, Erik; Frahm, Jana; Barta-Böszörményi, Anikó; Kluess, Jeannette; Kersten, Susanne; Schönfeld, Peter; Rothkötter, Hermann-Josef; Dänicke, Sven

2017-08-01

The aim of the present study was to examine the role of chronic deoxynivalenol (DON) exposition on the liver morphology and function in combination with pre- and post-hepatic lipopolysaccharide (LPS) stress in young pigs fed for 4 weeks with a DON-contaminated diet (4.59 mg/kg feed). At the end of the experiment, LPS (7.5 μg/kg BW) was administered for 1 h pre-hepatically (Vena portae hepatis) or post-hepatically (Vena jugularis). Liver morphology was macroscopically checked and showed haemorrhage in all LPS groups, significantly higher relative liver weights, accompanied by marked oedema in the gallbladder wall. Histological changes were judged by a modified histology activity index (HAI). Liver HAI score was significantly increased in all LPS groups compared to placebo, primarily due to neutrophil infiltration and haemorrhage. DON feed alone was without effect on the liver HAI. Liver function was characterized by (i) hepatic biochemical markers, (ii) mitochondrial respiration and (iii) Ca 2+ accumulation capacity of isolated mitochondria. Clinical chemical parameters characterizing liver function were initially (<3 h) slightly influenced by LPS. After 3 h, bilirubin and alkaline phosphatase were increased significantly, in DON-fed, jugular-infused LPS group. Respiration and Ca 2+ accumulation capacity of isolated liver mitochondria was not impaired by chronic DON exposure, acute LPS challenge or combined treatments. DON-contaminated feed did not change macroscopy and histology of the liver, but modified the function under LPS stress. The different function was not linked to modifications of liver mitochondria.

Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Lingling; Noncoding RNA Center, Yangzhou University, Yangzhou 225001; Zhao, Yingmin

Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feedermore » layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. - Highlights: • LPS alone in culture is required for the proliferation of murine myeloid tumor cells. • Bone marrow stromal cells as a feeder layer is also required for the proliferation of myeloid tumor cells. • However, the growth of myeloid tumor cells is inhibited when LPS and stromal cells are both available in culture. • Thus LPS can either facilitate or attenuate tumor growth through its direct effect or modulation of tumor microenvironment.« less

Genetic modification of Bacillus subtilis for production of D-chiro-inositol, an investigational drug candidate for treatment of type 2 diabetes and polycystic ovary syndrome.

PubMed

Yoshida, Ken-ichi; Yamaguchi, Masanori; Morinaga, Tetsuro; Ikeuchi, Maya; Kinehara, Masaki; Ashida, Hitoshi

2006-02-01

D-chiro-inositol (DCI) is a drug candidate for the treatment of type 2 diabetes and polycystic ovary syndrome, since it improves the efficiency with which the body uses insulin and also promotes ovulation. Here, we report genetic modification of Bacillus subtilis for production of DCI from myo-inositol (MI). The B. subtilis iolABCDEFGHIJ operon encodes enzymes for the multiple steps of the MI catabolic pathway. In the first and second steps, MI is converted to 2-keto-MI (2KMI) by IolG and then to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione by IolE. In this study, we identified iolI encoding inosose isomerase, which converts 2KMI to 1-keto-D-chiro-inositol (1KDCI), and found that IolG reduces 1KDCI to DCI. Inactivation of iolE in a mutant constitutively expressing the iol operon blocked the MI catabolic pathway to accumulate 2KMI, which was converted to DCI via the activity of IolI and IolG. The mutant was able to convert at least 6% of input MI in the culture medium to DCI.

Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide

PubMed Central

2004-01-01

Antibacterial peptide acylation, which mimics the structure of the natural lipopeptide polymyxin B, increases antimicrobial and endotoxin-neutralizing activities. The interaction of the lactoferricin-derived peptide LF11 and its N-terminally acylated analogue, lauryl-LF11, with different chemotypes of bacterial lipopolysaccharide (LPS Re, Ra and smooth S form) was investigated by biophysical means and was related to the peptides' biological activities. Both peptides exhibit high antibacterial activity against the three strains of Salmonella enterica differing in the LPS chemotype. Lauryl-LF11 has one order of magnitude higher activity against Re-type, but activity against Ra- and S-type bacteria is comparable with that of LF11. The alkyl derivative peptide lauryl-LF11 shows a much stronger inhibition of the LPS-induced cytokine induction in human mononuclear cells than LF11. Although peptide–LPS interaction is essentially of electrostatic nature, the lauryl-modified peptide displays a strong hydrophobic component. Such a feature might then explain the fact that saturation of the peptide binding takes place at a much lower peptide/LPS ratio for LF11 than for lauryl-LF11, and that an overcompensation of the negative LPS backbone charges is observed for lauryl-LF11. The influence of LF11 on the gel-to-liquid-crystalline phase-transition of LPS is negligible for LPS Re, but clearly fluidizing for LPS Ra. In contrast, lauryl-LF11 causes a cholesterol-like effect in the two chemotypes, fluidizing in the gel and rigidifying of the hydrocarbon chains in the liquid-crystalline phase. Both peptides convert the mixed unilamellar/non-lamellar aggregate structure of lipid A, the ‘endotoxic principle’ of LPS, into a multilamellar one. These data contribute to the understanding of the mechanisms of the peptide-mediated neutralization of endotoxin and effect of lipid modification of peptides. PMID:15344905

In Azospirillum brasilense, mutations in flmA or flmB genes affect polar flagellum assembly, surface polysaccharides, and attachment to maize roots.

PubMed

Rossi, Fernando Ariel; Medeot, Daniela Beatriz; Liaudat, Juan Pablo; Pistorio, Mariano; Jofré, Edgardo

2016-09-01

Azospirillum brasilense is a soil bacterium capable of promoting plant growth. Several surface components were previously reported to be involved in the attachment of A. brasilense to root plants. Among these components are the exopolysaccharide (EPS), lipopolysaccharide (LPS) and the polar flagellum. Flagellin from polar flagellum is glycosylated and it was suggested that genes involved in such a posttranslational modification are the same ones involved in the biosynthesis of sugars present in the O-antigen of the LPS. In this work, we report on the characterization of two homologs present in A. brasilense Cd, to the well characterized flagellin modification genes, flmA and flmB, from Aeromonas caviae. We show that mutations in either flmA or flmB genes of A. brasilense resulted in non-motile cells due to alterations in the polar flagellum assembly. Moreover, these mutations also affected the capability of A. brasilense cells to adsorb to maize roots and to produce LPS and EPS. By generating a mutant containing the polar flagellum affected in their rotation, we show the importance of the bacterial motility for the early colonization of maize roots. Copyright © 2016 Elsevier GmbH. All rights reserved.

Endogenous hydrogen sulfide regulates histone demethylase JMJD3-mediated inflammatory response in LPS-stimulated macrophages and in a mouse model of LPS-induced septic shock.

PubMed

Liu, Siyu; Wang, Xiling; Pan, Lilong; Wu, Weijun; Yang, Di; Qin, Ming; Jia, Wanwan; Xiao, Chenxi; Long, Fen; Ge, Junbo; Liu, Xinhua; Zhu, YiZhun

2018-03-01

Overproduction of inflammatory mediators contributes to uncontrolled inflammation during endotoxin shock. Cystathionine-γ-lyase (CSE), an enzyme involved in hydrogen sulfide (H 2 S) biosynthesis, has potential anti-inflammatory activity in a variety of inflammatory diseases. Jumonji domain-containing protein 3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase, has been implicated in macrophage activation, but its function in CSE-mediated anti-inflammatory activities remains unknown. In the present study CSE was found to be upregulated in macrophages and mouse lipopolysaccharide (LPS) challenge models. LPS stimulation also enhanced the activation of JMJD3 and decreased H3K27me3 levels. JMJD3 knockdown upregulated H3K27me3 levels and attenuated the LPS-mediated inflammatory response. CSE knockout amplified the inflammatory cascade by increasing JMJD3 expression in septic mice. Similarly, enhanced production of inflammatory mediators by macrophages was mitigated by CSE overexpression via inhibition of JMJD3 expression. This is the first report indicating that inflammation enhanced CSE/H 2 S system biosynthesis, that in turn attenuated the LPS-triggered inflammatory response by regulating JMJD3 expression. Thus, the CSE/H 2 S system represents an epigenetic-based modification mechanism to prevent uncontrolled inflammation. Copyright © 2017 Elsevier Inc. All rights reserved.

Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

PubMed

Zhu, Wen-Liang; Cui, Jin-Yu; Cui, Lan-Yu; Liang, Wei-Fan; Yang, Song; Zhang, Chong; Xing, Xin-Hui

2016-03-01

Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Morgan N.; Arkin, Adam P.; Alm, Eric J.

Operons are a major feature of all prokaryotic genomes, but how and why operon structures vary is not well understood. To elucidate the life-cycle of operons, we compared gene order between Escherichia coli K12 and its relatives and identified the recently formed and destroyed operons in E. coli. This allowed us to determine how operons form, how they become closely spaced, and how they die. Our findings suggest that operon evolution is driven by selection on gene expression patterns. First, both operon creation and operon destruction lead to large changes in gene expression patterns. For example, the removal of lysAmore » and ruvA from ancestral operons that contained essential genes allowed their expression to respond to lysine levels and DNA damage, respectively. Second, some operons have undergone accelerated evolution, with multiple new genes being added during a brief period. Third, although most operons are closely spaced because of a neutral bias towards deletion and because of selection against large overlaps, highly expressed operons tend to be widely spaced because of regulatory fine-tuning by intervening sequences. Although operon evolution seems to be adaptive, it need not be optimal: new operons often comprise functionally unrelated genes that were already in proximity before the operon formed.« less

Evolutionary dynamics of nematode operons: easy come, slow go.

PubMed

Qian, Wenfeng; Zhang, Jianzhi

2008-03-01

Operons are widespread in prokaryotes, but are uncommon in eukaryotes, except nematode worms, where approximately 15% of genes reside in over 1100 operons in the model organism Caenorhabditis elegans. It is unclear how operons have become abundant in nematode genomes. The "one-way street" hypothesis asserts that once formed by chance, operons are very difficult to break, because the breakage would leave downstream genes in an operon without a promoter, and hence, unexpressed. To test this hypothesis, we analyzed the presence and absence of C. elegans operons in Caenorhabditis briggsae, Caenorhabditis remanei, and Caenorhabditis brenneri, using Pristionchus pacificus and Brugia malayi as outgroups, and identified numerous operon gains and losses. Coupled with experimental examination of trans-splicing patterns, our comparative genomic analysis revealed diverse molecular mechanisms of operon losses, including inversion, insertion, and relocation, but the presence of internal promoters was not found to facilitate operon losses. In several cases, the data allowed inference of mechanisms by which downstream genes are expressed after operon breakage. We found that the rate of operon gain is approximately 3.3 times that of operon loss. Thus, the evolutionary dynamics of nematode operons is better described as "easy come, slow go," rather than a "one-way street." Based on a mathematic model of operon gains and losses and additional assumptions, we projected that the number of operons in C. elegans will continue to rise by 6%-18% in future evolution before reaching equilibrium between operon gains and losses.

Curcumin Attenuates Lipopolysaccharide-Induced Hepatic Lipid Metabolism Disorder by Modification of m6 A RNA Methylation in Piglets.

PubMed

Lu, Na; Li, Xingmei; Yu, Jiayao; Li, Yi; Wang, Chao; Zhang, Lili; Wang, Tian; Zhong, Xiang

2018-01-01

N 6 -methyladenosine (m 6 A) regulates gene expression and affects cellular metabolism. In this study, we checked whether the regulation of lipid metabolism by curcumin is associated with m 6 A RNA methylation. We investigated the effects of dietary curcumin supplementation on lipopolysaccharide (LPS)-induced liver injury and lipid metabolism disorder, and on m 6 A RNA methylation in weaned piglets. A total of 24 Duroc × Large White × Landrace piglets were randomly assigned to control, LPS, and CurL (LPS challenge and 200 mg/kg dietary curcumin) groups (n = 8/group). The results showed that curcumin reduced the increase in relative liver weight as well as the concentrations of aspartate aminotransferase and lactate dehydrogenase induced by LPS injection in the plasma and liver of weaning piglets (p < 0.05). The amounts of total cholesterol and triacylglycerols were decreased by curcumin compared to that by the LPS injection (p < 0.05). Additionally, curcumin reduced the expression of Bcl-2 and Bax mRNA, whereas it increased the p53 mRNA level in the liver (p < 0.05). Curcumin inhibited the enhancement of SREBP-1c and SCD-1 mRNA levels induced by LPS in the liver. Notably, dietary curcumin affected the expression of METTL3, METTL14, ALKBH5, FTO, and YTHDF2 mRNA, and increased the abundance of m 6 A in the liver of piglets. In conclusion, the protective effect of curcumin in LPS-induced liver injury and hepatic lipid metabolism disruption might be due to the increase in m 6 A RNA methylation. Our study provides mechanistic insights into the effect of curcumin in protecting against hepatic injury during inflammation and metabolic diseases. © 2018 AOCS.

The Lipopolysaccharide of Brucella abortus BvrS/BvrR Mutants Contains Lipid A Modifications and Has Higher Affinity for Bactericidal Cationic Peptides

PubMed Central

Manterola, Lorea; Moriyón, Ignacio; Moreno, Edgardo; Sola-Landa, Alberto; Weiss, David S.; Koch, Michel H. J.; Howe, Jörg; Brandenburg, Klaus; López-Goñi, Ignacio

2005-01-01

The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic β(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants. PMID:16077108

Operon Formation is Driven by Co-Regulation and Not by Horizontal Gene Transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Morgan N.; Huang, Katherine H.; Arkin, Adam P.

Although operons are often subject to horizontal gene transfer (HGT), non-HGT genes are particularly likely to be in operons. To resolve this apparent discrepancy and to determine whether HGT is involved in operon formation, we examined the evolutionary history of the genes and operons in Escherichia coli K12. We show that genes that have homologs in distantly related bacteria but not in close relatives of E. coli (indicating HGTi) form new operons at about the same rates as native genes. Furthermore, genes in new operons are no more likely than other genes to have phylogenetic trees that are inconsistent withmore » the species tree. In contrast, essential genes and ubiquitous genes without paralogs (genes believed to undergo HGT rarely) often form new operons. We conclude that HGT is not associated with operon formation, but instead promotes the prevalence of pre-existing operons. To explain operon formation, we propose that new operons reduce the amount of regulatory information required to specify optimal expression patterns. Consistent with this hypothesis, operons have greater amounts of conserved regulatory sequences than do individually transcribed genes.« less

Toll-like receptor-4 is a target for suppression of proliferation and chemoresistance in HepG2 hepatoblastoma cells.

PubMed

Hsiao, Chih-Cheng; Chen, Po-Han; Cheng, Cheng-I; Tsai, Ming-Shian; Chang, Chih-Yang; Lu, Shang-Chieh; Hsieh, Ming-Chu; Lin, Yu-Chun; Lee, Po-Huang; Kao, Ying-Hsien

2015-11-01

Toll-like receptor-4 (TLR4) is known to influence growth and migration of hepatocellular tumors; however, its role in hepatoblastoma remains poorly understood. This study investigated the regulatory role of TLR4 in proliferation and chemoresistance of HepG2 hepatoblastoma cells. Treatment with lipopolysaccharide (LPS), a TLR4 agonist, was found to significantly upregulate TLR4 expression in HepG2 cells, but not in malignant Huh-7 and Sk-Hep1 hepatocellular carcinoma cells. Additionally, IL-6 enhanced LPS-induced TLR4 upregulation. LPS-stimulated TLR4 activation increased proliferation, nitric oxide synthase (NOS) expression, and NO production in HepG2 cells. Chemotherapeutic agents, cisplatin and doxorubicin, effectively inhibited TLR4 expression in HepG2 cells. Characterization of LPS-induced signaling activation and blockade with kinase inhibitors revealed the involvement of Akt and MAPK pathways in LPS-enhanced NO release from, and proliferation of HepG2 cells. Mechanistically, gene modifications as a result of TLR4 transfection and siRNA-mediated knockdown further demonstrated a crucial role for TLR4 in the regulation of NOS expression, cell proliferation, and chemoresistance in HepG2 cells. These findings suggest that targeting TLR4 expression and its cognate signaling may modulate proliferation and chemosensitivity in hepatoblastoma cells and serve as a potential therapeutic target. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Chronic Ethanol Feeding Modulates Inflammatory Mediators, Activation of Nuclear Factor-κB, and Responsiveness to Endotoxin in Murine Kupffer Cells and Circulating Leukocytes

PubMed Central

Oppermann, Elsie; Jobin, Christian; Schleucher, Elke; Marzi, Ingo

2014-01-01

Chronic ethanol abuse is known to increase susceptibility to infections after injury, in part, by modification of macrophage function. Several intracellular signalling mechanisms are involved in the initiation of inflammatory responses, including the nuclear factor-κB (NF-κB) pathway. In this study, we investigated the systemic and hepatic effect of chronic ethanol feeding on in vivo activation of NF-κB in NF-κBEGFP reporter gene mice. Specifically, the study focused on Kupffer cell proinflammatory cytokines IL-6 and TNF-α and activation of NF-κB after chronic ethanol feeding followed by in vitro stimulation with lipopolysaccharide (LPS). We found that chronic ethanol upregulated NF-κB activation and increased hepatic and systemic proinflammatory cytokine levels. Similarly, LPS-stimulated IL-1β release from whole blood was significantly enhanced in ethanol-fed mice. However, LPS significantly increased IL-6 and TNF-α levels. These results demonstrate that chronic ethanol feeding can improve the responsiveness of macrophage LPS-stimulated IL-6 and TNF-α production and indicate that this effect may result from ethanol-induced alterations in intracellular signalling through NF-κB. Furthermore, LPS and TNF-α stimulated the gene expression of different inflammatory mediators, in part, in a NF-κB-dependent manner. PMID:24623963

Design, synthesis, and structure-activity relationship study of halogen containing 2-benzylidene-1-indanone derivatives for inhibition of LPS-stimulated ROS production in RAW 264.7 macrophages.

PubMed

Shrestha, Aarajana; Jin Oh, Hye; Kim, Mi Jin; Pun, Nirmala Tilija; Magar, Til Bahadur Thapa; Bist, Ganesh; Choi, Hongseok; Park, Pil-Hoon; Lee, Eung-Seok

2017-06-16

As a continuous effort to discover new potential anti-inflammatory agents, we systematically designed and synthesized sixty-one 2-benzylidene-1-indanone derivatives with structural modification of chalcone, and evaluated their inhibitory activity on LPS-stimulated ROS production in RAW 264.7 macrophages. Systematic structure-activity relationship study revealed that hydroxyl group in C-5, C-6, or C-7 position of indanone moiety, and ortho-, meta-, or para-fluorine, trifluoromethyl, trifluoromethoxy, and bromine functionalities in phenyl ring are important for inhibition of ROS production in LPS-stimulated RAW 264.7 macrophages. Among all the tested compounds, 6-hydroxy-2-(2-(trifluoromethoxy) benzylidene)-2,3-dihydro-1H-inden-1-one (compound 44) showed the strongest inhibitory activity of ROS production. Further studies on the mode of action revealed that compound 44 potently suppressed LPS-stimulated ROS production via modulation of NADPH oxidase. The findings of this work could be useful to design 2-benzylidene-indanone based lead compounds as novel anti-inflammatory agents. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

A global analysis of adaptive evolution of operons in cyanobacteria.

PubMed

Memon, Danish; Singh, Abhay K; Pakrasi, Himadri B; Wangikar, Pramod P

2013-02-01

Operons are an important feature of prokaryotic genomes. Evolution of operons is hypothesized to be adaptive and has contributed significantly towards coordinated optimization of functions. Two conflicting theories, based on (i) in situ formation to achieve co-regulation and (ii) horizontal gene transfer of functionally linked gene clusters, are generally considered to explain why and how operons have evolved. Furthermore, effects of operon evolution on genomic traits such as intergenic spacing, operon size and co-regulation are relatively less explored. Based on the conservation level in a set of diverse prokaryotes, we categorize the operonic gene pair associations and in turn the operons as ancient and recently formed. This allowed us to perform a detailed analysis of operonic structure in cyanobacteria, a morphologically and physiologically diverse group of photoautotrophs. Clustering based on operon conservation showed significant similarity with the 16S rRNA-based phylogeny, which groups the cyanobacterial strains into three clades. Clade C, dominated by strains that are believed to have undergone genome reduction, shows a larger fraction of operonic genes that are tightly packed in larger sized operons. Ancient operons are in general larger, more tightly packed, better optimized for co-regulation and part of key cellular processes. A sub-clade within Clade B, which includes Synechocystis sp. PCC 6803, shows a reverse trend in intergenic spacing. Our results suggest that while in situ formation and vertical descent may be a dominant mechanism of operon evolution in cyanobacteria, optimization of intergenic spacing and co-regulation are part of an ongoing process in the life-cycle of operons.

Detecting uber-operons in prokaryotic genomes.

PubMed

Che, Dongsheng; Li, Guojun; Mao, Fenglou; Wu, Hongwei; Xu, Ying

2006-01-01

We present a study on computational identification of uber-operons in a prokaryotic genome, each of which represents a group of operons that are evolutionarily or functionally associated through operons in other (reference) genomes. Uber-operons represent a rich set of footprints of operon evolution, whose full utilization could lead to new and more powerful tools for elucidation of biological pathways and networks than what operons have provided, and a better understanding of prokaryotic genome structures and evolution. Our prediction algorithm predicts uber-operons through identifying groups of functionally or transcriptionally related operons, whose gene sets are conserved across the target and multiple reference genomes. Using this algorithm, we have predicted uber-operons for each of a group of 91 genomes, using the other 90 genomes as references. In particular, we predicted 158 uber-operons in Escherichia coli K12 covering 1830 genes, and found that many of the uber-operons correspond to parts of known regulons or biological pathways or are involved in highly related biological processes based on their Gene Ontology (GO) assignments. For some of the predicted uber-operons that are not parts of known regulons or pathways, our analyses indicate that their genes are highly likely to work together in the same biological processes, suggesting the possibility of new regulons and pathways. We believe that our uber-operon prediction provides a highly useful capability and a rich information source for elucidation of complex biological processes, such as pathways in microbes. All the prediction results are available at our Uber-Operon Database: http://csbl.bmb.uga.edu/uber, the first of its kind.

Detecting uber-operons in prokaryotic genomes

PubMed Central

Che, Dongsheng; Li, Guojun; Mao, Fenglou; Wu, Hongwei; Xu, Ying

2006-01-01

We present a study on computational identification of uber-operons in a prokaryotic genome, each of which represents a group of operons that are evolutionarily or functionally associated through operons in other (reference) genomes. Uber-operons represent a rich set of footprints of operon evolution, whose full utilization could lead to new and more powerful tools for elucidation of biological pathways and networks than what operons have provided, and a better understanding of prokaryotic genome structures and evolution. Our prediction algorithm predicts uber-operons through identifying groups of functionally or transcriptionally related operons, whose gene sets are conserved across the target and multiple reference genomes. Using this algorithm, we have predicted uber-operons for each of a group of 91 genomes, using the other 90 genomes as references. In particular, we predicted 158 uber-operons in Escherichia coli K12 covering 1830 genes, and found that many of the uber-operons correspond to parts of known regulons or biological pathways or are involved in highly related biological processes based on their Gene Ontology (GO) assignments. For some of the predicted uber-operons that are not parts of known regulons or pathways, our analyses indicate that their genes are highly likely to work together in the same biological processes, suggesting the possibility of new regulons and pathways. We believe that our uber-operon prediction provides a highly useful capability and a rich information source for elucidation of complex biological processes, such as pathways in microbes. All the prediction results are available at our Uber-Operon Database: , the first of its kind. PMID:16682449

Operon-mapper: A Web Server for Precise Operon Identification in Bacterial and Archaeal Genomes.

PubMed

Taboada, Blanca; Estrada, Karel; Ciria, Ricardo; Merino, Enrique

2018-06-19

Operon-mapper is a web server that accurately, easily, and directly predicts the operons of any bacterial or archaeal genome sequence. The operon predictions are based on the intergenic distance of neighboring genes as well as the functional relationships of their protein-coding products. To this end, Operon-mapper finds all the ORFs within a given nucleotide sequence, along with their genomic coordinates, orthology groups, and functional relationships. We believe that Operon-mapper, due to its accuracy, simplicity and speed, as well as the relevant information that it generates, will be a useful tool for annotating and characterizing genomic sequences. http://biocomputo.ibt.unam.mx/operon_mapper/.

The effect of loss of O-antigen ligase on phagocytic susceptibility of motile and non-motile Pseudomonas aeruginosa.

PubMed

Demirdjian, Sally; Schutz, Kristin; Wargo, Matthew J; Lam, Joseph S; Berwin, Brent

2017-12-01

The bacterial pathogen Pseudomonas aeruginosa undergoes adaptation and selection over the course of chronic respiratory tract infections which results in repeatedly-observed phenotypic changes that are proposed to enable its persistence. Two of the clinically significant P. aeruginosa phenotypic changes are loss of flagellar motility and modifications to LPS structure, including loss of O-antigen expression. The effect of loss of O-antigen, frequently described as conversion from smooth to rough LPS, and the combined effect of loss of motility and O-antigen on phagocytic susceptibility by immune cells remain unknown. To address this, we generated genetic deletion mutants of waaL, which encodes the O-antigen ligase responsible for linking O-antigen to lipid A-core oligosaccharide, in both motile and non-motile P. aeruginosa strains. With the use of these bacterial strains we provide the first demonstration that, despite a progressive selection for P. aeruginosa with rough LPS during chronic pulmonary infections, loss of the LPS O-antigen does not confer phagocytic resistance in vitro. However, use of the waaLmotABmotCD mutant revealed that loss of motility confers resistance to phagocytosis regardless of the smooth or rough LPS phenotype. These findings reveal how the O-antigen of P. aeruginosa can influence bacterial clearance during infection and expand our current knowledge about the impact of bacterial phenotypic changes during chronic infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural modifications of Helicobacter pylori lipopolysaccharide: An idea for how to live in peace

PubMed Central

Chmiela, Magdalena; Miszczyk, Eliza; Rudnicka, Karolina

2014-01-01

In this review, we discuss the findings and concepts underlying the “persistence mechanisms” of Helicobacter pylori (H. pylori), a spiral-shaped, Gram-negative rod bacterium that was discovered as a gastric pathogen by Marshall and Warren in 1984. H. pylori colonizes the gastric mucosa of nearly half of the human population. Infections appear in early childhood and, if not treated, persist for life. The presence or absence of symptoms and their severity depend on multiple bacterial components, host susceptibility and environmental factors, which allow H. pylori to switch between pathogenicity and commensalism. Many studies have shown that H. pylori components may facilitate the colonization process and the immune response of the host during the course of H. pylori infection. These H. pylori-driven interactions might result from positive or negative modulation. Among the negative immunomodulators, a prominent position is occupied by a vacuolating toxin A (VacA) and cytotoxin-associated gene A (CagA) protein. However, in light of the recent studies that are presented in this review, it is necessary to enrich this panel with H. pylori lipopolysaccharide (LPS). Together with CagA and VacA, LPS suppresses the elimination of H. pylori bacteria from the gastric mucosa by interfering with the activity of innate and adaptive immune cells, diminishing the inflammatory response, and affecting the adaptive T lymphocyte response, thus facilitating the development of chronic infections. The complex strategy of H. pylori bacteria for survival in the gastric mucosa of the host involves both structural modifications of LPS lipid A to diminish its endotoxic properties and the expression and variation of Lewis determinants, arranged in O-specific chains of H. pylori LPS. By mimicking host components, this phenomenon leaves these bacteria “invisible” to immune cells. Together, these mechanisms allow H. pylori to survive and live for many years within their hosts. PMID:25110419

Lipopolysaccharide loss produces partial colistin dependence and collateral sensitivity to azithromycin, rifampicin and vancomycin in Acinetobacter baumannii.

PubMed

García-Quintanilla, Meritxell; Carretero-Ledesma, Marta; Moreno-Martínez, Patricia; Martín-Peña, Reyes; Pachón, Jerónimo; McConnell, Michael J

2015-12-01

Treatment options for multidrug-resistant (MDR) strains of Acinetobacter baumannii that acquire resistance to colistin are limited. Acinetobacter baumannii can become highly resistant to colistin through complete loss of lipopolysaccharide (LPS) owing to mutations in the genes encoding the first three enzymes involved in lipid A biosynthesis (lpxA, lpxC and lpxD). The objective of this study was to characterise the susceptibility to 15 clinically relevant antibiotics and 6 antimicrobial peptides (AMPs) of MDR A. baumannii clinical isolates that acquired colistin resistance due to mutations in lpxA, lpxC and lpxD as well as their colistin-susceptible counterparts. A dramatic increase in antibiotic susceptibility (≥16-fold increase) was observed upon LPS loss for azithromycin, rifampicin and vancomycin, whereas a moderate increase in susceptibility was seen for amikacin, ceftazidime, imipenem, cefepime and meropenem. Importantly, concentrations ranging from 8 mg/L to 32 mg/L of the six AMPs were able to reduce bacterial viability by ≥3 log10 in growth curve assays. We also demonstrate that colistin resistance results in partial colistin dependence for growth in LPS-deficient strains containing mutations in lpxA, lpxC and lpxD, but not when colistin resistance occurs via LPS modification due to mutations in the PmrA/B two-component system. The results of this study indicate that loss of LPS expression results in collateral sensitivity to azithromycin, rifampicin and vancomycin, and that the six AMPs tested retain activity against LPS-deficient strains, indicating that these antibiotics may be viable treatment options for infections caused by these strains. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Lipopolysaccharide-induced sickness behaviour evaluated in different models of anxiety and innate fear in rats.

PubMed

Bassi, Gabriel S; Kanashiro, Alexandre; Santin, Francele M; de Souza, Glória E P; Nobre, Manoel J; Coimbra, Norberto C

2012-04-01

The fact that there is a complex and bidirectional communication between the immune and nervous systems has been well demonstrated. Lipopolysaccharide (LPS), a component of gram-negative bacteria, is widely used to systematically stimulate the immune system and generate profound physiological and behavioural changes, also known as 'sickness behaviour' (e.g. anhedonia, lethargy, loss of appetite, anxiety, sleepiness). Different ethological tools have been used to analyse the behavioural modifications induced by LPS; however, many researchers analysed only individual tests, a single LPS dose or a unique ethological parameter, thus leading to disagreements regarding the data. In the present study, we investigated the effects of different doses of LPS (10, 50, 200 and 500 μg/kg, i.p.) in young male Wistar rats (weighing 180-200 g; 8-9 weeks old) on the ethological and spatiotemporal parameters of the elevated plus maze, light-dark box, elevated T maze, open-field tests and emission of ultrasound vocalizations. There was a dose-dependent increase in anxiety-like behaviours caused by LPS, forming an inverted U curve peaked at LPS 200 μg/kg dose. However, these anxiety-like behaviours were detected only by complementary ethological analysis (stretching, grooming, immobility responses and alarm calls), and these reactions seem to be a very sensitive tool in assessing the first signs of sickness behaviour. In summary, the present work clearly showed that there are resting and alertness reactions induced by opposite neuroimmune mechanisms (neuroimmune bias) that could lead to anxiety behaviours, suggesting that misunderstanding data could occur when only few ethological variables or single doses of LPS are analysed. Finally, it is hypothesized that this bias is an evolutionary tool that increases animals' security while the body recovers from a systemic infection. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

Selfish operons: the evolutionary impact of gene clustering in prokaryotes and eukaryotes.

PubMed

Lawrence, J

1999-12-01

The Selfish Operon Model postulates that the organization of bacterial genes into operons is beneficial to the constituent genes in that proximity allows horizontal cotransfer of all genes required for a selectable phenotype; eukaryotic operons formed for very different reasons. Horizontal transfer of selfish operons most probably promotes bacterial diversification.

Evidence against the selfish operon theory.

PubMed

Pál, Csaba; Hurst, Laurence D

2004-06-01

According to the selfish operon hypothesis, the clustering of genes and their subsequent organization into operons is beneficial for the constituent genes because it enables the horizontal gene transfer of weakly selected, functionally coupled genes. The majority of these are expected to be non-essential genes. From our analysis of the Escherichia coli genome, we conclude that the selfish operon hypothesis is unlikely to provide a general explanation for clustering nor can it account for the gene composition of operons. Contrary to expectations, essential genes with related functions have an especially strong tendency to cluster, even if they are not in operons. Moreover, essential genes are particularly abundant in operons.

Roles of Salmonella typhimurium umuDC and samAB in UV mutagenesis and UV sensitivity.

PubMed Central

Nohmi, T; Yamada, M; Watanabe, M; Murayama, S Y; Sofuni, T

1992-01-01

Expression of the umuDC operon is required for UV mutagenesis and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons; the samAB operon is located in a 60-MDa cryptic plasmid, while the S. typhimurium umuDC (umuDCST) operon resides in a chromosome. The roles of these two umuDC-like operons in UV mutagenesis and UV sensitivity of S. typhimurium were investigated. A pBR322-derived plasmid carrying the samAB operon more efficiently restored UV mutability to a umuD44 strain and a umuC122::Tn5 strain of E. coli than a plasmid carrying the umuDCST operon did. When the umuDCST operon was specifically deleted from the chromosome of S. typhimurium TA2659, the resulting strain was not UV mutable and was more sensitive to the killing effect of UV irradiation than the parent strain was. Curing of the 60-MDa cryptic plasmid carrying the samAB operon did not influence the UV mutability of strain TA2659 but did increase its resistance to UV killing. A pSC101-derived plasmid carrying the samAB operon did not restore UV mutability to a umuD44 strain of E. coli, whereas pBR322- or pBluescript-derived plasmids carrying the samAB operon efficiently did restore UV mutability. We concluded that the umuDCST operon plays a major role in UV mutagenesis in S. typhimurium and that the ability of the samAB operon to promote UV mutagenesis is strongly affected by gene dosage. Possible reasons for the poor ability of samAB to promote UV mutagenesis when it is present on low-copy-number plasmids are discussed. Images PMID:1400244

Local and systemic response to intramammary lipopolysaccharide challenge during long-term manipulated plasma glucose and insulin concentrations in dairy cows.

PubMed

Vernay, M C M B; Wellnitz, O; Kreipe, L; van Dorland, H A; Bruckmaier, R M

2012-05-01

The metabolic load during periods of high milk production in dairy cows causes a variety of changes of metabolite blood concentrations including dramatically decreased glucose levels. These changes supposedly impair the immune system. The goal of this study was, therefore, to evaluate adaptations of the cow's immune system in response to an intramammary lipopolysaccharide (LPS) stimulation during a 3-d modification of plasma glucose and insulin induced by different clamp infusions. Seventeen midlactating dairy cows received a hypoglycemic hyperinsulinemic clamp induced by insulin infusion (HypoG; n=5), a euglycemic hyperinsulinemic clamp induced by insulin and glucose infusion (EuG; n=6), or infusion of saline solution (NaCl; n=6) for 56 h. At 48 h of infusion, 2 udder quarters were challenged with 200 μg of Escherichia coli LPS. At 48 h of infusion (immediately before LPS challenge), tumor necrosis factor α, lactoferrin, and serum amyloid A (SAA) mRNA abundance was increased in HypoG and Il-1β mRNA abundance was decreased in EuG. After LPS challenge, plasma glucose concentration did not decrease, although plasma insulin increased simultaneously in all groups either due to enhanced endogenous release (NaCl) or due to increased insulin infusion rate (HypoG; EuG). Plasma cortisol, rectal temperatures, and milk somatic cell count of challenged quarters increased, whereas plasma nonesterified fatty acid concentrations were similarly decreased across treatments. In mammary biopsies, increased mRNA expression of tumor necrosis factor α, IL-1β, IL-8, and IL-10, and SAA were observed in LPS-treated quarters of all groups, with a more pronounced increase in IL-1β, IL-10, and SAA expression in EuG. Nuclear factor-κB mRNA expression was upregulated in NaCl and EuG but not in HypoG in response to LPS. Lactoferrin, toll-like receptor 4, and cyclooxygenase-2 mRNA expression was increased in LPS-treated quarters of EuG only, and 5-lipoxygenase mRNA expression was decreased in LPS-treated quarters only in treatments HypoG and NaCl. In conclusion, intramammary LPS induces local and systemic inflammatory responses, as well as systemic insulin resistance. The observed treatment differences of the mammary mRNA expression of several immune parameters both before and after LPS challenge indicate a direct influence of changed glucose and insulin concentrations during the course of lactation on the immune defense against mastitis pathogens. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Lipopolysaccharide inhibits or accelerates biomedical titanium corrosion depending on environmental acidity

PubMed Central

Yu, Fei; Addison, Owen; Baker, Stephen J; Davenport, Alison J

2015-01-01

Titanium and its alloys are routinely used as biomedical implants and are usually considered to be corrosion resistant under physiological conditions. However, during inflammation, chemical modifications of the peri-implant environment including acidification occur. In addition certain biomolecules including lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls and driver of inflammation have been shown to interact strongly with Ti and modify its corrosion resistance. Gram-negative microbes are abundant in biofilms which form on dental implants. The objective was to investigate the influence of LPS on the corrosion properties of relevant biomedical Ti substrates as a function of environmental acidity. Inductively coupled plasma mass spectrometry was used to quantify Ti dissolution following immersion testing in physiological saline for three common biomedical grades of Ti (ASTM Grade 2, Grade 4 and Grade 5). Complementary electrochemical tests including anodic and cathodic polarisation experiments and potentiostatic measurements were also conducted. All three Ti alloys were observed to behave similarly and ion release was sensitive to pH of the immersion solution. However, LPS significantly inhibited Ti release under the most acidic conditions (pH 2), which may develop in localized corrosion sites, but promoted dissolution at pH 4–7, which would be more commonly encountered physiologically. The observed pattern of sensitivity to environmental acidity of the effect of LPS on Ti corrosion has not previously been reported. LPS is found extensively on the surfaces of skin and mucosal penetrating Ti implants and the findings are therefore relevant when considering the chemical stability of Ti implant surfaces in vivo. PMID:25634122

Synthesis and biological evaluation of 5-carbamoyl-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors.

PubMed

Goto, Taiji; Shiina, Akiko; Yoshino, Toshiharu; Mizukami, Kiyoshi; Hirahara, Kazuki; Suzuki, Osamu; Sogawa, Yoshitaka; Takahashi, Tomoko; Mikkaichi, Tsuyoshi; Nakao, Naoki; Takahashi, Mizuki; Hasegawa, Masashi; Sasaki, Shigeki

2013-11-15

5-Carbamoyl-2-phenylpyrimidine derivative 2 has been identified as a phosphodiesterase 4 (PDE4) inhibitor with moderate PDE4B inhibitory activity (IC50=200 nM). Modification of the carboxylic acid moiety of 2 gave N-neopentylacetamide derivative 10f, which had high in vitro PDE4B inhibitory activity (IC50=8.3 nM) and in vivo efficacy against lipopolysaccharide (LPS)-induced pulmonary neutrophilia in mice (ID50=16 mg/kg, ip). Furthermore, based on the X-ray crystallography of 10f bound to the human PDE4B catalytic domain, we designed 7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one derivative 39 which has a fused bicyclic lactam scaffold. Compound 39 exhibited excellent inhibitory activity against LPS-induced tumor necrosis factor alpha (TNF-α) production in mouse splenocytes (IC50=0.21 nM) and in vivo anti-inflammatory activity against LPS-induced pulmonary neutrophilia in mice (41% inhibition at a dose of 1.0 mg/kg, i.t.). Copyright © 2013 Elsevier Ltd. All rights reserved.

The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB.

PubMed

Schneider, Monika; Zimmermann, Albert G; Roberts, Reid A; Zhang, Lu; Swanson, Karen V; Wen, Haitao; Davis, Beckley K; Allen, Irving C; Holl, Eda K; Ye, Zhengmao; Rahman, Adeeb H; Conti, Brian J; Eitas, Timothy K; Koller, Beverly H; Ting, Jenny P-Y

2012-09-01

Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo, macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3(-/-) mice. LPS-treated Nlrc3(-/-) macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3(-/-) mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.

Chemical compositions and properties of Schinus areira L. essential oil on airway inflammation and cardiovascular system of mice and rabbits.

PubMed

Bigliani, María C; Rossetti, Víctor; Grondona, Ezequiel; Lo Presti, Silvina; Paglini, Patricia M; Rivero, Virginia; Zunino, María P; Ponce, Andrés A

2012-07-01

The main purpose was to investigate the effects of essential plant-oil of Schinus areira L. on hemodynamic functions in rabbits, as well as myocardial contractile strength and airways inflammation associated to bacterial endotoxin lipopolysaccharide (LPS) in mice. This study shows the important properties of the essential oil (EO) of S. areira studied and these actions on lung with significant inhibition associated to LPS, all of which was assessed in mice bronchoalveolar lavage fluid and evidenced by stability of the percentage of alveolar macrophages, infiltration of polymorphonuclear leukocytes and tumor necrosis factor-α concentration, and without pathway modifications in conjugated dienes activity. Clinical status (morbidity or mortality), macroscopic morphology and lung/body weight index were unaffected by the administration of the EO S. areira. Furthermore, the ex vivo analysis of isolated hearts demonstrated the negative inotropic action of the EO of S. areira in a mice model, and in rabbits changes in the hemodynamic parameters, such as a reduction of systolic blood pressure. We conclude that EO S. areira could be responsible for modifications on the cardiovascular and/or airway parameters. Copyright © 2012 Elsevier Ltd. All rights reserved.

Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing.

PubMed

Conway, Tyrrell; Creecy, James P; Maddox, Scott M; Grissom, Joe E; Conkle, Trevor L; Shadid, Tyler M; Teramoto, Jun; San Miguel, Phillip; Shimada, Tomohiro; Ishihama, Akira; Mori, Hirotada; Wanner, Barry L

2014-07-08

We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future. Copyright © 2014 Conway et al.

Comparison of the overlapping frd and ampC operons of Escherichia coli with the corresponding DNA sequences in other gram-negative bacteria.

PubMed

Bergström, S; Lindberg, F P; Olsson, O; Normark, S

1983-09-01

Specific DNA probes from Escherichia coli K-12 were used to analyze the sequence divergence of the frd and ampC operons in various species of gram-negative bacteria. These operons code for the fumarate reductase complex and the chromosomal beta-lactamase, respectively. We demonstrate that the two operons show the same general pattern of divergence, although the frd operon is considerably more conserved than is the ampC operon. The major exception is Salmonella typhimurium LT2, which shows a strong homology to the E. coli frd probe but none to the E. coli ampC probe. The operons from Citrobacter freundii and Shigella sonnei were cloned and characterized by physical mapping, Southern hybridization, and protein synthesis in minicells. In S. sonnei, as in E. coli K-12, the frd and ampC operons overlap (T. Grundström and B. Jaurin, Proc. Natl. Acad. Sci. U.S.A. 79:1111-1115, 1982). Only minor discrepancies between the two operons were found over the entire frd-ampC region. In C. freundii, the ampC and frd operons do not overlap, being separated by about 1,100 base pairs. Presumably the inducible property of the C. freundii chromosomal beta-lactamase is encoded by this 1,100-base-pair DNA segment.

Interplay of Noisy Gene Expression and Dynamics Explains Patterns of Bacterial Operon Organization

NASA Astrophysics Data System (ADS)

Igoshin, Oleg

2011-03-01

Bacterial chromosomes are organized into operons -- sets of genes co-transcribed into polycistronic messenger RNA. Hypotheses explaining the emergence and maintenance of operons include proportional co-regulation, horizontal transfer of intact ``selfish'' operons, emergence via gene duplication, and co-production of physically interacting proteins to speed their association. We hypothesized an alternative: operons can reduce or increase intrinsic gene expression noise in a manner dependent on the post-translational interactions, thereby resulting in selection for or against operons in depending on the network architecture. We devised five classes of two-gene network modules and show that the effects of operons on intrinsic noise depend on class membership. Two classes exhibit decreased noise with co-transcription, two others reveal increased noise, and the remaining one does not show a significant difference. To test our modeling predictions we employed bioinformatic analysis to determine the relationship gene expression noise and operon organization. The results confirm the overrepresentation of noise-minimizing operon architectures and provide evidence against other hypotheses. Our results thereby suggest a central role for gene expression noise in selecting for or maintaining operons in bacterial chromosomes. This demonstrates how post-translational network dynamics may provide selective pressure for organizing bacterial chromosomes, and has practical consequences for designing synthetic gene networks. This work is supported by National Institutes of Health grant 1R01GM096189-01.

Operon Conservation and the Evolution of trans-Splicing in the Phylum Nematoda

PubMed Central

Guiliano, David B; Blaxter, Mark L

2006-01-01

The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage. PMID:17121468

Glycopeptide Resistance vanA Operons in Paenibacillus Strains Isolated from Soil

PubMed Central

Guardabassi, Luca; Perichon, Bruno; van Heijenoort, Jean; Blanot, Didier; Courvalin, Patrice

2005-01-01

The sequence and gene organization of the van operons in vancomycin (MIC of >256 μg/ml)- and teicoplanin (MIC of ≥32 μg/ml)-resistant Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B isolated from soil were determined. Both operons had regulatory (vanR and vanS), resistance (vanH, vanA, and vanX), and accessory (vanY, vanZ, and vanW) genes homologous to the corresponding genes in enterococcal vanA and vanB operons. The vanAPT operon in P. thiaminolyticus PT-2B1 had the same gene organization as that of vanA operons whereas vanAPA in P. apiarius PA-B2B resembled vanB operons due to the presence of vanW upstream from the vanHAX cluster but was closer to vanA operons in sequence. Reference P. apiarius strains NRRL B-4299 and NRRL B-4188 were found to harbor operons indistinguishable from vanAPA by PCR mapping, restriction fragment length polymorphism, and partial sequencing, suggesting that this operon was species specific. As in enterococci, resistance was inducible by glycopeptides and associated with the synthesis of pentadepsipeptide peptidoglycan precursors ending in d-Ala-d-Lac, as demonstrated by d,d-dipeptidase activities, high-pressure liquid chromatography, and mass spectrometry. The precursors differed from those in enterococci by the presence of diaminopimelic acid instead of lysine in the peptide chain. Altogether, the results are compatible with the notion that van operons in soil Paenibacillus strains and in enterococci have evolved from a common ancestor. PMID:16189102

Glycopeptide resistance vanA operons in Paenibacillus strains isolated from soil.

PubMed

Guardabassi, Luca; Perichon, Bruno; van Heijenoort, Jean; Blanot, Didier; Courvalin, Patrice

2005-10-01

The sequence and gene organization of the van operons in vancomycin (MIC of >256 microg/ml)- and teicoplanin (MIC of > or =32 microg/ml)-resistant Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B isolated from soil were determined. Both operons had regulatory (vanR and vanS), resistance (vanH, vanA, and vanX), and accessory (vanY, vanZ, and vanW) genes homologous to the corresponding genes in enterococcal vanA and vanB operons. The vanA(PT) operon in P. thiaminolyticus PT-2B1 had the same gene organization as that of vanA operons whereas vanA(PA) in P. apiarius PA-B2B resembled vanB operons due to the presence of vanW upstream from the vanHAX cluster but was closer to vanA operons in sequence. Reference P. apiarius strains NRRL B-4299 and NRRL B-4188 were found to harbor operons indistinguishable from vanA(PA) by PCR mapping, restriction fragment length polymorphism, and partial sequencing, suggesting that this operon was species specific. As in enterococci, resistance was inducible by glycopeptides and associated with the synthesis of pentadepsipeptide peptidoglycan precursors ending in D-Ala-D-Lac, as demonstrated by D,D-dipeptidase activities, high-pressure liquid chromatography, and mass spectrometry. The precursors differed from those in enterococci by the presence of diaminopimelic acid instead of lysine in the peptide chain. Altogether, the results are compatible with the notion that van operons in soil Paenibacillus strains and in enterococci have evolved from a common ancestor.

Insights into arsenic multi-operons expression and resistance mechanisms in Rhodopseudomonas palustris CGA009

PubMed Central

Zhao, Chungui; Zhang, Yi; Chan, Zhuhua; Chen, Shicheng; Yang, Suping

2015-01-01

Arsenic (As) is widespread in the environment and causes numerous health problems. Rhodopseudomonas palustris has been regarded as a good model organism for studying arsenic detoxification since it was first demonstrated to methylate environmental arsenic by conversion to soluble or gaseous methylated species. However, the detailed arsenic resistance mechanisms remain unknown though there are at least three arsenic-resistance operons (ars1, ars2, and ars3) in R. palustris. In this study, we investigated how arsenic multi-operons contributed to arsenic detoxification in R. palustris. The expression of ars2 or ars3 operons increased with increasing environmental arsenite (As(III)) concentrations (up to 1.0 mM) while transcript of ars1 operon was not detected in the middle log-phase (55 h). ars2 operon was actively expressed even at the low concentration of As(III) (0.01 μM), whereas the ars3 operon was expressed at 1.0 μM of As(III), indicating that there was a differential regulation mechanism for the three arsenic operons. Furthermore, ars2 and ars3 operons were maximally transcribed in the early log-phase where ars2 operon was 5.4-fold higher than that of ars3 operon. A low level of ars1 transcript was only detected at 43 h (early log-phase). Arsenic speciation analysis demonstrated that R. palustris could reduce As(V) to As(III). Collectively, strain CGA009 detoxified arsenic by using arsenic reduction and methylating arsenic mechanism, while the latter might occur with the presence of higher concentrations of arsenic. PMID:26441915

Partially Glycosylated Dendrimers Block MD-2 and Prevent TLR4-MD-2-LPS Complex Mediated Cytokine Responses

PubMed Central

Barata, Teresa S.; Teo, Ian; Brocchini, Steve; Zloh, Mire; Shaunak, Sunil

2011-01-01

The crystal structure of the TLR4-MD-2-LPS complex responsible for triggering powerful pro-inflammatory cytokine responses has recently become available. Central to cell surface complex formation is binding of lipopolysaccharide (LPS) to soluble MD-2. We have previously shown, in biologically based experiments, that a generation 3.5 PAMAM dendrimer with 64 peripheral carboxylic acid groups acts as an antagonist of pro-inflammatory cytokine production after surface modification with 8 glucosamine molecules. We have also shown using molecular modelling approaches that this partially glycosylated dendrimer has the flexibility, cluster density, surface electrostatic charge, and hydrophilicity to make it a therapeutically useful antagonist of complex formation. These studies enabled the computational study of the interactions of the unmodified dendrimer, glucosamine, and of the partially glycosylated dendrimer with TLR4 and MD-2 using molecular docking and molecular dynamics techniques. They demonstrate that dendrimer glucosamine forms co-operative electrostatic interactions with residues lining the entrance to MD-2's hydrophobic pocket. Crucially, dendrimer glucosamine interferes with the electrostatic binding of: (i) the 4′phosphate on the di-glucosamine of LPS to Ser118 on MD-2; (ii) LPS to Lys91 on MD-2; (iii) the subsequent binding of TLR4 to Tyr102 on MD-2. This is followed by additional co-operative interactions between several of the dendrimer glucosamine's carboxylic acid branches and MD-2. Collectively, these interactions block the entry of the lipid chains of LPS into MD-2's hydrophobic pocket, and also prevent TLR4-MD-2-LPS complex formation. Our studies have therefore defined the first nonlipid-based synthetic MD-2 antagonist using both animal model-based studies of pro-inflammatory cytokine responses and molecular modelling studies of a whole dendrimer with its target protein. Using this approach, it should now be possible to computationally design additional macromolecular dendrimer based antagonists for other Toll Like Receptors. They could be useful for treating a spectrum of infectious, inflammatory and malignant diseases. PMID:21738462

Hypermutation in derepressed operons of Escherichia coli K12

PubMed Central

Wright, Barbara E.; Longacre, Angelika; Reimers, Jacqueline M.

1999-01-01

This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation. Derepression of the leu operon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response). A quantitative correlation was established between leuB mRNA abundance and leuB− reversion rates. To further demonstrate that derepression increased mutation rates, the chromosomal leu operon was placed under the control of the inducible tac promoter. When the leu operon was induced by isopropyl-d-thiogalactoside, both leuB mRNA abundance and leuB− reversion rates increased. These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulating leu operon expression and that higher rates of mutation are specifically associated with the derepressed leu operon. PMID:10220423

Teaching the Big Ideas of Biology with Operon Models

ERIC Educational Resources Information Center

Cooper, Robert A.

2015-01-01

This paper presents an activity that engages students in model-based reasoning, requiring them to predict the behavior of the trp and lac operons under different environmental conditions. Students are presented six scenarios for the "trp" operon and five for the "lac" operon. In most of the scenarios, specific mutations have…

A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

PubMed Central

Tian, Tian; Salis, Howard M.

2015-01-01

Natural and engineered genetic systems require the coordinated expression of proteins. In bacteria, translational coupling provides a genetically encoded mechanism to control expression level ratios within multi-cistronic operons. We have developed a sequence-to-function biophysical model of translational coupling to predict expression level ratios in natural operons and to design synthetic operons with desired expression level ratios. To quantitatively measure ribosome re-initiation rates, we designed and characterized 22 bi-cistronic operon variants with systematically modified intergenic distances and upstream translation rates. We then derived a thermodynamic free energy model to calculate de novo initiation rates as a result of ribosome-assisted unfolding of intergenic RNA structures. The complete biophysical model has only five free parameters, but was able to accurately predict downstream translation rates for 120 synthetic bi-cistronic and tri-cistronic operons with rationally designed intergenic regions and systematically increased upstream translation rates. The biophysical model also accurately predicted the translation rates of the nine protein atp operon, compared to ribosome profiling measurements. Altogether, the biophysical model quantitatively predicts how translational coupling controls protein expression levels in synthetic and natural bacterial operons, providing a deeper understanding of an important post-transcriptional regulatory mechanism and offering the ability to rationally engineer operons with desired behaviors. PMID:26117546

15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-{alpha} by decreasing levels of acetylated histone H3 and H4 at its promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Engdahl, Ryan; Monroy, M. Alexandra; Temple University School of Medicine, Department of Anatomy and Cell Biology, 3400 North Broad Street, Philadelphia, PA 19140

2007-07-20

Prostaglandin metabolite 15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPAR{gamma}. We investigated the ability of 15d-PGJ2 to inhibit TNF-{alpha} gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 {mu}M) inhibited LPS-stimulated TNF-{alpha} mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPAR{gamma} ligand, GW1929, failed to inhibit LPS-induced TNF-{alpha} mRNA expression nor did a PPAR{gamma} antagonist, GW9662, alter the repression of TNF-{alpha} mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPAR{gamma}-independent inhibition of TNF-{alpha} mRNA in THP-1more » cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-{alpha} promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-{alpha} promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-{alpha} promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-{alpha} promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-{alpha} promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-{alpha} transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter.« less

Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1

PubMed Central

Jiang, Bei; Xiao, Bo; Liu, Linde; Ge, Yihe; Hu, Xiaomei

2016-01-01

Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1. PMID:26735915

Genome-wide analysis of trans-splicing in the nematode Pristionchus pacificus unravels conserved gene functions for germline and dauer development in divergent operons.

PubMed

Sinha, Amit; Langnick, Claudia; Sommer, Ralf J; Dieterich, Christoph

2014-09-01

Discovery of trans-splicing in multiple metazoan lineages led to the identification of operon-like gene organization in diverse organisms, including trypanosomes, tunicates, and nematodes, but the functional significance of such operons is not completely understood. To see whether the content or organization of operons serves similar roles across species, we experimentally defined operons in the nematode model Pristionchus pacificus. We performed affinity capture experiments on mRNA pools to specifically enrich for transcripts that are trans-spliced to either the SL1- or SL2-spliced leader, using spliced leader-specific probes. We obtained distinct trans-splicing patterns from the analysis of three mRNA pools (total mRNA, SL1 and SL2 fraction) by RNA-seq. This information was combined with a genome-wide analysis of gene orientation and spacing. We could confirm 2219 operons by RNA-seq data out of 6709 candidate operons, which were predicted by sequence information alone. Our gene order comparison of the Caenorhabditis elegans and P. pacificus genomes shows major changes in operon organization in the two species. Notably, only 128 out of 1288 operons in C. elegans are conserved in P. pacificus. However, analysis of gene-expression profiles identified conserved functions such as an enrichment of germline-expressed genes and higher expression levels of operonic genes during recovery from dauer arrest in both species. These results provide support for the model that a necessity for increased transcriptional efficiency in the context of certain developmental processes could be a selective constraint for operon evolution in metazoans. Our method is generally applicable to other metazoans to see if similar functional constraints regulate gene organization into operons. © 2014 Sinha et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Ancient Origin of the Tryptophan Operon and the Dynamics of Evolutionary Change†

PubMed Central

Xie, Gary; Keyhani, Nemat O.; Bonner; Jensen, Roy A.

2003-01-01

The seven conserved enzymatic domains required for tryptophan (Trp) biosynthesis are encoded in seven genetic regions that are organized differently (whole-pathway operons, multiple partial-pathway operons, and dispersed genes) in prokaryotes. A comparative bioinformatics evaluation of the conservation and organization of the genes of Trp biosynthesis in prokaryotic operons should serve as an excellent model for assessing the feasibility of predicting the evolutionary histories of genes and operons associated with other biochemical pathways. These comparisons should provide a better understanding of possible explanations for differences in operon organization in different organisms at a genomics level. These analyses may also permit identification of some of the prevailing forces that dictated specific gene rearrangements during the course of evolution. Operons concerned with Trp biosynthesis in prokaryotes have been in a dynamic state of flux. Analysis of closely related organisms among the Bacteria at various phylogenetic nodes reveals many examples of operon scission, gene dispersal, gene fusion, gene scrambling, and gene loss from which the direction of evolutionary events can be deduced. Two milestone evolutionary events have been mapped to the 16S rRNA tree of Bacteria, one splitting the operon in two, and the other rejoining it by gene fusion. The Archaea, though less resolved due to a lesser genome representation, appear to exhibit more gene scrambling than the Bacteria. The trp operon appears to have been an ancient innovation; it was already present in the common ancestor of Bacteria and Archaea. Although the operon has been subjected, even in recent times, to dynamic changes in gene rearrangement, the ancestral gene order can be deduced with confidence. The evolutionary history of the genes of the pathway is discernible in rough outline as a vertical line of descent, with events of lateral gene transfer or paralogy enriching the analysis as interesting features that can be distinguished. As additional genomes are thoroughly analyzed, an increasingly refined resolution of the sequential evolutionary steps is clearly possible. These comparisons suggest that present-day trp operons that possess finely tuned regulatory features are under strong positive selection and are able to resist the disruptive evolutionary events that may be experienced by simpler, poorly regulated operons. PMID:12966138

Cyclophilin B attenuates the expression of TNF-α in lipopolysaccharide-stimulated macrophages through the induction of B cell lymphoma-3.

PubMed

Marcant, Adeline; Denys, Agnès; Melchior, Aurélie; Martinez, Pierre; Deligny, Audrey; Carpentier, Mathieu; Allain, Fabrice

2012-08-15

Extracellular cyclophilin A (CyPA) and CyPB have been well described as chemotactic factors for various leukocyte subsets, suggesting their contribution to inflammatory responses. Unlike CyPA, CyPB accumulates in extracellular matrixes, from which it is released by inflammatory proteases. Hence, we hypothesized that it could participate in tissue inflammation by regulating the activity of macrophages. In the current study, we confirmed that CyPB initiated in vitro migration of macrophages, but it did not induce production of proinflammatory cytokines. In contrast, pretreatment of macrophages with CyPB attenuated the expression of inflammatory mediators induced by LPS stimulation. The expression of TNF-α mRNA was strongly reduced after exposure to CyPB, but it was not accompanied by significant modification in LPS-induced activation of MAPK and NF-κB pathways. LPS activation of a reporter gene under the control of TNF-α gene promoter was also markedly decreased in cells treated with CyPB, suggesting a transcriptional mechanism of inhibition. Consistent with this hypothesis, we found that CyPB induced the expression of B cell lymphoma-3 (Bcl-3), which was accompanied by a decrease in the binding of NF-κB p65 to the TNF-α promoter. As expected, interfering with the expression of Bcl-3 restored cell responsiveness to LPS, thus confirming that CyPB acted by inhibiting initiation of TNF-α gene transcription. Finally, we found that CyPA was not efficient in attenuating the production of TNF-α from LPS-stimulated macrophages, which seemed to be due to a modest induction of Bcl-3 expression. Collectively, these findings suggest an unexpected role for CyPB in attenuation of the responses of proinflammatory macrophages.

Burkholderia thailandensis oacA mutants facilitate the expression of Burkholderia mallei-like O polysaccharides.

PubMed

Brett, Paul J; Burtnick, Mary N; Heiss, Christian; Azadi, Parastoo; DeShazer, David; Woods, Donald E; Gherardini, Frank C

2011-02-01

Previous studies have shown that the O polysaccharides (OPS) expressed by Burkholderia mallei are similar to those produced by Burkholderia thailandensis except that they lack the 4-O-acetyl modifications on their 6-deoxy-α-l-talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designated oacA, expressed by B. thailandensis that accounts for this phenomenon. Utilizing the B. thailandensis and B. mallei lipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by the oacA mutant, B. thailandensis ZT0715, were antigenically similar to those produced by B. mallei ATCC 23344. In addition, immunoblot analyses demonstrated that when B. mallei ATCC 23344 was complemented in trans with oacA, it synthesized B. thailandensis-like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by the oacA mutant reacted strongly with the B. mallei LPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4-O acetylation and 2-O methylation of B. thailandensis OPS antigens and that ZT0715 may provide a safe and cost-effective source of B. mallei-like OPS to facilitate the synthesis of glanders subunit vaccine candidates.

Problem-Solving Test: Tryptophan Operon Mutants

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2010-01-01

This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

Macrophage genetic reprogramming during chronic peritonitis is augmented by LPS pretreatment.

PubMed

Kanaan, Ziad; Gardner, Sarah; Carruba, Christopher; Mattingly, Jameson; Druen, Devin; Cheadle, William G

2012-06-15

Persistent and tertiary chronic peritonitis is a clinically challenging problem especially in those who are critically ill. This could be attributed to a state of immune-paralysis, known as microbial tolerance. Microbial tolerance is the diminished pro-inflammatory protein response following repeated stimulation by numerous pathogen-associated molecular patterns (PAMPs) of varying origins, which we have shown in this novel model of chronic peritonitis. We aimed in this study to investigate the molecular mechanisms behind microbial tolerance and the early innate immune response resolution in this model. C57BL/6 mice were pretreated intra-peritoneally (IP) with saline or endotoxin LPS 10 mg/kg (LPS 10). Following pretreatment, peritonitis was induced 24 h later injecting 10(3)Klebsiella pneumonia CFU IP. Gentamicin was administered 4 h prior to infection and BID thereafter. Peritoneal exudate cells (PEC) were obtained through peritoneal lavage and RNA was isolated (n = 3) at 4, 24, and 48 h following infection. SA Biosciences© RT2-Profiler PCR array mouse Toll-like receptor signaling pathway (PAMM_018A) data were further analyzed by Ingenuity Pathway Inc. analysis (IPA). Of the 89 genes studied, 26 were significantly up-regulated (fold change > 1.2 and P value < 0.05) in the saline pretreated group at 4, 24, and 48 h after infection. There were no down-regulated genes. In the LPS-pretreated group, 35 genes were significantly up-regulated; of these genes, 13 were not increased in the saline pretreated infected mice. This left 22 up-regulated genes in both infected groups. At 4 h, 6 of these 22 genes (CHUK, HMGB1, HSPD1, IRAK2, LY96, and TLR4) were further 2-fold increased in the LPS pretreatment group compared with the saline pretreatment group. Only IRAK2 was 2-fold increased at 24 h. By 48 h, no LPS effect was seen. When applying IPA analysis, six main canonical pathways were constantly dysregulated in the same significance order in both the saline and LPS group at 4, 24, and 48 h. These were: Toll-like receptor and NF-κB signaling, hepatic cholestasis, interleukin-6, and LPS-mediated MAPK signaling pathways, and pattern recognition receptors of bacterial pathway. Peritonitis increased PEC gene expression associated with sepsis and a pro-inflammatory response, which was further augmented by LPS pretreatment over 24 h only. Prior exposure to LPS did not induce PEC gene tolerance to subsequent infection with Klebsiella at the mRNA level. Post-transcriptional modification as microRNA down-regulation of inflammatory cytokines could possibly explain such phenomena. Published by Elsevier Inc.

Gene context conservation of a higher order than operons.

PubMed

Lathe, W C; Snel, B; Bork, P

2000-10-01

Operons, co-transcribed and co-regulated contiguous sets of genes, are poorly conserved over short periods of evolutionary time. The gene order, gene content and regulatory mechanisms of operons can be very different, even in closely related species. Here, we present several lines of evidence which suggest that, although an operon and its individual genes and regulatory structures are rearranged when comparing the genomes of different species, this rearrangement is a conservative process. Genomic rearrangements invariably maintain individual genes in very specific functional and regulatory contexts. We call this conserved context an uber-operon.

A phylogenomic analysis of the Actinomycetales mce operons.

PubMed

Casali, Nicola; Riley, Lee W

2007-02-26

The genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins. The M. tuberculosis mce loci each include two yrbE and six mce genes, which have homology to ABC transporter permeases and substrate-binding proteins, respectively. Operons with an identical structure were identified in all Mycobacterium species examined, as well as in five other Actinomycetales genera. Some of the Actinomycetales mce operons include an mkl gene, which encodes an ATPase resembling those of ABC uptake transporters. The phylogenetic profile of Mkl orthologs exactly matched that of the Mce and YrbE proteins. Through topology and motif analyses of YrbE homologs, we identified a region within the penultimate cytoplasmic loop that may serve as the site of interaction with the putative cognate Mkl ATPase. Homologs of the exported proteins encoded adjacent to the M. tuberculosis mce operons were detected in a conserved chromosomal location downstream of the majority of Actinomycetales operons. Operons containing linked mkl, yrbE and mce genes, resembling the classic organization of an ABC importer, were found to be common in Gram-negative bacteria and appear to be associated with changes in properties of the cell surface. Evidence presented suggests that the mce operons of Actinomycetales species and related operons in Gram-negative bacteria encode a subfamily of ABC uptake transporters with a possible role in remodeling the cell envelope.

High accuracy operon prediction method based on STRING database scores.

PubMed

Taboada, Blanca; Verde, Cristina; Merino, Enrique

2010-07-01

We present a simple and highly accurate computational method for operon prediction, based on intergenic distances and functional relationships between the protein products of contiguous genes, as defined by STRING database (Jensen,L.J., Kuhn,M., Stark,M., Chaffron,S., Creevey,C., Muller,J., Doerks,T., Julien,P., Roth,A., Simonovic,M. et al. (2009) STRING 8-a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res., 37, D412-D416). These two parameters were used to train a neural network on a subset of experimentally characterized Escherichia coli and Bacillus subtilis operons. Our predictive model was successfully tested on the set of experimentally defined operons in E. coli and B. subtilis, with accuracies of 94.6 and 93.3%, respectively. As far as we know, these are the highest accuracies ever obtained for predicting bacterial operons. Furthermore, in order to evaluate the predictable accuracy of our model when using an organism's data set for the training procedure, and a different organism's data set for testing, we repeated the E. coli operon prediction analysis using a neural network trained with B. subtilis data, and a B. subtilis analysis using a neural network trained with E. coli data. Even for these cases, the accuracies reached with our method were outstandingly high, 91.5 and 93%, respectively. These results show the potential use of our method for accurately predicting the operons of any other organism. Our operon predictions for fully-sequenced genomes are available at http://operons.ibt.unam.mx/OperonPredictor/.

Solving a discrete model of the lac operon using Z3

NASA Astrophysics Data System (ADS)

Gutierrez, Natalia A.

2014-05-01

A discrete model for the Lcac Operon is solved using the SMT-solver Z3. Traditionally the Lac Operon is formulated in a continuous math model. This model is a system of ordinary differential equations. Here, it was considerated as a discrete model, based on a Boolean red. The biological problem of Lac Operon is enunciated as a problem of Boolean satisfiability, and it is solved using an STM-solver named Z3. Z3 is a powerful solver that allows understanding the basic dynamic of the Lac Operon in an easier and more efficient way. The multi-stability of the Lac Operon can be easily computed with Z3. The code that solves the Boolean red can be written in Python language or SMT-Lib language. Both languages were used in local version of the program as online version of Z3. For future investigations it is proposed to solve the Boolean red of Lac Operon using others SMT-solvers as cvc4, alt-ergo, mathsat and yices.

Recognition of LPS by TLR4: Potential for Anti-Inflammatory Therapies

PubMed Central

Nijland, Reindert; Hofland, Tom; van Strijp, Jos A. G.

2014-01-01

LPS molecules of marine bacteria show structures distinct from terrestrial bacteria, due to the different environment that marine bacteria live in. Because of these different structures, lipid A molecules from marine bacteria are most often poor stimulators of the Toll-like receptor 4 (TLR4) pathway. Due to their low stimulatory potential, these lipid A molecules are suggested to be applicable as antagonists of TLR4 signaling in sepsis patients, where this immune response is amplified and unregulated. Antagonizing lipid A molecules might be used for future therapies against sepsis, therapies that currently do not exist. In this review, we will discuss these differences in lipid A structures and their recognition by the immune system. The modifications present in marine lipid A structures are described, and their potential as LPS antagonists will be discussed. Finally, since clinical trials built on antagonizing lipid A molecules have proven unsuccessful, we propose to also focus on different aspects of the TLR4 signaling pathway when searching for new potential drugs. Furthermore, we put forward the notion that bacteria probably already produce inhibitors of TLR4 signaling, making these bacterial products interesting molecules to investigate for future sepsis therapies. PMID:25056632

Novel role of the LPS core glycosyltransferase WapH for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis.

PubMed

Benforte, Florencia C; Colonnella, Maria A; Ricardi, Martiniano M; Solar Venero, Esmeralda C; Lizarraga, Leonardo; López, Nancy I; Tribelli, Paula M

2018-01-01

Psychrotroph microorganisms have developed cellular mechanisms to cope with cold stress. Cell envelopes are key components for bacterial survival. Outer membrane is a constituent of Gram negative bacterial envelopes, consisting of several components, such as lipopolysaccharides (LPS). In this work we investigated the relevance of envelope characteristics for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis by analyzing a mini Tn5 wapH mutant strain, encoding a core LPS glycosyltransferase. Our results showed that wapH strain is impaired to grow under low temperature but not for cold survival. The mutation in wapH, provoked a strong aggregative phenotype and modifications of envelope nanomechanical properties such as lower flexibility and higher turgor pressure, cell permeability and surface area to volume ratio (S/V). Changes in these characteristics were also observed in the wild type strain grown at different temperatures, showing higher cell flexibility but lower turgor pressure under cold conditions. Cold shock experiments indicated that an acclimation period in the wild type is necessary for cell flexibility and S/V ratio adjustments. Alteration in cell-cell interaction capabilities was observed in wapH strain. Mixed cells of wild type and wapH strains, as well as those of the wild type strain grown at different temperatures, showed a mosaic pattern of aggregation. These results indicate that wapH mutation provoked marked envelope alterations showing that LPS core conservation appears as a novel essential feature for active growth under cold conditions.

A phylogenomic analysis of the Actinomycetales mce operons

PubMed Central

Casali, Nicola; Riley, Lee W

2007-01-01

Background The genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins. Results The M. tuberculosis mce loci each include two yrbE and six mce genes, which have homology to ABC transporter permeases and substrate-binding proteins, respectively. Operons with an identical structure were identified in all Mycobacterium species examined, as well as in five other Actinomycetales genera. Some of the Actinomycetales mce operons include an mkl gene, which encodes an ATPase resembling those of ABC uptake transporters. The phylogenetic profile of Mkl orthologs exactly matched that of the Mce and YrbE proteins. Through topology and motif analyses of YrbE homologs, we identified a region within the penultimate cytoplasmic loop that may serve as the site of interaction with the putative cognate Mkl ATPase. Homologs of the exported proteins encoded adjacent to the M. tuberculosis mce operons were detected in a conserved chromosomal location downstream of the majority of Actinomycetales operons. Operons containing linked mkl, yrbE and mce genes, resembling the classic organization of an ABC importer, were found to be common in Gram-negative bacteria and appear to be associated with changes in properties of the cell surface. Conclusion Evidence presented suggests that the mce operons of Actinomycetales species and related operons in Gram-negative bacteria encode a subfamily of ABC uptake transporters with a possible role in remodeling the cell envelope. PMID:17324287

Sequence and features of the tryptophan operon of Vibrio parahemolyticus.

PubMed

Crawford, I P; Han, C Y; Silverman, M

1991-01-01

The nucleotide sequence of the trp operon of the marine enteric bacterium Vibrio parahemolyticus is presented. The gene order E, G, D, C(F), B, A is identical to that of other enterics. The structural genes of the operon are preceded by a long leader region encoding a 41-residue peptide containing five tryptophan residues. The organization of the leader region suggests that transcription of the operon is subject to attenuation control. The promoter-operator region of the V. parahemolyticus trp operon is almost identical to the corresponding promoter-operator of E. coli. The similarities suggest that promoter strength and operator function are identical in the two species, and that transcription initiation is regulated by repression. The operon appears to lack the internal promoter within trpD that is common in terrestrial enteric species.

Biofilms Formed by Gram-Negative Bacteria Undergo Increased Lipid A Palmitoylation, Enhancing In Vivo Survival

PubMed Central

Chalabaev, Sabina; Chauhan, Ashwini; Novikov, Alexey; Iyer, Pavithra; Szczesny, Magdalena; Beloin, Christophe; Caroff, Martine

2014-01-01

ABSTRACT Bacterial biofilm communities are associated with profound physiological changes that lead to novel properties compared to the properties of individual (planktonic) bacteria. The study of biofilm-associated phenotypes is an essential step toward control of deleterious effects of pathogenic biofilms. Here we investigated lipopolysaccharide (LPS) structural modifications in Escherichia coli biofilm bacteria, and we showed that all tested commensal and pathogenic E. coli biofilm bacteria display LPS modifications corresponding to an increased level of incorporation of palmitate acyl chain (palmitoylation) into lipid A compared to planktonic bacteria. Genetic analysis showed that lipid A palmitoylation in biofilms is mediated by the PagP enzyme, which is regulated by the histone-like protein repressor H-NS and the SlyA regulator. While lipid A palmitoylation does not influence bacterial adhesion, it weakens inflammatory response and enhances resistance to some antimicrobial peptides. Moreover, we showed that lipid A palmitoylation increases in vivo survival of biofilm bacteria in a clinically relevant model of catheter infection, potentially contributing to biofilm tolerance to host immune defenses. The widespread occurrence of increased lipid A palmitoylation in biofilms formed by all tested bacteria suggests that it constitutes a new biofilm-associated phenotype in Gram-negative bacteria. PMID:25139899

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

PubMed

Iuchi, S; Cole, S T; Lin, E C

1990-01-01

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.

LacR Is a Repressor of lacABCD and LacT Is an Activator of lacTFEG, Constituting the lac Gene Cluster in Streptococcus pneumoniae

PubMed Central

Afzal, Muhammad; Shafeeq, Sulman

2014-01-01

Comparison of the transcriptome of Streptococcus pneumoniae strain D39 grown in the presence of either lactose or galactose with that of the strain grown in the presence of glucose revealed the elevated expression of various genes and operons, including the lac gene cluster, which is organized into two operons, i.e., lac operon I (lacABCD) and lac operon II (lacTFEG). Deletion of the DeoR family transcriptional regulator lacR that is present downstream of the lac gene cluster revealed elevated expression of lac operon I even in the absence of lactose. This suggests a function of LacR as a transcriptional repressor of lac operon I, which encodes enzymes involved in the phosphorylated tagatose pathway in the absence of lactose or galactose. Deletion of lacR did not affect the expression of lac operon II, which encodes a lactose-specific phosphotransferase. This finding was further confirmed by β-galactosidase assays with PlacA-lacZ and PlacT-lacZ in the presence of either lactose or glucose as the sole carbon source in the medium. This suggests the involvement of another transcriptional regulator in the regulation of lac operon II, which is the BglG-family transcriptional antiterminator LacT. We demonstrate the role of LacT as a transcriptional activator of lac operon II in the presence of lactose and CcpA-independent regulation of the lac gene cluster in S. pneumoniae. PMID:24951784

Combined Chromatin and Expression Analysis Reveals Specific Regulatory Mechanisms within Cytokine Genes in the Macrophage Early Immune Response

PubMed Central

Emanuelsson, Olof; Sennblad, Bengt; Pirmoradian Najafabadi, Mohammad; Folkersen, Lasse; Mälarstig, Anders; Lagergren, Jens; Eriksson, Per; Hamsten, Anders; Odeberg, Jacob

2012-01-01

Macrophages play a critical role in innate immunity, and the expression of early response genes orchestrate much of the initial response of the immune system. Macrophages undergo extensive transcriptional reprogramming in response to inflammatory stimuli such as Lipopolysaccharide (LPS). To identify gene transcription regulation patterns involved in early innate immune responses, we used two genome-wide approaches - gene expression profiling and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. We examined the effect of 2 hrs LPS stimulation on early gene expression and its relation to chromatin remodeling (H3 acetylation; H3Ac) and promoter binding of Sp1 and RNA polymerase II phosphorylated at serine 5 (S5P RNAPII), which is a marker for transcriptional initiation. Our results indicate novel and alternative gene regulatory mechanisms for certain proinflammatory genes. We identified two groups of up-regulated inflammatory genes with respect to chromatin modification and promoter features. One group, including highly up-regulated genes such as tumor necrosis factor (TNF), was characterized by H3Ac, high CpG content and lack of TATA boxes. The second group, containing inflammatory mediators (interleukins and CCL chemokines), was up-regulated upon LPS stimulation despite lacking H3Ac in their annotated promoters, which were low in CpG content but did contain TATA boxes. Genome-wide analysis showed that few H3Ac peaks were unique to either +/−LPS condition. However, within these, an unpacking/expansion of already existing H3Ac peaks was observed upon LPS stimulation. In contrast, a significant proportion of S5P RNAPII peaks (approx 40%) was unique to either condition. Furthermore, data indicated a large portion of previously unannotated TSSs, particularly in LPS-stimulated macrophages, where only 28% of unique S5P RNAPII peaks overlap annotated promoters. The regulation of the inflammatory response appears to occur in a very specific manner at the chromatin level for specific genes and this study highlights the level of fine-tuning that occurs in the immune response. PMID:22384210

Cloning and Molecular Analysis of a Mannitol Operon of Phosphoenolpyruvate-dependent Phosphotransferase (PTS) type From Vibrio cholerae O395

PubMed Central

Kumar, Sanath; Smith, Kenneth P.; Floyd, Jody L.; Varela, Manuel F.

2010-01-01

A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395 and its activity studied in Escherichia coli. The 3.9 kb operon comprising of three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EIIMtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD) and a mannitol operon repressor (MtlR). The transport of [3H]mannitol by the cloned mannitol operon in E. coli was 13.8±1.4 nmol/min/mg protein. The insertional inactivation of EIIMtl abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon. PMID:21184218

Quantitative approaches to the study of bistability in the lac operon of Escherichia coli.

PubMed

Santillán, Moisés; Mackey, Michael C

2008-08-06

In this paper, the history and importance of the lac operon in the development of molecular and systems biology are briefly reviewed. We start by presenting a description of the regulatory mechanisms in this operon, taking into account the most recent discoveries. Then we offer a survey of the history of the lac operon, including the discovery of its main elements and the subsequent influence on the development of molecular and systems biology. Next the bistable behaviour of the operon is discussed, both with respect to its discovery and its molecular origin. A review of the literature in which this bistable phenomenon has been studied from a mathematical modelling viewpoint is then given. We conclude with some brief remarks.

A mutation in a new gene bglJ, activates the bgl operon in Escherichia coli K-12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giel, M.; Desnoyer, M.; Lopilato, J.

1996-06-01

A new mutation , bglJ4, has been characterized that results in the expression of the silent bgl operon. The bgl operon encodes proteins necessary for the transport and utilization of the aromatic {beta}-glucosides arbutin and salicin. A variety of mutations activate the operon and result in a Bgl{sup +} phenotype. Activating mutations are located upstream of the bgl promoter and in genes located elsewhere on the chromosome. Mutations outside of the bgl operon occur in the genes encoding DNA gyrase and in the gene encoding the nucleoid associated protein H-NS. The mutation described here, bglJ4, has been mapped to amore » new locus at min 99 on the Escherichia coli K-12 genetic map. The putative protein encoded by the bglJ gene has homology to a family of transcriptional activators. Evidence is presented that increased expression of the bglJ product is needed for activation of the bgl operon. 56 refs., 3 figs., 3 tabs.« less

Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa.

PubMed

Cursino, Luciana; Galvani, Cheryl D; Athinuwat, Dusit; Zaini, Paulo A; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

2011-10-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

Nicotinamide: a vitamin able to shift macrophage differentiation toward macrophages with restricted inflammatory features.

PubMed

Weiss, Ronald; Schilling, Erik; Grahnert, Anja; Kölling, Valeen; Dorow, Juliane; Ceglarek, Uta; Sack, Ulrich; Hauschildt, Sunna

2015-11-01

The differentiation of human monocytes into macrophages is influenced by environmental signals. Here we asked in how far nicotinamide (NAM), a vitamin B3 derivative known to play a major role in nicotinamide adenine dinucleotide (NAD)-mediated signaling events, is able to modulate monocyte differentiation into macrophages developed in the presence of granulocyte macrophage colony-stimulating factor (GM-MØ) or macrophage colony-stimulating factor (M-MØ). We found that GM-MØ undergo biochemical, morphological and functional modifications in response to NAM, whereas M-MØ were hardly affected. GM-MØ exposed to NAM acquired an M-MØ-like structure while the LPS-induced production of pro-inflammatory cytokines and COX-derived eicosanoids were down-regulated. In contrast, NAM had no effect on the production of IL-10 or the cytochrome P450-derived eicosanoids. Administration of NAM enhanced intracellular NAD concentrations; however, it did not prevent the LPS-mediated drain on NAD pools. In search of intracellular molecular targets of NAM known to be involved in LPS-induced cytokine and eicosanoid synthesis, we found NF-κB activity to be diminished. In conclusion, our data show that vitamin B3, when present during the differentiation of monocytes into GM-MØ, interferes with biochemical pathways resulting in strongly reduced pro-inflammatory features. © The Author(s) 2015.

Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells.

PubMed

Jones, Jane T; Qian, Xi; van der Velden, Jos L J; Chia, Shi Biao; McMillan, David H; Flemer, Stevenson; Hoffman, Sidra M; Lahue, Karolyn G; Schneider, Robert W; Nolin, James D; Anathy, Vikas; van der Vliet, Albert; Townsend, Danyelle M; Tew, Kenneth D; Janssen-Heininger, Yvonne M W

2016-08-01

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Modification of acute and late-phase allergic responses to ovalbumin with lipopolysaccharide.

PubMed

Tulic, Mark K; Holt, Patrick G; Sly, Peter D

2002-10-01

We have previously shown that lipopolysaccharide (LPS) exposure in sensitised animals 18 h after ovalbumin (OVA) challenge inhibits OVA-induced airway hyper-responsiveness (AHR). In the present study, we investigated the effect of LPS on OVA-induced acute and late-phase allergic responses in sensitised rats when challenged with OVA. Rats were sensitised with OVA and 11 days later challenged with 1% OVA in the presence or absence of LPS (0.5-50 microg/ml) given in the same nebulizer. Acute responses to OVA were measured each minute for 30 min after challenge. In a separate group of animals, late-phase responses to OVA were determined at 24 h. At the end of each study, Evans blue dye was injected and animals sacrificed 30 min later. Bronchoalveolar lavage was obtained to monitor inflammatory cell migration and microvascular leakage. OVA challenge in sensitised animals produced an acute response with changes in lung mechanics peaking 10.0 +/- 0.9 min after OVA and returning to baseline within 30 min. This was followed 24 h later by increased responses to methacholine chloride (MCh), inflammatory cell influx and increased Evans blue leakage into the lungs. Presence of 5 or 50 microg/ml LPS in the nebulizer during OVA challenge altered the kinetics of the acute-phase response, with an immediate decrease in lung function (time to peak decreased from 10.3 +/- 1.2 to 1.8 +/- 0.2 and 2.2 +/- 0.3 min, respectively: p < 0.001, n = 6) and a dose-dependent attenuation of late-phase AHR, cellular influx (n = 5, p < 0.001) and Evans blue leakage (n = 5, p < 0.001) at 24 h. In summary, co-administration of OVA with LPS modifies both the acute and late-phase responses to the allergen, inducing an earlier acute change in lung function and a dose-dependent inhibition of late-phase responses to the allergen. Copyright 2002 S. Karger AG, Basel

Functional Analysis of d-Alanylation of Lipoteichoic Acid in the Probiotic Strain Lactobacillus rhamnosus GG▿

PubMed Central

Vélez, Mónica Perea; Verhoeven, Tine L. A.; Draing, Christian; Von Aulock, Sonja; Pfitzenmaier, Markus; Geyer, Armin; Lambrichts, Ivo; Grangette, Corinne; Pot, Bruno; Vanderleyden, Jos; De Keersmaecker, Sigrid C. J.

2007-01-01

Lipoteichoic acid (LTA) is a macroamphiphile molecule which performs several functions in gram-positive bacteria, such as maintenance of cell wall homeostasis. d-Alanylation of LTA requires the proteins encoded by the dlt operon, and this process is directly related to the charge properties of this polymer strongly contributing to its function. The insertional inactivation of dltD of the probiotic strain Lactobacillus rhamnosus GG (ATCC 53103) resulted in the complete absence of d-alanyl esters in the LTA as confirmed by nuclear magnetic resonance analysis. This was reflected in modifications of the bacterial cell surface properties. The dltD strain showed 2.4-fold-increased cell length, a low survival capacity in response to gastric juice challenge, an increased sensitivity to human beta-defensin-2, an increased rate of autolysis, an increased capacity to initiate growth in the presence of an anionic detergent, and a decreased capacity to initiate growth in the presence of cationic peptides compared to wild-type results. However, in vitro experiments revealed no major differences for adhesion to human intestinal epithelial cells, biofilm formation, and immunomodulation. These properties are considered to be important for probiotics. The role of the dlt operon in lactobacilli is discussed in view of these results. PMID:17434999

A Novel Method for Accurate Operon Predictions in All SequencedProkaryotes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Morgan N.; Huang, Katherine H.; Alm, Eric J.

2004-12-01

We combine comparative genomic measures and the distance separating adjacent genes to predict operons in 124 completely sequenced prokaryotic genomes. Our method automatically tailors itself to each genome using sequence information alone, and thus can be applied to any prokaryote. For Escherichia coli K12 and Bacillus subtilis, our method is 85 and 83% accurate, respectively, which is similar to the accuracy of methods that use the same features but are trained on experimentally characterized transcripts. In Halobacterium NRC-1 and in Helicobacterpylori, our method correctly infers that genes in operons are separated by shorter distances than they are in E.coli, andmore » its predictions using distance alone are more accurate than distance-only predictions trained on a database of E.coli transcripts. We use microarray data from sixphylogenetically diverse prokaryotes to show that combining intergenic distance with comparative genomic measures further improves accuracy and that our method is broadly effective. Finally, we survey operon structure across 124 genomes, and find several surprises: H.pylori has many operons, contrary to previous reports; Bacillus anthracis has an unusual number of pseudogenes within conserved operons; and Synechocystis PCC6803 has many operons even though it has unusually wide spacings between conserved adjacent genes.« less

Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem

PubMed Central

Valdivia-Anistro, Jorge A.; Eguiarte-Fruns, Luis E.; Delgado-Sapién, Gabriela; Márquez-Zacarías, Pedro; Gasca-Pineda, Jaime; Learned, Jennifer; Elser, James J.; Olmedo-Alvarez, Gabriela; Souza, Valeria

2016-01-01

The ribosomal RNA (rrn) operon is a key suite of genes related to the production of protein synthesis machinery and thus to bacterial growth physiology. Experimental evidence has suggested an intrinsic relationship between the number of copies of this operon and environmental resource availability, especially the availability of phosphorus (P), because bacteria that live in oligotrophic ecosystems usually have few rrn operons and a slow growth rate. The Cuatro Ciénegas Basin (CCB) is a complex aquatic ecosystem that contains an unusually high microbial diversity that is able to persist under highly oligotrophic conditions. These environmental conditions impose a variety of strong selective pressures that shape the genome dynamics of their inhabitants. The genus Bacillus is one of the most abundant cultivable bacterial groups in the CCB and usually possesses a relatively large number of rrn operon copies (6–15 copies). The main goal of this study was to analyze the variation in the number of rrn operon copies of Bacillus in the CCB and to assess their growth-related properties as well as their stoichiometric balance (N and P content). We defined 18 phylogenetic groups within the Bacilli clade and documented a range of from six to 14 copies of the rrn operon. The growth dynamic of these Bacilli was heterogeneous and did not show a direct relation to the number of operon copies. Physiologically, our results were not consistent with the Growth Rate Hypothesis, since the copies of the rrn operon were decoupled from growth rate. However, we speculate that the diversity of the growth properties of these Bacilli as well as the low P content of their cells in an ample range of rrn copy number is an adaptive response to oligotrophy of the CCB and could represent an ecological mechanism that allows these taxa to coexist. These findings increase the knowledge of the variability in the number of copies of the rrn operon in the genus Bacillus and give insights about the physiology of this bacterial group under extreme oligotrophic conditions. PMID:26779143

A quantitative study of the benefits of co-regulation using the spoIIA operon as an example

PubMed Central

Iber, Dagmar

2006-01-01

The distribution of most genes is not random, and functionally linked genes are often found in clusters. Several theories have been put forward to explain the emergence and persistence of operons in bacteria. Careful analysis of genomic data favours the co-regulation model, where gene organization into operons is driven by the benefits of coordinated gene expression and regulation. Direct evidence that coexpression increases the individual's fitness enough to ensure operon formation and maintenance is, however, still lacking. Here, a previously described quantitative model of the network that controls the transcription factor σF during sporulation in Bacillus subtilis is employed to quantify the benefits arising from both organization of the sporulation genes into the spoIIA operon and from translational coupling. The analysis shows that operon organization, together with translational coupling, is important because of the inherent stochastic nature of gene expression, which skews the ratios between protein concentrations in the absence of co-regulation. The predicted impact of different forms of gene regulation on fitness and survival agrees quantitatively with published sporulation efficiencies. PMID:16924264

A quantitative study of the benefits of co-regulation using the spoIIA operon as an example.

PubMed

Iber, Dagmar

2006-01-01

The distribution of most genes is not random, and functionally linked genes are often found in clusters. Several theories have been put forward to explain the emergence and persistence of operons in bacteria. Careful analysis of genomic data favours the co-regulation model, where gene organization into operons is driven by the benefits of coordinated gene expression and regulation. Direct evidence that coexpression increases the individual's fitness enough to ensure operon formation and maintenance is, however, still lacking. Here, a previously described quantitative model of the network that controls the transcription factor sigma(F) during sporulation in Bacillus subtilis is employed to quantify the benefits arising from both organization of the sporulation genes into the spoIIA operon and from translational coupling. The analysis shows that operon organization, together with translational coupling, is important because of the inherent stochastic nature of gene expression, which skews the ratios between protein concentrations in the absence of co-regulation. The predicted impact of different forms of gene regulation on fitness and survival agrees quantitatively with published sporulation efficiencies.

Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120.

PubMed

Frías, José E; Flores, Enrique

2015-07-01

Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria assimilate nitrate, but regulation of the nitrate assimilation system varies in different cyanobacterial groups. In the N2-fixing, heterocyst-forming cyanobacteria, the nirA operon, which includes the structural genes for the nitrate assimilation system, is expressed in the presence of nitrate or nitrite if ammonium is not available to the cells. Here we studied the genes required for production of an active nitrate reductase, providing information on the nitrate-dependent induction of the operon, and found evidence for possible protein-protein interactions in the maturation of nitrate reductase and nitrite reductase. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Synergic role of the two ars operons in arsenic tolerance in Pseudomonas putida KT2440.

PubMed

Fernández, Matilde; Udaondo, Zulema; Niqui, José-Luis; Duque, Estrella; Ramos, Juan-Luis

2014-10-01

The chromosome of Pseudomonas putida KT2440 carries two clusters of genes, denoted ars1 and ars2, that are annotated as putative arsenic resistance operons. In this work, we present evidence that both operons encode functional arsenic-response regulatory genes as well as arsenic extrusion systems that confer resistance to both arsenite [As(III)] and arsenate [As(V)]. Transcriptional fusions of P(ars1) and P(ars2) to lacZ revealed that expression of both operons was induced by arsenite and arsenate. We generated single mutants in ars1 and ars2, which showed lower resistance to arsenic than the wild-type strain. A double ars1/ars2 was found to be highly sensitive to arsenic. Minimum inhibitory concentrations (MICs) for single mutants decreased two- to fourfold with respect to the parental strain, while in the double mutant the MIC decreased 128-fold for arsenite and 32-fold for arsenate. Bioinformatic analysis revealed that the ars2 resistance operon is part of the core genome of P. putida, while the ars1 operon appears to only occur in the KT2440 strain, suggesting that ars1 was acquired by horizontal gene transfer. The presence of ars1 in KT2440 may explain why it exhibits higher resistance to arsenic than other P. putida strains, which bear only the ars2 operon.

OperomeDB: A Database of Condition-Specific Transcription Units in Prokaryotic Genomes.

PubMed

Chetal, Kashish; Janga, Sarath Chandra

2015-01-01

Background. In prokaryotic organisms, a substantial fraction of adjacent genes are organized into operons-codirectionally organized genes in prokaryotic genomes with the presence of a common promoter and terminator. Although several available operon databases provide information with varying levels of reliability, very few resources provide experimentally supported results. Therefore, we believe that the biological community could benefit from having a new operon prediction database with operons predicted using next-generation RNA-seq datasets. Description. We present operomeDB, a database which provides an ensemble of all the predicted operons for bacterial genomes using available RNA-sequencing datasets across a wide range of experimental conditions. Although several studies have recently confirmed that prokaryotic operon structure is dynamic with significant alterations across environmental and experimental conditions, there are no comprehensive databases for studying such variations across prokaryotic transcriptomes. Currently our database contains nine bacterial organisms and 168 transcriptomes for which we predicted operons. User interface is simple and easy to use, in terms of visualization, downloading, and querying of data. In addition, because of its ability to load custom datasets, users can also compare their datasets with publicly available transcriptomic data of an organism. Conclusion. OperomeDB as a database should not only aid experimental groups working on transcriptome analysis of specific organisms but also enable studies related to computational and comparative operomics.

Uptake of chitosan-derived D-glucosamine oligosaccharides in Streptomyces coelicolor A3(2).

PubMed

Viens, Pascal; Dubeau, Marie-Pierre; Kimura, Akane; Desaki, Yoshitake; Shinya, Tomonori; Shibuya, Naoto; Saito, Akihiro; Brzezinski, Ryszard

2015-05-01

The csnR gene, localized at the beginning of an operon, csnR-K, which organization is conserved through many actinomycete genomes, was previously shown to repress the transcription of the chitosanase gene csnA in Streptomyces lividans. However, knowledge on the function of the whole csnR-K operon in the metabolism of chitosan (an N-deacetylated derivative of chitin) remained limited. Mutants of S. coelicolor A3(2) harboring partial or total deletions of the csnR-K operon were analyzed for their capacity to uptake glucosamine oligosaccharides (GlcN)n. The csnR-K operon was autoregulated by CsnR repressor and its transcription was inducible by GlcN oligosaccharides. The operon controlled the uptake of GlcN oligosaccharides in S. coelicolor A3(2), with a minor contribution to the consumption of monomeric GlcN but not chitin-related N-acetylated derivatives. The deletion of the whole operon abolished the uptake of GlcN oligosaccharides. The CsnEFG transporter encoded by this operon is the front door for the assimilation of chitosan-derived hydrolysis products in S. coelicolor A3(2). The ATP-binding component MsiK was essential for CsnEFG transport function. Also, deletion of msiK abolished the induction of csnA transcription by GlcN oligosaccharides. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

An operon for production of bioactive gibberellin A4 phytohormone with wide distribution in the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola.

PubMed

Nagel, Raimund; Turrini, Paula C G; Nett, Ryan S; Leach, Jan E; Verdier, Valérie; Van Sluys, Marie-Anne; Peters, Reuben J

2017-05-01

Phytopathogens have developed elaborate mechanisms to attenuate the defense response of their host plants, including convergent evolution of complex pathways for production of the GA phytohormones, which were actually first isolated from the rice fungal pathogen Gibberella fujikuroi. The rice bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) has been demonstrated to contain a biosynthetic operon with cyclases capable of producing the universal GA precursor ent-kaurene. Genetic (knock-out) studies indicate that the derived diterpenoid serves as a virulence factor for this rice leaf streak pathogen, serving to reduce the jasmonic acid-mediated defense response. Here the functions of the remaining genes in the Xoc operon are elucidated and the distribution of the operon in X. oryzae is investigated in over 100 isolates. The Xoc operon leads to production of the bioactive GA 4 , an additional step beyond production of the penultimate precursor GA 9 mediated by the homologous operons recently characterized from rhizobia. Moreover, this GA biosynthetic operon was found to be widespread in Xoc (> 90%), but absent in the other major X. oryzae pathovar. These results indicate selective pressure for production of GA 4 in the distinct lifestyle of Xoc, and the importance of GA to both fungal and bacterial pathogens of rice. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

An operon for production of bioactive gibberellin A4 phytohormone with wide distribution in the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola

PubMed Central

Nagel, Raimund; Turrini, Paula C. G.; Nett, Ryan S.; Leach, Jan E.; Verdier, Valérie; Van Sluys, Marie-Anne; Peters, Reuben J.

2016-01-01

Summary Phytopathogens have developed elaborate mechanisms to attenuate the defense response of their host plants, including convergent evolution of complex pathways for production of the gibberellin (GA) phytohormones, which were actually first isolated from the rice fungal pathogen Gibberella fujikuroi. The rice bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) has been demonstrated to contain a biosynthetic operon with cyclases capable of producing the universal GA precursor ent-kaurene. Genetic (knock-out) studies indicate that the derived diterpenoid serves as a virulence factor for this rice leaf streak pathogen, serving to reduce the jasmonic acid (JA) mediated defense response.Here the function of the remaining genes in the Xoc operon are elucidated and the distribution of the operon in X. oryzae investigated in over 100 isolates.The Xoc operon leads to production of the bioactive GA4, an additional step beyond production of the penultimate precursor GA9 mediated by the homologous operons recently characterized from rhizobia. Moreover, this GA biosynthetic operon was found to be widespread in Xoc (>90%), but absent in the other major oryzae pathovar.These results indicate selective pressure for production of GA4 in the distinct lifestyle of Xoc, and the importance of GA to both fungal and bacterial pathogens of rice. PMID:28134995

A Numbers Game: Ribosome Densities, Bacterial Growth, and Antibiotic-Mediated Stasis and Death

PubMed Central

McCall, Ingrid C.; Perrot, Véronique; Weiss, Howard; Ovesepian, Armen; Baquero, Fernando

2017-01-01

ABSTRACT We postulate that the inhibition of growth and low rates of mortality of bacteria exposed to ribosome-binding antibiotics deemed bacteriostatic can be attributed almost uniquely to these drugs reducing the number of ribosomes contributing to protein synthesis, i.e., the number of effective ribosomes. We tested this hypothesis with Escherichia coli K-12 MG1655 and constructs that had been deleted for 1 to 6 of the 7 rRNA (rrn) operons. In the absence of antibiotics, constructs with fewer rrn operons have lower maximum growth rates and longer lag phases than those with more ribosomal operons. In the presence of the ribosome-binding “bacteriostatic” antibiotics tetracycline, chloramphenicol, and azithromycin, E. coli strains with 1 and 2 rrn operons are killed at a substantially higher rate than those with more rrn operons. This increase in the susceptibility of E. coli with fewer rrn operons to killing by ribosome-targeting bacteriostatic antibiotics is not reflected in their greater sensitivity to killing by the bactericidal antibiotic ciprofloxacin, which does not target ribosomes, but also to killing by gentamicin, which does. Finally, when such strains are exposed to these ribosome-targeting bacteriostatic antibiotics, the time before these bacteria start to grow again when the drugs are removed, referred to as the post-antibiotic effect (PAE), is markedly greater for constructs with fewer rrn operons than for those with more rrn operons. We interpret the results of these other experiments reported here as support for the hypothesis that the reduction in the effective number of ribosomes due to binding to these structures provides a sufficient explanation for the action of bacteriostatic antibiotics that target these structures. PMID:28174311

Regulation of Lactobacillus casei Sorbitol Utilization Genes Requires DNA-Binding Transcriptional Activator GutR and the Conserved Protein GutM▿

PubMed Central

Alcántara, Cristina; Sarmiento-Rubiano, Luz Adriana; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar; Yebra, María J.

2008-01-01

Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encoding Lactobacillus casei gutF gene revealed that they constitute a sorbitol (glucitol) utilization operon. The gutRM genes encode putative regulators, while the gutCBA genes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTSGut). The gut operon is transcribed as a polycistronic gutFRMCBA messenger, the expression of which is induced by sorbitol and repressed by glucose. gutR encodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIBGat domain) and a mannitol/fructose-specific EIIA-like domain (EIIAMtl domain). Its inactivation abolished gut operon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying a gutB mutation expressed the gut operon constitutively, but they failed to transport sorbitol, indicating that EIIBCGut negatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from the gut promoter. A sequence comparison with the presumed promoter region of gut operons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of the L. casei gut operon is therefore likely to be operative in other firmicutes. Finally, gutM codes for a conserved protein of unknown function present in all sequenced gut operons. A gutM mutant, the first constructed in a firmicute, showed drastically reduced gut operon expression and sorbitol uptake, indicating a regulatory role also for GutM. PMID:18676710

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed Central

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-01-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis. PMID:8892842

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-11-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.

Incorporation of a horizontally transferred gene into an operon during cnidarian evolution.

PubMed

Dana, Catherine E; Glauber, Kristine M; Chan, Titus A; Bridge, Diane M; Steele, Robert E

2012-01-01

Genome sequencing has revealed examples of horizontally transferred genes, but we still know little about how such genes are incorporated into their host genomes. We have previously reported the identification of a gene (flp) that appears to have entered the Hydra genome through horizontal transfer. Here we provide additional evidence in support of our original hypothesis that the transfer was from a unicellular organism, and we show that the transfer occurred in an ancestor of two medusozoan cnidarian species. In addition we show that the gene is part of a bicistronic operon in the Hydra genome. These findings identify a new animal phylum in which trans-spliced leader addition has led to the formation of operons, and define the requirements for evolution of an operon in Hydra. The identification of operons in Hydra also provides a tool that can be exploited in the construction of transgenic Hydra strains.

mar Operon Involved in Multidrug Resistance of Enterobacter aerogenes

PubMed Central

Chollet, Renaud; Bollet, Claude; Chevalier, Jacqueline; Malléa, Monique; Pagès, Jean-Marie; Davin-Regli, Anne

2002-01-01

We determined the sequence of the entire marRAB operon in Enterobacter aerogenes. It is functionally and structurally analogous to the Escherichia coli operon. The overexpression of E. aerogenes MarA induces a multidrug resistance phenotype in a susceptible strain, demonstrated by a noticeable resistance to various antibiotics, a decrease in immunodetected porins, and active efflux of norfloxacin. PMID:11897595

Functional Analysis of the Magnetosome Island in Magnetospirillum gryphiswaldense: The mamAB Operon Is Sufficient for Magnetite Biomineralization

PubMed Central

Lohße, Anna; Ullrich, Susanne; Katzmann, Emanuel; Borg, Sarah; Wanner, Gerd; Richter, Michael; Voigt, Birgit; Schweder, Thomas; Schüler, Dirk

2011-01-01

Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only <25% of the region comprising four major operons could be associated with magnetite biomineralization, which correlated with high expression of these genes and their conservation among magnetotactic bacteria. Whereas only deletion of the mamAB operon resulted in the complete loss of magnetic particles, deletion of the conserved mms6, mamGFDC, and mamXY operons led to severe defects in morphology, size and organization of magnetite crystals. However, strains in which these operons were eliminated together retained the ability to synthesize small irregular crystallites, and weakly aligned in magnetic fields. This demonstrates that whereas the mamGFDC, mms6 and mamXY operons have crucial and partially overlapping functions for the formation of functional magnetosomes, the mamAB operon is the only region of the MAI, which is necessary and sufficient for magnetite biomineralization. Our data further reduce the known minimal gene set required for magnetosome formation and will be useful for future genome engineering approaches. PMID:22043287

Parallel Evolution and Horizontal Gene Transfer of the pst Operon in Firmicutes from Oligotrophic Environments

PubMed Central

Moreno-Letelier, Alejandra; Olmedo, Gabriela; Eguiarte, Luis E.; Martinez-Castilla, Leon; Souza, Valeria

2011-01-01

The high affinity phosphate transport system (pst) is crucial for phosphate uptake in oligotrophic environments. Cuatro Cienegas Basin (CCB) has extremely low P levels and its endemic Bacillus are closely related to oligotrophic marine Firmicutes. Thus, we expected the pst operon of CCB to share the same evolutionary history and protein similarity to marine Firmicutes. Orthologs of the pst operon were searched in 55 genomes of Firmicutes and 13 outgroups. Phylogenetic reconstructions were performed for the pst operon and 14 concatenated housekeeping genes using maximum likelihood methods. Conserved domains and 3D structures of the phosphate-binding protein (PstS) were also analyzed. The pst operon of Firmicutes shows two highly divergent clades with no correlation to the type of habitat nor a phylogenetic congruence, suggesting horizontal gene transfer. Despite sequence divergence, the PstS protein had a similar 3D structure, which could be due to parallel evolution after horizontal gene transfer events. PMID:21461370

Decaffeination and measurement of caffeine content by addicted Escherichia coli with a refactored N-demethylation operon from Pseudomonas putida CBB5.

PubMed

Quandt, Erik M; Hammerling, Michael J; Summers, Ryan M; Otoupal, Peter B; Slater, Ben; Alnahhas, Razan N; Dasgupta, Aurko; Bachman, James L; Subramanian, Mani V; Barrick, Jeffrey E

2013-06-21

The widespread use of caffeine (1,3,7-trimethylxanthine) and other methylxanthines in beverages and pharmaceuticals has led to significant environmental pollution. We have developed a portable caffeine degradation operon by refactoring the alkylxanthine degradation (Alx) gene cluster from Pseudomonas putida CBB5 to function in Escherichia coli. In the process, we discovered that adding a glutathione S-transferase from Janthinobacterium sp. Marseille was necessary to achieve N 7 -demethylation activity. E. coli cells with the synthetic operon degrade caffeine to the guanine precursor, xanthine. Cells deficient in de novo guanine biosynthesis that contain the refactored operon are ″addicted″ to caffeine: their growth density is limited by the availability of caffeine or other xanthines. We show that the addicted strain can be used as a biosensor to measure the caffeine content of common beverages. The synthetic N-demethylation operon could be useful for reclaiming nutrient-rich byproducts of coffee bean processing and for the cost-effective bioproduction of methylxanthine drugs.

Ureaplasma Species Differentially Modulate Pro- and Anti-Inflammatory Cytokine Responses in Newborn and Adult Human Monocytes Pushing the State Toward Pro-Inflammation

PubMed Central

Glaser, Kirsten; Silwedel, Christine; Fehrholz, Markus; Waaga-Gasser, Ana M.; Henrich, Birgit; Claus, Heike; Speer, Christian P.

2017-01-01

Background: Ureaplasma species have been associated with chorioamnionitis and preterm birth and have been implicated in the pathogenesis of neonatal short and long-term morbidity. However, being mostly commensal bacteria, controversy remains on the pro-inflammatory capacity of Ureaplasma. Discussions are ongoing on the incidence and impact of prenatal, perinatal, and postnatal infection. The present study addressed the impact of Ureaplasma isolates on monocyte-driven inflammation. Methods: Cord blood monocytes of term neonates and adult monocytes, either native or LPS-primed, were cultured with Ureaplasma urealyticum (U. urealyticum) serovar 8 (Uu8) and Ureaplasma parvum serovar 3 (Up3). Using qRT-PCR, cytokine flow cytometry, and multi-analyte immunoassay, we assessed mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, IL-12p40, IL-10, and IL-1 receptor antagonist (IL-1ra) as well as Toll-like receptor (TLR) 2 and TLR4. Results: Uu8 and Up3 induced mRNA expression and protein release of TNF-α, IL-1β and IL-8 in term neonatal and adult monocytes (p < 0.01 and p < 0.05). Intracellular protein expression of TNF-α, IL-1β and IL-8 in Ureaplasma-stimulated cells paralleled those results. Ureaplasma-induced cytokine levels did not significantly differ from LPS-mediated levels except for lower intracellular IL-1β in adult monocytes (Uu8: p < 0.05). Remarkably, ureaplasmas did not induce IL-12p40 response and promoted lower amounts of anti-inflammatory IL-10 and IL-1ra than LPS, provoking a cytokine imbalance more in favor of pro-inflammation (IL-1β/IL-10, IL-8/IL-10 and IL-8/IL-1ra: p < 0.01, vs. LPS). In contrast to LPS, both isolates induced TLR2 mRNA in neonatal and adult cells (p < 0.001 and p < 0.05) and suppressed TLR4 mRNA in adult monocytes (p < 0.05). Upon co-stimulation, Uu8 and Up3 inhibited LPS-induced intracellular IL-1β (p < 0.001 and p < 0.05) and IL-8 in adult monocytes (p < 0.01), while LPS-induced neonatal cytokines were maintained or aggravated (p < 0.05). Conclusion: Our data demonstrate a considerable pro-inflammatory capacity of Ureaplasma isolates in human monocytes. Stimulating pro-inflammatory cytokine responses while hardly inducing immunomodulatory and anti-inflammatory cytokines, ureaplasmas might push monocyte immune responses toward pro-inflammation. Inhibition of LPS-induced cytokines in adult monocytes in contrast to sustained inflammation in term neonatal monocytes indicates a differential modulation of host immune responses to a second stimulus. Modification of TLR2 and TLR4 expression may shape host susceptibility to inflammation. PMID:29234642

Insertional Mutations in the Hydrogenase vhc and frc Operons Encoding Selenium-Free Hydrogenases in Methanococcus voltae

PubMed Central

Berghofer, Y.; Klein, A.

1995-01-01

Methanococcus voltae, which contains four different gene groups that encode [NiFe]-hydrogenases, was transformed with integration vectors to achieve polar inactivation of two of the four hydrogenase operons that encode the selenium-free enzymes Vhc and Frc. Transformants which were selected by their acquired puromycin resistance showed site-specific insertions in either the vhc or frc operon by single crossover events. Southern hybridization revealed tandem integrations of whole vectors in the vhc operon, whereas only one vector copy was found in the frc operon. Northern (RNA) hybridizations showed a pac transcript of defined size, indicating strong termination in front of the hydrogenase genes downstream. In spite of the apparent abolition of expression of selenium-free hydrogenases through these polar insertions, they were not lethal to cells upon growth in selenium-deprived minimal medium, which we had previously shown to strongly induce transcription of the respective operons in M. voltae. Instead, like wild-type control cultures, transformants responded to selenium deprivation only with a reduction in growth rate. We conclude that loss of the potential to express a selenium-free hydrogenase can nevertheless be balanced by very small amounts of selenium hydrogenases under laboratory conditions in which the hydrogen supply is not likely to be a limiting growth factor. PMID:16535019

Characterization of the regulation of a plant polysaccharide utilization operon and its role in biofilm formation in Bacillus subtilis

PubMed Central

Habib, Cameron; Yu, Yiyang; Gozzi, Kevin; Ching, Carly; Shemesh, Moshe

2017-01-01

The soil bacterium Bacillus subtilis is often found in association with plants in the rhizosphere. Previously, plant polysaccharides have been shown to stimulate formation of root-associated multicellular communities, or biofilms, in this bacterium, yet the underlying mechanism is not fully understood. A five-gene gan operon (ganSPQAB) in B. subtilis has recently been shown to be involved in utilization of the plant-derived polysaccharide galactan. Despite these findings, molecular details about the regulation of the operon and the role of the operon in biofilm formation remain elusive. In this study, we performed comprehensive genetic analyses on the regulation of the gan operon. We show that this operon is regulated both by a LacI-like transcription repressor (GanR), which directly binds to pairs of inverted DNA repeats in the promoter region of the operon, and by the catabolite control protein A (CcpA). Derepression can be triggered by the presence of the inducer β-1,4-galactobiose, a hydrolysis product of galactan, or in situ when B. subtilis cells are associated with plant roots. In addition to the transcriptional regulation, the encoded ß-galactosidase GanA (by ganA), which hydrolyzes ß-1,4-galactobiose into galactose, is inhibited at the enzymatic level by the catalytic product galactose. Thus, the galactan utilization pathway is under complex regulation involving both positive and negative feedback mechanisms in B. subtilis. We discuss about the biological significance of such complex regulation as well as a hypothesis of biofilm induction by galactan via multiple mechanisms. PMID:28617843

Isoprenoid Pyrophosphate-Dependent Transcriptional Regulation of Carotenogenesis in Corynebacterium glutamicum

PubMed Central

Henke, Nadja A.; Heider, Sabine A. E.; Hannibal, Silvin; Wendisch, Volker F.; Peters-Wendisch, Petra

2017-01-01

Corynebacterium glutamicum is a natural producer of the C50 carotenoid decaprenoxanthin. The crtEcg0722crtBIYEb operon comprises most of its genes for terpenoid biosynthesis. The MarR-type regulator encoded upstream and in divergent orientation of the carotenoid biosynthesis operon has not yet been characterized. This regulator, named CrtR in this study, is encoded in many actinobacterial genomes co-occurring with terpenoid biosynthesis genes. CrtR was shown to repress the crt operon of C. glutamicum since DNA microarray experiments revealed that transcript levels of crt operon genes were increased 10 to 70-fold in its absence. Transcriptional fusions of a promoter-less gfp gene with the crt operon and crtR promoters confirmed that CrtR represses its own gene and the crt operon. Gel mobility shift assays with purified His-tagged CrtR showed that CrtR binds to a region overlapping with the −10 and −35 promoter sequences of the crt operon. Isoprenoid pyrophosphates interfered with binding of CrtR to its target DNA, a so far unknown mechanism for regulation of carotenogenesis. The molecular details of protein-ligand interactions remain to be studied. Decaprenoxanthin synthesis by C. glutamicum wild type was enhanced 10 to 30-fold upon deletion of crtR and was decreased 5 to 6-fold as result of crtR overexpression. Moreover, deletion of crtR was shown as metabolic engineering strategy to improve production of native and non-native carotenoids including lycopene, β-carotene, C.p. 450 and sarcinaxanthin. PMID:28484430

Extrachromosomal Nucleolus-Like Compartmentalization by a Plasmid-Borne Ribosomal RNA Operon and Its Role in Nucleoid Compaction.

PubMed

Mata Martin, Carmen; Sun, Zhe; Zhou, Yan Ning; Jin, Ding Jun

2018-01-01

In the fast-growing Escherichia coli cells, RNA polymerase (RNAP) molecules are concentrated and form foci at clusters of ribosomal RNA (rRNA) operons resembling eukaryotic nucleolus. The bacterial nucleolus-like organization, spatially compartmentalized at the surface of the compact bacterial chromosome (nucleoid), serves as transcription factories for rRNA synthesis and ribosome biogenesis, which influences the organization of the nucleoid. Unlike wild type that has seven rRNA operons in the genome in a mutant that has six (Δ6 rrn ) rRNA operons deleted in the genome, there are no apparent transcription foci and the nucleoid becomes uncompacted, indicating that formation of RNAP foci requires multiple copies of rRNA operons clustered in space and is critical for nucleoid compaction. It has not been determined, however, whether a multicopy plasmid-borne rRNA operon (p rrnB ) could substitute the multiple chromosomal rRNA operons for the organization of the bacterial nucleolus-like structure in the mutants of Δ6 rrn and Δ7 rrn that has all seven rRNA operons deleted in the genome. We hypothesized that extrachromosomal nucleolus-like structures are similarly organized and functional in trans from p rrnB in these mutants. In this report, using multicolor images of three-dimensional superresolution Structured Illumination Microscopy (3D-SIM), we determined the distributions of both RNAP and NusB that are a transcription factor involved in rRNA synthesis and ribosome biogenesis, p rrnB clustering, and nucleoid structure in these two mutants in response to environmental cues. Our results found that the extrachromosomal nucleolus-like organization tends to be spatially located at the poles of the mutant cells. In addition, formation of RNAP foci at the extrachromosomal nucleolus-like structure condenses the nucleoid, supporting the idea that active transcription at the nucleolus-like organization is a driving force in nucleoid compaction.

Relatively slow stochastic gene-state switching in the presence of positive feedback significantly broadens the region of bimodality through stabilizing the uninduced phenotypic state.

PubMed

Ge, Hao; Wu, Pingping; Qian, Hong; Xie, Xiaoliang Sunney

2018-03-01

Within an isogenic population, even in the same extracellular environment, individual cells can exhibit various phenotypic states. The exact role of stochastic gene-state switching regulating the transition among these phenotypic states in a single cell is not fully understood, especially in the presence of positive feedback. Recent high-precision single-cell measurements showed that, at least in bacteria, switching in gene states is slow relative to the typical rates of active transcription and translation. Hence using the lac operon as an archetype, in such a region of operon-state switching, we present a fluctuating-rate model for this classical gene regulation module, incorporating the more realistic operon-state switching mechanism that was recently elucidated. We found that the positive feedback mechanism induces bistability (referred to as deterministic bistability), and that the parameter range for its occurrence is significantly broadened by stochastic operon-state switching. We further show that in the absence of positive feedback, operon-state switching must be extremely slow to trigger bistability by itself. However, in the presence of positive feedback, which stabilizes the induced state, the relatively slow operon-state switching kinetics within the physiological region are sufficient to stabilize the uninduced state, together generating a broadened parameter region of bistability (referred to as stochastic bistability). We illustrate the opposite phenotype-transition rate dependence upon the operon-state switching rates in the two types of bistability, with the aid of a recently proposed rate formula for fluctuating-rate models. The rate formula also predicts a maximal transition rate in the intermediate region of operon-state switching, which is validated by numerical simulations in our model. Overall, our findings suggest a biological function of transcriptional "variations" among genetically identical cells, for the emergence of bistability and transition between phenotypic states.

Prediction of operon-like gene clusters in the Arabidopsis thaliana genome based on co-expression analysis of neighboring genes.

PubMed

Wada, Masayoshi; Takahashi, Hiroki; Altaf-Ul-Amin, Md; Nakamura, Kensuke; Hirai, Masami Y; Ohta, Daisaku; Kanaya, Shigehiko

2012-07-15

Operon-like arrangements of genes occur in eukaryotes ranging from yeasts and filamentous fungi to nematodes, plants, and mammals. In plants, several examples of operon-like gene clusters involved in metabolic pathways have recently been characterized, e.g. the cyclic hydroxamic acid pathways in maize, the avenacin biosynthesis gene clusters in oat, the thalianol pathway in Arabidopsis thaliana, and the diterpenoid momilactone cluster in rice. Such operon-like gene clusters are defined by their co-regulation or neighboring positions within immediate vicinity of chromosomal regions. A comprehensive analysis of the expression of neighboring genes therefore accounts a crucial step to reveal the complete set of operon-like gene clusters within a genome. Genome-wide prediction of operon-like gene clusters should contribute to functional annotation efforts and provide novel insight into evolutionary aspects acquiring certain biological functions as well. We predicted co-expressed gene clusters by comparing the Pearson correlation coefficient of neighboring genes and randomly selected gene pairs, based on a statistical method that takes false discovery rate (FDR) into consideration for 1469 microarray gene expression datasets of A. thaliana. We estimated that A. thaliana contains 100 operon-like gene clusters in total. We predicted 34 statistically significant gene clusters consisting of 3 to 22 genes each, based on a stringent FDR threshold of 0.1. Functional relationships among genes in individual clusters were estimated by sequence similarity and functional annotation of genes. Duplicated gene pairs (determined based on BLAST with a cutoff of E<10(-5)) are included in 27 clusters. Five clusters are associated with metabolism, containing P450 genes restricted to the Brassica family and predicted to be involved in secondary metabolism. Operon-like clusters tend to include genes encoding bio-machinery associated with ribosomes, the ubiquitin/proteasome system, secondary metabolic pathways, lipid and fatty-acid metabolism, and the lipid transfer system. Copyright © 2012 Elsevier B.V. All rights reserved.

The complete nucleotide sequence of the glnALG operon of Escherichia coli K12.

PubMed Central

Miranda-Ríos, J; Sánchez-Pescador, R; Urdea, M; Covarrubias, A A

1987-01-01

The nucleotide sequence of the E. coli glnALG operon has been determined. The glnL (ntrB) and glnG (ntrC) genes present a high homology, at the nucleotide and aminoacid levels, with the corresponding genes of Klebsiella pneumoniae. The predicted aminoacid sequence for glutamine synthetase allowed us to locate some of the enzyme domains. The structure of this operon is discussed. PMID:2882477

Escherichia coli mutant with altered respiratory control of the frd operon.

PubMed Central

Iuchi, S; Kuritzkes, D R; Lin, E C

1985-01-01

In wild-type Escherichia coli, fumarate reductase encoded by the frd operon is inducible by its substrate in the absence of molecular oxygen and nitrate. Synthesis of this enzyme under permissive conditions requires the fnr+ gene product, which is believed to be a pleiotropic regulatory protein that activates transcription. A spontaneous mutant was isolated in which the expression of the frd operon no longer depended on the presence of fumarate or the fnr+ gene product. Aerobic repression of the operon was abolished, but nitrate repression remained intact. Transductional analysis showed that the mutation was closely linked to the frd locus. The mutant phenotype strongly suggests that repression by molecular oxygen and nitrate is mediated by different mechanisms. PMID:3882660

Structure, Regulation, and Putative Function of the Arginine Deiminase System of Streptococcus suis

PubMed Central

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative β-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression. Furthermore, comparing an arcA knockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that the arcABC operon of S. suis contributes to survival under acidic conditions. PMID:16385025

Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis.

PubMed

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative beta-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression. Furthermore, comparing an arcA knockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that the arcABC operon of S. suis contributes to survival under acidic conditions.

Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro

PubMed Central

Darwin, Andrew J.; Ziegelhoffer, Eva C.; Kiley, Patricia J.; Stewart, Valley

1998-01-01

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro. PMID:9696769

The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply*

PubMed Central

Eisenhut, Marion; Georg, Jens; Klähn, Stephan; Sakurai, Isamu; Mustila, Henna; Zhang, Pengpeng; Hess, Wolfgang R.; Aro, Eva-Mari

2012-01-01

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (Ci), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the QB site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by Ci limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in Ci conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon. PMID:22854963

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

PubMed Central

Quintard, Kévin; Dewitte, Amélie; Reboul, Angéline; Madec, Edwige; Bontemps-Gallo, Sébastien; Dondeyne, Jacqueline; Marceau, Michaël; Simonet, Michel

2015-01-01

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary. PMID:26150539

Discovery of an operon that participates in agmatine metabolism and regulates biofilm formation in Pseudomonas aeruginosa

PubMed Central

Williams, Bryan J.; Du, Rui-Hong; Calcutt, M. Wade; Abdolrasulnia, Rasul; Christman, Brian W.; Blackwell, Timothy S.

2013-01-01

Summary Agmatine is the decarboxylation product of arginine and a number of bacteria have devoted enzymatic pathways for its metabolism. Pseudomonas aeruginosa harbours the aguBA operon that metabolizes agmatine to putrescine, which can be subsequently converted into other polyamines or shunted into the TCA cycle for energy production. We discovered an alternate agmatine operon in the P. aeruginosa strain PA14 named agu2ABCA′ that contains two genes for agmatine deiminases (agu2A and agu2A′). This operon was found to be present in 25% of clinical P. aeruginosa isolates. Agu2A′ contains a twin-arginine translocation signal at its N-terminus and site-directed mutagenesis and cell fractionation experiments confirmed this protein is secreted to the periplasm. Analysis of the agu2ABCA′ promoter demonstrates that agmatine induces expression of the operon during the stationary phase of growth and during biofilm growth and agu2ABCA′ provides only weak complementation of aguBA, which is induced during log phase. Biofilm assays of mutants of all three agmatine deiminase genes in PA14 revealed that deletion of agu2ABCA′, specifically its secreted product Agu2A′, reduces biofilm production of PA14 following addition of exogenous agmatine. Together, these findings reveal a novel role for the agu2ABCA′ operon in the biofilm development of P. aeruginosa. PMID:20149107

The transcriptional terminator sequences downstream of the covR gene terminate covR/S operon transcription to generate covR monocistronic transcripts in Streptococcus pyogenes.

PubMed

Chiang-Ni, Chuan; Tsou, Chih-Cheng; Lin, Yee-Shin; Chuang, Woei-Jer; Lin, Ming-T; Liu, Ching-Chuan; Wu, Jiunn-Jong

2008-12-31

CovR/S is an important two component regulatory system, which regulates about 15% of the gene expression in Streptococcus pyogenes. The covR/S locus was identified as an operon generating an RNA transcript around 2.5-kb in size. In this study, we found the covR/S operon produced three RNA transcripts (around 2.5-, 1.0-, and 0.8-kb in size). Using RNA transcriptional terminator sequence prediction and transcriptional terminator analysis, we identified two atypical rho-independent terminator sequences downstream of the covR gene and showed these terminator sequences terminate RNA transcription efficiently. These results indicate that covR/S operon generates covR/S transcript and monocistronic covR transcripts.

Post-ruminal branched-chain amino acid supplementation and intravenous lipopolysaccharide infusion alters blood metabolites, rumen fermentation, and nitrogen balance of beef steers.

PubMed

Löest, C A; Gilliam, G G; Waggoner, J W; Turner, J L

2018-04-27

Steers exposed to an endotoxin may require additional branched-chain AA (BCAA) to support an increase in synthesis of immune proteins. This study evaluated effects of bacterial lipopolysaccharide (LPS) and BCAA supplementation on blood metabolites and N balance of 20 ruminally-cannulated steers (177 ± 4.2 kg BW). The experiment was a randomized block design, with 14-d adaptation to metabolism stalls and diet (DM fed = 1.5% BW) and 6-d collection. Treatments were a 2 × 2 factorial of LPS (0 vs 1.0 to 1.5 μg/kg BW; -LPS vs +LPS) and BCAA (0 vs 35 g/d; -BCAA vs +BCAA). The LPS in 100 mL sterile saline was infused (1 mL/min via i.v. catheter) on d 15. The BCAA in an essential AA solution were abomasally infused (900 mL/d) 3 times daily in equal portions beginning on d 7. Blood, rumen fluid, and rectal temperature were collected on d 15 at h 0, 2, 4, 8, 12, and 24 after LPS infusion. Feces and urine were collected from d 16 to 20. Rectal temperatures were greater for +LPS vs. -LPS steers at 4 h and lower at 8 h after LPS infusion (LPS h, P < 0.01). Serum cortisol and plasma urea N were greater for +LPS than -LPS steers at 2 (cortisol only), 4, 8, 12, and 24 h after LPS infusion (LPS × h, P < 0.01). Serum cortisol was greater for +BCAA than -BCAA steers at 12 h after LPS infusion (BCAA × h, P < 0.05). Serum glucose was greater for +LPS than -LPS steers at 2 h after LPS infusion (LPS × h, P < 0.01). Plasma Ile, Leu, and Val were lower, and plasma His was greater in +LPS than -LPS steers (LPS, P < 0.05). Plasma Lys, Met, Thr, and Trp of +LPS steers was lower than -LPS steers at 4 (Thr only), 8 (Lys and Trp only), 12, and 24 h after infusion (LPS × h, P < 0.05). Plasma Ile, Leu, and Val were greater (BCAA, P < 0.01), and Met, His, Phe, Thr, and Trp were lower for +BCAA than -BCAA steers at 0 h and 24 h after LPS infusion (BCAA × h, P ≤ 0.05). Steers receiving +LPS had lower rumen pH at 8 h, greater total VFA at 8 h, and lower rumen NH3 at 24 h after LPS infusion compared with -LPS steers (LPS × h, P ≤ 0.04). Total tract passage rates, DM, OM, NDF, ADF, and N intake, fecal N, digested N, and retained N were lower (P < 0.05) for +LPS than -LPS steers. Total N supply (dietary plus infused) and fecal N were greater (P < 0.05) for +BCAA vs -BCAA steers. The absence of LPS × BCAA interactions (P ≥ 0.20) for N balance indicated that post-ruminal supplementation of BCAA did not alleviate the negative effects of endotoxin on N utilization by growing steers.

Mosaic Structure and Molecular Evolution of the Leukotoxin Operon (lktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi

PubMed Central

Davies, Robert L.; Campbell, Susan; Whittam, Thomas S.

2002-01-01

The mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) was investigated by nucleotide sequence comparison of the lktC, lktB, and lktD genes in 23 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains. Sequence variation in the lktA gene has been described previously (R. L. Davies et al., J. Bacteriol. 183:1394–1404, 2001). The leukotoxin operon of M. haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events. However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of M. haemolytica. The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including M. glucosida and P. trehalosi. In contrast, the lktD gene is highly conserved in M. haemolytica. The lktC, lktA, and lktB genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains. These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of M. haemolytica. Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms. The 5′ and 3′ ends of the operon are highly conserved in M. haemolytica, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction. Although the lktA and lktB genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement. The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites. PMID:11741868

Mutations affecting transport of the hexitols D-mannitol, D-glucitol, and galactitol in Escherichia coli K-12: isolation and mapping.

PubMed Central

Lengeler, J

1975-01-01

Mutants of Escherichia coli K-12 unable to grow on any of the three naturally occurring hexitols D-manitol, D-glucitol, and galactitol and, among these specifically, mutants with altered transport and phosphorylating activity have been isolated. Different isolation procedures have been utilized, including suicide by D-[3H]mannitol, chemotaxis, and resistance to the toxic hexitol analogue 2-deoxy-arabino-hexitol. Mutations thus obtained have been mapped in four distinct operons. (i) Mutations affecting an enzyme II-complexmt1 activity of the phosphoenolpyruvate-dependent phosphotransferase system all map in gene mtlA. This gene has previously been shown (Solomon and Lin, 1972) to be part of an operon, mtl, located at 71 min on the E. coli linkage map containing, in addition to mtlA, the cis-dominant regulatory gene mtlC and mtlD, the structural gene for the enzyme D-mannitol-1-phosphate dehydrogenase. The gene order in this operon, induced by D-mannitol, is mtlC A D. (ii) Mutations in gene gutA affecting a second enzyme II-complexgut of the phosphotransferase system map at 51 min, clustered in operon gutC A D together with the cis-dominant regulatory gene gutC and the structural gene gutD for the enzyme D-glucitol-6-phosphate dehydrogenase. The gut operon, previously called sbl or srl, is induced by D-glucitol. (iii) Mutations affecting the transport and catabolism of galactitol are clustered in a third operon, gatC A D, located at 40.5 min. This operon again contains a cis-dominant regulatory gene, gatC, the structural gene gatD for galactitol-1-phosphate dehydrogenase, and gene gatA coding for a thrid hexitol-specific enzyme II-complexgat. Other genes coding for two additional enzymes involved in galactitol catabolism apparently are not linked to gatC A D. (iv) A fourth class of mutants pleiotropically negative for hexitol growth and transport maps in the pts operon. Triple-negative mutants (mtlA gutA gatA) do not have further transport or phosphorylating activity for any of the three hexitols. PMID:1100602

Anti-inflammatory effects of phenolic crude extracts from five fractions of Corchorus Olitorius L.

PubMed

Yan, Yeong-Yu; Wang, Yue-Wen; Chen, Su-Lin; Zhuang, Shu-Ru; Wang, Chin-Kun

2013-06-01

Corchorus olitorius L. is grown in Taiwan during summer. Tender leaves are crushed and washed by running water before eating. Five fractions including crude phenolic extracts (using 80 per cent aqueous acetone) of whole plant, leaf, stem, washed leaf (WL) and dried water washing material (WW) were used in this study. Linoleic acid autoxidation inhibitions on all fractions were higher than that on α-tocopherol. Except for WL and WW, other fractions also showed DPPH radical scavenging efficiency. The effect of all fractions on the regulation of inflammatory responses in lipopolysaccharide (LPS)-stimulated J774A.1 macrophage cells was investigated. All fractions diminished LPS-induced protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Nitric oxide (NO) and prostaglandin E2 (PGE(2)), downstream products, were also suppressed in dose-dependent manners, except for WL and WW. Oxidative modification and loss of leaf phenolics after kneading and washing greatly affected DPPH radical scavenging and inflammatory responses. Copyright © 2012 Elsevier Ltd. All rights reserved.

Disaggregation of lipopolysaccharide by albumin, hemoglobin or high-density lipoprotein, forming complexes that prime neutrophils for enhanced release of superoxide.

PubMed

Komatsu, Toshiya; Aida, Yoshitomi; Fukuda, Takao; Sanui, Terukazu; Hiratsuka, Shunji; Pabst, Michael J; Nishimura, Fusanori

2016-04-01

We studied the interaction of LPS with albumin, hemoglobin or high-density lipoprotein (HDL), and whether the interaction affected the activity of LPS on neutrophils. These proteins disaggregated LPS, depending upon temperature and LPS:protein ratio. Albumin-treated LPS was absorbed by immobilized anti-albumin antibody and was eluted with Triton X-100, indicating that LPS formed a hydrophobic complex with albumin. Rd mutant LPS was not disaggregated by the proteins, and did not form a complex with the proteins. But triethylamine-treated Rd mutant LPS formed complexes. When LPS was incubated with an equal concentration of albumin and with polymyxin B (PMXB), PMXB-LPS-protein three-way complexes were formed. After removal of PMXB, the complexes consisted of 11-15 LPS monomers bound to one albumin or hemoglobin molecule. LPS primed neutrophils for enhanced release of formyl peptide-stimulated superoxide, in a serum- and LPS-binding protein (LBP)-dependent manner. Although LPS plus LBP alone did not prime neutrophils, albumin-, hemoglobin- or HDL-treated LPS primed neutrophils when added with LBP. Triethylamine-treated Rd mutant LPS primed neutrophils only when incubated with one of the proteins and with LBP. Thus, in addition to LBP, disaggregation and complex formation of LPS with one of these proteins is required for LPS to prime neutrophils. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Sirt1 S-nitrosylation induces acetylation of HMGB1 in LPS-activated RAW264.7 cells and endotoxemic mice.

PubMed

Kim, Young Min; Park, Eun Jung; Kim, Hye Jung; Chang, Ki Churl

2018-06-18

Excessive inflammation plays a detrimental role in endotoxemia. A recent study indicated that alarmins such as high mobility group box 1 (HMGB1) have drawn attention as therapeutic targets of sepsis. Post-translational modification (i.e., acetylation of lysine residues) of HMGB1 leads to the release of HMGB1 into the cellular space, operating as a warning signal that induces inflammation. Sirtuin 1 (SIRT1) has been shown to negatively regulate HMGB1 hyperacetylation and its extracellular release in sepsis. Therefore, we hypothesized that the S-nitrosylation (SNO) of SIRT1 may disrupt the ability of SIRT1 to negatively regulate the hyperacetylation of HMGB1. As long as the S-nitrosylation of SIRT1 occurs during septic conditions, it may worsen the situation. We found that the activity of SIRT1 decreased as the SNO-SIRT1 levels increased, resulting in HMGB1 release by LPS in RAW264.7 cells. Both the iNOS inhibitor (1400 W) and silencing iNOS significantly inhibited SNO-SIRT1, allowing increases in SIRT1 activity that decreased the HMGB1 release by LPS. SNAP, a NO donor, significantly increased both SNO-SIRT1 levels and the HMGB1 release that was accompanied by decreased sirt1 activity. However, sirtinol, a Sirt1 inhibitor, by itself decreased Sirt1 activity compared to that of the control, so that it did not affect already increased SNO-SIRT levels by SNAP. Most importantly, in lung tissues of LPS-endotoxic mice, significantly increased levels of SNO-SIRT were found, which was inhibited by 1400 W treatment. Plasma nitrite and HMGB1 levels were significantly higher than those in the sham controls, and the elevated levels were significantly lowered in the presence of 1400 W. We concluded that the S-nitrosylation of Sirt1 under endotoxic conditions may uninhibit the acetylation of HMGB1 and its extracellular release. Copyright © 2018 Elsevier Inc. All rights reserved.

Genetic Characterization of a Sinorhizobium meliloti Chromosomal Region Involved in Lipopolysaccharide Biosynthesis

PubMed Central

Lagares, Antonio; Hozbor, Daniela F.; Niehaus, Karsten; Otero, Augusto J. L. Pich; Lorenzen, Jens; Arnold, Walter; Pühler, Alfred

2001-01-01

The genetic characterization of a 5.5-kb chromosomal region of Sinorhizobium meliloti 2011 that contains lpsB, a gene required for the normal development of symbiosis with Medicago spp., is presented. The nucleotide sequence of this DNA fragment revealed the presence of six genes: greA and lpsB, transcribed in the forward direction; and lpsE, lpsD, lpsC, and lrp, transcribed in the reverse direction. Except for lpsB, none of the lps genes were relevant for nodulation and nitrogen fixation. Analysis of the transcriptional organization of lpsB showed that greA and lpsB are part of separate transcriptional units, which is in agreement with the finding of a DNA stretch homologous to a “nonnitrogen” promoter consensus sequence between greA and lpsB. The opposite orientation of lpsB with respect to its first downstream coding sequence, lpsE, indicated that the altered LPS and the defective symbiosis of lpsB mutants are both consequences of a primary nonpolar defect in a single gene. Global sequence comparisons revealed that the greA-lpsB and lrp genes of S. meliloti have a genetic organization similar to that of their homologous loci in R. leguminosarum bv. viciae. In particular, high sequence similarity was found between the translation product of lpsB and a core-related biosynthetic mannosyltransferase of R. leguminosarum bv. viciae encoded by the lpcC gene. The functional relationship between these two genes was demonstrated in genetic complementation experiments in which the S. meliloti lpsB gene restored the wild-type LPS phenotype when introduced into lpcC mutants of R. leguminosarum. These results support the view that S. meliloti lpsB also encodes a mannosyltransferase that participates in the biosynthesis of the LPS core. Evidence is provided for the presence of other lpsB-homologous sequences in several members of the family Rhizobiaceae. PMID:11157937

Lipopolysaccharide modulation of a CD14-like molecule on porcine alveolar macrophages

NASA Technical Reports Server (NTRS)

Kielian, T. L.; Ross, C. R.; McVey, D. S.; Chapes, S. K.; Blecha, F.; Spooner, B. S. (Principal Investigator)

1995-01-01

Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.

Induction of surfactin production in Bacillus subtilis by gsp, a gene located upstream of the gramicidin S operon in Bacillus brevis.

PubMed Central

Borchert, S; Stachelhaus, T; Marahiel, M A

1994-01-01

The deduced amino acid sequence of the gsp gene, located upstream of the 5' end of the gramicidin S operon (grs operon) in Bacillus brevis, showed a high degree of similarity to the sfp gene product, which is located downstream of the srfA operon in B. subtilis. The gsp gene complemented in trans a defect in the sfp gene (sfpO) and promoted production of the lipopeptide antibiotic surfactin. The functional homology of Gsp and Sfp and the sequence similarity of these two proteins to EntD suggest that the three proteins represent a new class of proteins involved in peptide secretion, in support of a hypothesis published previously (T. H. Grossman, M. Tuckman, S. Ellestad, and M. S. Osburne, J. Bacteriol. 175:6203-6211, 1993). Images PMID:7512553

Quantifying translational coupling in E. coli synthetic operons using RBS modulation and fluorescent reporters.

PubMed

Levin-Karp, Ayelet; Barenholz, Uri; Bareia, Tasneem; Dayagi, Michal; Zelcbuch, Lior; Antonovsky, Niv; Noor, Elad; Milo, Ron

2013-06-21

Translational coupling is the interdependence of translation efficiency of neighboring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. Translational coupling was observed in prokaryotic operons several decades ago, but the quantitative range of modulation translational coupling leads to and the factors governing this modulation were only partially characterized. In this study, we systematically quantify and characterize translational coupling in E. coli synthetic operons using a library of plasmids carrying fluorescent reporter genes that are controlled by a set of different ribosome binding site (RBS) sequences. The downstream gene expression level is found to be enhanced by the upstream gene expression via translational coupling with the enhancement level varying from almost no coupling to over 10-fold depending on the upstream gene's sequence. Additionally, we find that the level of translational coupling in our system is similar between the second and third locations in the operon. The coupling depends on the distance between the stop codon of the upstream gene and the start codon of the downstream gene. This study is the first to systematically and quantitatively characterize translational coupling in a synthetic E. coli operon. Our analysis will be useful in accurate manipulation of gene expression in synthetic biology and serves as a step toward understanding the mechanisms involved in translational expression modulation.

A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

PubMed

Mancini, Stefano; Abicht, Helge K; Gonskikh, Yulia; Solioz, Marc

2015-02-01

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments. © 2014 John Wiley & Sons Ltd.

Repeated exposure to intra-amniotic LPS partially protects against adverse effects of intravenous LPS in preterm lambs.

PubMed

Gisslen, Tate; Hillman, Noah H; Musk, Gabrielle C; Kemp, Matthew W; Kramer, Boris W; Senthamaraikannan, Paranthaman; Newnham, John P; Jobe, Alan H; Kallapur, Suhas G

2014-02-01

Histologic chorioamnionitis, frequently associated with preterm births and adverse outcomes, results in prolonged exposure of preterm fetuses to infectious agents and pro-inflammatory mediators, such as LPS. Endotoxin tolerance-type effects were demonstrated in fetal sheep following repetitive systemic or intra-amniotic (i.a.) exposures to LPS, suggesting that i.a. LPS exposure would cause endotoxin tolerance to a postnatal systemic dose of LPS in preterm sheep. In this study, randomized pregnant ewes received either two i.a. injections of LPS or saline prior to preterm delivery. Following operative delivery, the lambs were treated with surfactant, ventilated, and randomized to receive either i.v. LPS or saline at 30  min of age. Physiologic variables and indicators of systemic and lung inflammation were measured. Intravenous LPS decreased blood neutrophils and platelets values following i.a. saline compared to that after i.a. LPS. Intra-amniotic LPS prevented blood pressure from decreasing following the i.v. LPS, but also caused an increased oxygen index. Intra-amniotic LPS did not cause endotoxin tolerance as assessed by cytokine expression in the liver, lung or plasma, but increased myeloperoxidase-positive cells in the lung. The different compartments of exposure to LPS (i.a. vs i.v.) are unique to the fetal to newborn transition. Intra-amniotic LPS incompletely tolerized fetal lambs to postnatal i.v. LPS.

Influence of the feedback loops in the trp operon of B. subtilis on the system dynamic response and noise amplitude.

PubMed

Zamora-Chimal, Criseida; Santillán, Moisés; Rodríguez-González, Jesús

2012-10-07

In this paper we introduce a mathematical model for the tryptophan operon regulatory pathway in Bacillus subtilis. This model considers the transcription-attenuation, and the enzyme-inhibition regulatory mechanisms. Special attention is paid to the estimation of all the model parameters from reported experimental data. With the aid of this model we investigate, from a mathematical-modeling point of view, whether the existing multiplicity of regulatory feedback loops is advantageous in some sense, regarding the dynamic response and the biochemical noise in the system. The tryptophan operon dynamic behavior is studied by means of deterministic numeric simulations, while the biochemical noise is analyzed with the aid of stochastic simulations. The model feasibility is tested comparing its stochastic and deterministic results with experimental reports. Our results for the wildtype and for a couple of mutant bacterial strains suggest that the enzyme-inhibition feedback loop, dynamically accelerates the operon response, and plays a major role in the reduction of biochemical noise. Also, the transcription-attenuation feedback loop makes the trp operon sensitive to changes in the endogenous tryptophan level, and increases the amplitude of the biochemical noise. Copyright © 2012 Elsevier Ltd. All rights reserved.

Transcriptional and posttranscriptional regulation of Bacillus sp. CDB3 arsenic-resistance operon ars1

PubMed Central

Yu, Xuefei; Zheng, Wei; Bhat, Somanath; Aquilina, J. Andrew

2015-01-01

Bacillus sp. CDB3 possesses a novel eight-gene ars cluster (ars1, arsRYCDATorf7orf8) with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons. PMID:26355338

Neoadjuvant radiation in primary extremity liposarcoma: correlation of MRI features with histopathology.

PubMed

Wortman, Jeremy R; Tirumani, Sree Harsha; Tirumani, Harika; Shinagare, Atul B; Jagannathan, Jyothi P; Hornick, Jason L; Ramaiya, Nikhil H

2016-05-01

To evaluate MRI features of response of primary extremity liposarcoma (LPS) to neoadjuvant radiation therapy (RT) with histopathologic correlation. In this IRB-approved study including 125 patients with extremity LPS treated with neoadjuvant RT from 2000 to 2013, MRI of the primary tumour in 18 patients (5 pleomorphic LPS, 13 myxoid LPS) before and after RT were reviewed by two radiologists by consensus. Histopathology of the surgical specimens was reviewed by a pathologist with expertise in sarcomas. In the pleomorphic LPS cohort, 3/5 tumours increased in size; 3/5 decreased in enhancing component; and 3/5 increased in peritumoral oedema, intratumoral haemorrhage, and necrosis. In the myxoid LPS cohort, 12/13 tumours decreased in size, 8/13 decreased in enhancing component, and 5/13 increased in internal fat following RT. Histopathology showed ≥50% residual tumour in 1/5 pleomorphic LPS and 2/13 myxoid LPS. Hyalinization/necrosis of ≥75% was noted in 4/5 pleomorphic LPS and 11/13 myxoid LPS. Cytodifferentiation was noted in 1/5 pleomorphic and 9/13 myxoid LPS. While pleomorphic LPS showed an increase in size, peritumoral oedema, intratumoral haemorrhage, and necrosis on MRI following neoadjuvant RT, myxoid LPS showed a decrease in size and enhancement with an increase in internal fat. • Pleomorphic LPS commonly increase in size and necrosis on MRI following RT. • Myxoid LPS commonly decrease in size and enhancement on MRI following RT. • Myxoid LPS often increase in fatty component on MRI following RT.

Kukoamine B promotes TLR4-independent lipopolysaccharide uptake in murine hepatocytes.

PubMed

Yang, Dong; Zheng, Xinchuan; Wang, Ning; Fan, Shijun; Yang, Yongjun; Lu, Yongling; Chen, Qian; Liu, Xin; Zheng, Jiang

2016-09-06

Free bacterial lipopolysaccharide (LPS) is generally removed from the bloodstream through hepatic uptake via TLR4, the LPS pattern recognition receptor, but mechanisms for internalization and clearance of conjugated LPS are less clear. Kukoamine B (KB) is a novel cationic alkaloid that interferes with LPS binding to TLR4. In this study, KB accelerated blood clearance of LPS. KB also enhanced LPS distribution in the hepatic tissues of C57 BL/6 mice, along with LPS uptake in primary hepatocytes and HepG2 cells. By contrast, KB inhibited LPS internalization in Kupffer and RAW 264.7 cells. Loss of TLR4 did not affect LPS uptake into KB-treated hepatocytes. We also detected selective upregulation of the asialoglycoprotein receptor (ASGPR) upon KB treatment, and ASGPR colocalized with KB in cultured hepatocytes. Molecular docking showed that KB bound to ASGPR in a manner similar to GalNAc, a known ASGPR agonist. GalNAc dose-dependently reduced KB internalization, suggesting it competes with KB for ASGPR binding, and ASGPR knockdown also impaired LPS uptake into hepatocytes. Finally, while KB enhanced LPS uptake, it was protective against LPS-induced inflammation and hepatocyte injury. Our study provides a new mechanism for conjugated LPS hepatic uptake induced by the LPS neutralizer KB and mediated by membrane ASGPR binding.

Effect of chocolate and Propolfenol on rabbit spermatogenesis and sperm quality following bacterial lipopolysaccharide treatment.

PubMed

Collodel, Giulia; Moretti, Elena; Del Vecchio, Maria Teresa; Biagi, Marco; Cardinali, Raffaella; Mazzi, Lucia; Brecchia, Gabriele; Maranesi, Margherita; Manca, Daniela; Castellini, Cesare

2014-08-01

The aims of the study were to evaluate the effects of chocolate and propolis-enriched diets on rabbit spermatogenesis, sperm motility, and ultrastructure following bacterial lipopolysaccharide (LPS) treatment. Thirty-two New Zealand White rabbits were divided into four groups. The LPS-Propolfenol(®) group received propolis (500 mg/kg/day) in their diet for 15 days, while the LPS-chocolate group was fed 70% cacao chocolate (1 g/1 kg/day) for the same period. Following the diet treatments, rabbits in the LPS-Propolfenol(®) and LPS-chocolate groups, and an LPS group received a single intraperitoneal dose of 50 μg/kg LPS, and the control group received only saline. Kinematic sperm traits were evaluated with a computer assisted sperm analyzer (CASA) system, and ultrastructural characteristics were examined by transmission electron microscopy (TEM). Testicular and epididymal tissues were observed by light microscopy and TEM and multiplex real time reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to detect and quantify toll-like receptor-4 (TLR-4) gene expression. The values of the analyzed semen parameters of rabbits treated with LPS-Propolfenol(®) and LPS-chocolate did not show any variations compared with the control group, but they were lower in rabbits treated only with LPS. Alterations observed in the testicular tissue of LPS treated-rabbits were not detected in specimens from the LPS-chocolate and LPS-Propolfenol(®) groups, which showed normal spermatogenesis. The TLR-4 mRNA expression was similar in controls, in LPS treated, and in LPS-chocolate groups, but it was significantly (p < 0.01) decreased in LPS-Propolfenol(®) rabbits. In conclusion, a chocolate and propolis-enriched diet showed a protective effect on the spermatogenetic process of buck rabbits following LPS treatment.

Positions of Trp Codons in the Leader Peptide-Coding Region of the at Operon Influence Anti-Trap Synthesis and trp Operon Expression in Bacillus licheniformis▿

PubMed Central

Levitin, Anastasia; Yanofsky, Charles

2010-01-01

Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria, trp operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNATrp. Tryptophan-activated TRAP binds to trp operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNATrp. In this paper, we focus on explaining the differences in organization and regulatory functions of the at operon's leader peptide-coding region, rtpLP, of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B. licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at operon leader peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed to allow a mild charged-tRNATrp deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis. PMID:20061467

The groESL Chaperone Operon of Lactobacillus johnsonii†

PubMed Central

Walker, D. Carey; Girgis, Hany S.; Klaenhammer, Todd R.

1999-01-01

The Lactobacillus johnsonii VPI 11088 groESL operon was localized on the chromosome near the insertion element IS1223. The operon was initially cloned as a series of three overlapping PCR fragments, which were sequenced and used to design primers to amplify the entire operon. The amplified fragment was used as a probe to recover the chromosomal copy of the groESL operon from a partial library of L. johnsonii VPI 11088 (NCK88) DNA, cloned in the shuttle vector pTRKH2. The 2,253-bp groESL fragment contained three putative open reading frames, two of which encoded the ubiquitous GroES and GroEL chaperone proteins. Analysis of the groESL promoter region revealed three transcription initiation sites, as well as three sets of inverted repeats (IR) positioned between the transcription and translation start sites. Two of the three IR sets bore significant homology to the CIRCE elements, implicated in negative regulation of the heat shock response in many bacteria. Northern analysis and primer extension revealed that multiple temperature-sensitive promoters preceded the groESL chaperone operon, suggesting that stress protein production in L. johnsonii is strongly regulated. Maximum groESL transcription activity was observed following a shift to 55°C, and a 15 to 30-min exposure of log-phase cells to this temperature increased the recovery of freeze-thawed L. johnsonii VPI 11088. These results suggest that a brief, preconditioning heat shock can be used to trigger increased chaperone production and provide significant cross-protection from the stresses imposed during the production of frozen culture concentrates. PMID:10388700

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

USGS Publications Warehouse

Cardenas, W.; Dankert, J.R.; Jenkins, J.A.

2004-01-01

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P < 0.05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by LPS, thereby indicting commone innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of invertbrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic compounds.

Regulation of the glv Operon in Bacillus subtilis: YfiA (GlvR) Is a Positive Regulator of the Operon That Is Repressed through CcpA and cre

PubMed Central

Yamamoto, Hiroki; Serizawa, Masakuni; Thompson, John; Sekiguchi, Junichi

2001-01-01

Maltose metabolism and the regulation of the glv operon of Bacillus subtilis, comprising three genes, glvA (6-phospho-α-glucosidase), yfiA (now designated glvR), and glvC (EIICB transport protein), were investigated. Maltose dissimilation was dependent primarily upon the glv operon, and insertional inactivation of either glvA, glvR, or glvC markedly inhibited growth on the disaccharide. A second system (MalL) contributed to a minor extent to maltose metabolism. Northern blotting revealed two transcripts corresponding to a monocistronic mRNA of glvA and a polycistronic mRNA of glvA-glvR-glvC. Primer extension analysis showed that both transcripts started at the same base (G) located 26 bp upstream of the 5′ end of glvA. When glvR was placed under control of the spac promoter, expression of the glv operon was dependent upon the presence of isopropyl-β-d-thiogalactopyranoside (IPTG). In regulatory studies, the promoter sequence of the glv operon was fused to lacZ and inserted into the amyE locus, and the resultant strain (AMGLV) was then transformed with a citrate-controlled glvR plasmid, pHYCM2VR. When cultured in Difco sporulation medium containing citrate, this transformant [AMGLV(pHYCM2VR)] expressed LacZ activity, but synthesis of LacZ was repressed by glucose. In an isogenic strain, [AMGLVCR(pHYCM2VR)], except for a mutation in the sequence of a catabolite-responsive element (cre), LacZ activity was expressed in the presence of citrate and glucose. Insertion of a citrate-controlled glvR plasmid at the amyE locus of ccpA+ and ccpA mutant organisms yielded strains AMCMVR and AMCMVRCC, respectively. In the presence of both glucose and citrate, AMCMVR failed to express the glv operon, whereas under the same conditions high-level expression of both mRNA transcripts was found in strain AMCMVRCC. Collectively, our findings suggest that GlvR (the product of the glvR gene) is a positive regulator of the glv operon and that glucose exerts its effect via catabolite repression requiring both CcpA and cre. PMID:11489864

Improved immunogenicity of tetanus toxoid by Brucella abortus S19 LPS adjuvant.

PubMed

Mohammadi, Mohsen; Kianmehr, Zahra; Kaboudanian Ardestani, Sussan; Gharegozlou, Behnaz

2014-09-01

Adjuvants are used to increase the immunogenicity of new generation vaccines, especially those based on recombinant proteins. Despite immunostimulatory properties, the use of bacterial lipopolysaccharide (LPS) as an adjuvant has been hampered due to its toxicity and pyrogenicity. Brucella abortus LPS is less toxic and has no pyrogenic properties compared to LPS from other gram negative bacteria. To evaluate the adjuvant effect of B. abortus (vaccine strain, S19) LPS for tetanus toxoid antigen (TT) and to investigate the protective effect of different tetanus vaccine preparations. LPS was extracted and purified from B. abortus S19 and KDO, glycan, phosphate content, and protein contamination were measured. Adipic acid dihydrazide (ADH) was used as a linker for conjugation of TT to LPS. Different amounts of B. abortus LPS, TT, TT conjugated with LPS, and TT mixed with LPS or complete Freund's adjuvant (CFA) were injected into mice and antibody production against TT was measured. The protective effect of induced antibodies was determined by LD50. Immunization of mice with TT+LPS produced the highest anti-TT antibody titer in comparison to the group immunized with TT without any adjuvant or the groups immunized with TT-LPS or TT+CFA. Tetanus toxid-S19 LPS also produced a 100% protective effect against TT in immunized mice. These data indicate that B. abortus LPS enhances the immune responses to TT and suggest the possible use of B. abortus LPS as an adjuvant in vaccine preparations.

Dedifferentiated liposarcoma with "homologous" lipoblastic (pleomorphic liposarcoma-like) differentiation: clinicopathologic and molecular analysis of a series suggesting revised diagnostic criteria.

PubMed

Mariño-Enríquez, Adrián; Fletcher, Christopher D M; Dal Cin, Paola; Hornick, Jason L

2010-08-01

Dedifferentiated liposarcoma (LPS) is a malignant adipocytic neoplasm defined as the transition from well-differentiated LPS to a nonlipogenic sarcoma. Heterologous differentiation is seen in 5% to 10% of dedifferentiated LPS, usually with myogenic or osteo/chondrosarcomatous elements. Adipocytic differentiation in the dedifferentiated component is incompatible with the current definition of dedifferentiated LPS. Pleomorphic LPS is a high-grade sarcoma containing lipoblasts. At least in areas, pleomorphic LPS can be indistinguishable from dedifferentiated LPS, except for the presence of lipoblasts in pleomorphic LPS and well-differentiated LPS areas in dedifferentiated LPS. We evaluated 12 unusual liposarcomas: 11 cases with pleomorphic LPS-like morphology affecting patients with concomitant or previous well-differentiated/dedifferentiated LPS, and 1 case resembling inflammatory "MFH" with scattered lipoblasts. Clinical and histologic features were reviewed. Immunohistochemistry for MDM2 and CDK4 was carried out. Amplification of 12q13 to q15 was studied by FISH analysis of the HMGA2 locus. The tumors arose in the retroperitoneum (7), proximal lower extremity (3), chest wall (1), and neck (1) of 9 males and 3 females (median age 66 y; range 49 to 76). Size ranged from 9 to 32 cm (median 23 cm). In 3 cases, there was an abrupt transition between well-differentiated LPS and sheets of pleomorphic lipoblasts, indistinguishable from pleomorphic LPS. Four cases consisted of otherwise typical dedifferentiated LPS (with adjacent well-differentiated LPS), except for the presence of lipoblasts in the high-grade component. One case contained both nonlipogenic spindle cell areas and an inflammatory "MFH"-like component with numerous admixed lipoblasts. Four cases were composed exclusively of pleomorphic LPS-like areas developing in 1 of the recurrences or metastases of a prior typical dedifferentiated LPS. Two cases also showed heterologous smooth muscle differentiation. MDM2 and CDK4 were positive in both the dedifferentiated LPS and pleomorphic LPS-like components in 12/12 and 11/12 cases, respectively. FISH analysis showed high-level amplification of 12q14.3 in all 8 cases successfully tested. Karyotypes were available for 3 cases and showed ring and giant marker chromosomes. Follow-up, available for 11 patients, ranged from 19 to 196 months (median 36 mo). Seven patients developed local recurrences (multiple in 3), and 3 developed lung metastases. Thus far, 5 patients have died of disease, 3 are alive with recurrent or metastatic disease, and 3 are alive with no evidence of disease. We conclude that dedifferentiated LPS can show lipoblastic differentiation in the high-grade component, resulting in areas indistinguishable from pleomorphic LPS. The available clinical and molecular data support the notion of "homologous" lipoblastic differentiation in dedifferentiated LPS, rather than mixed-type LPS.

LPS receptor CD14 participates in release of TNF-alpha in RAW 264.7 and peritoneal cells but not in kupffer cells.

PubMed

Lichtman, S N; Wang, J; Lemasters, J J

1998-07-01

Lipopolysaccharide (LPS) is a bacterial polymer that stimulates macrophages to release tumor necrosis factor-alpha (TNF-alpha). In macrophages (RAW 264.7 and peritoneal cells), LPS binds to the CD14 surface receptor as the first step toward signaling. Liver macrophages, Kupffer cells, are the most numerous fixed-tissue macrophage in the body. The presence of CD14 on Kupffer cells and its role in LPS stimulation of TNF-alpha were examined. TNF-alpha release by Kupffer cells after LPS stimulation was the same in the presence and absence of serum. RAW 264.7 and peritoneal cells, which utilize the CD14 receptor, released significantly less TNF-alpha after LPS stimulation in the absence of serum because of the absence of LPS-binding protein. Phosphatidylinositol-phospholipase C treatment, which cleaves the CD14 receptor, decreased LPS-stimulated TNF-alpha release by RAW 264.7 cells but not by Kupffer cells. Deacylated LPS (dLPS) competes with LPS at the CD14 receptor when incubated in a ratio of 100:1 (dLPS/LPS). Such competition blocked LPS-stimulated TNF-alpha release from RAW 264.7 cells but not from Kupffer cells. Western and fluorescence-activated cell sorter analysis directly demonstrated the presence of CD14 on RAW 264.7 cells and murine peritoneal cells but showed only minimal amounts of CD14 in murine Kupffer cells. LPS stimulation did not increase the amount of CD14 detectable on mouse Kupffer cells. CD14 expression is very low in Kupffer cells, and LPS-stimulated TNF-alpha release is independent of CD14 in these cells.

The three-dimensional structure of TrmB, a transcriptional regulator of dual function in the hyperthermophilic archaeon Pyrococcus furiosus in complex with sucrose

PubMed Central

Krug, Michael; Lee, Sung-Jae; Boos, Winfried; Diederichs, Kay; Welte, Wolfram

2013-01-01

TrmB is a repressor that binds maltose, maltotriose, and sucrose, as well as other α-glucosides. It recognizes two different operator sequences controlling the TM (Trehalose/Maltose) and the MD (Maltodextrin) operon encoding the respective ABC transporters and sugar-degrading enzymes. Binding of maltose to TrmB abrogates repression of the TM operon but maintains the repression of the MD operon. On the other hand, binding of sucrose abrogates repression of the MD operon but maintains repression of the TM operon. The three-dimensional structure of TrmB in complex with sucrose was solved and refined to a resolution of 3.0 Å. The structure shows the N-terminal DNA binding domain containing a winged-helix-turn-helix (wHTH) domain followed by an amphipathic helix with a coiled-coil motif. The latter promotes dimerization and places the symmetry mates of the putative recognition helix in the wHTH motif about 30 Å apart suggesting a canonical binding to two successive major grooves of duplex palindromic DNA. This suggests that the structure resembles the conformation of TrmB recognizing the pseudopalindromic TM promoter but not the conformation recognizing the nonpalindromic MD promoter. PMID:23576322

Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic

PubMed Central

Mirkovic, Nemanja; Polovic, Natalija; Vukotic, Goran; Jovcic, Branko; Miljkovic, Marija; Radulovic, Zorica; Diep, Dzung B.

2016-01-01

Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins. Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes, lmgA, lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes, lmgF, lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization–time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid. PMID:26896142

A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

PubMed

Elliott, T

1992-01-01

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.

Concomitant Exposure to Ovalbumin and Endotoxin Augments Airway Inflammation but Not Airway Hyperresponsiveness in a Murine Model of Asthma

PubMed Central

Mac Sharry, John; Shalaby, Karim H.; Marchica, Cinzia; Farahnak, Soroor; Chieh-Li, Tien; Lapthorne, Susan; Qureshi, Salman T.; Shanahan, Fergus; Martin, James G.

2014-01-01

Varying concentrations of lipopolysaccharide (LPS) in ovalbumin (OVA) may influence the airway response to allergic sensitization and challenge. We assessed the contribution of LPS to allergic airway inflammatory responses following challenge with LPS-rich and LPS-free commercial OVA. BALB/c mice were sensitized with LPS-rich OVA and alum and then underwent challenge with the same OVA (10 µg intranasally) or an LPS-free OVA. Following challenge, bronchoalveolar lavage (BAL), airway responsiveness to methacholine and the lung regulatory T cell population (Treg) were assessed. Both OVA preparations induced BAL eosinophilia but LPS-rich OVA also evoked BAL neutrophilia. LPS-free OVA increased interleukin (IL)-2, IL-4 and IL-5 whereas LPS-rich OVA additionally increased IL-1β, IL-12, IFN-γ, TNF-α and KC. Both OVA-challenged groups developed airway hyperresponsiveness. TLR4-deficient mice challenged with either OVA preparation showed eosinophilia but not neutrophilia and had increased IL-5. Only LPS-rich OVA challenged mice had increased lung Tregs and LPS-rich OVA also induced in vitro Treg differentiation. LPS-rich OVA also induced a Th1 cytokine response in human peripheral blood mononuclear cells.We conclude that LPS-rich OVA evokes mixed Th1, Th2 and innate immune responses through the TLR-4 pathway, whereas LPS-free OVA evokes only a Th2 response. Contaminating LPS is not required for induction of airway hyperresponsiveness but amplifies the Th2 inflammatory response and is a critical mediator of the neutrophil, Th1 and T regulatory cell responses to OVA. PMID:24968337

Prevalence of lymphoplasmacytic synovitis in dogs with naturally occurring cranial cruciate ligament rupture.

PubMed

Erne, Jay B; Goring, Robert L; Kennedy, Fidelma A; Schoenborn, William C

2009-08-15

To determine the prevalence of lymphoplasmacytic synovitis (LPS) in dogs with naturally occurring cranial cruciate ligament (CCL) rupture and compare clinical, radiographic, cytologic, and histologic findings in dogs with and without LPS. Cross-sectional study. 110 dogs with naturally occurring CCL rupture. Histologic examination of synovial biopsy specimens obtained at the time of surgical treatment was used to identify dogs with LPS. Clinical, radiographic, cytologic, and histologic findings were compared between dogs with and without LPS. 56 (51%) dogs had histologic evidence of LPS. There were no significant differences in age, body weight, duration of lameness, severity of lameness, severity of radiographic signs of degenerative joint disease, extent of CCL rupture (partial vs complete), or gross appearance of the medial meniscus between dogs with and without LPS. Mean tibial plateau angle was significantly lower in dogs with LPS than in dogs without LPS, and dogs with LPS were significantly more likely to have neutrophils in their synovial fluid. Lymphocytes were seen in synovial fluid from a single dog with LPS. Results suggested that LPS was common in dogs with naturally occurring CCL rupture. However, only minor clinical, radiographic, cytologic, and histologic differences were identified between dogs with and without LPS.

Lipopolysaccharide binding protein enhances the responsiveness of alveolar macrophages to bacterial lipopolysaccharide. Implications for cytokine production in normal and injured lungs.

PubMed Central

Martin, T R; Mathison, J C; Tobias, P S; Letúrcq, D J; Moriarty, A M; Maunder, R J; Ulevitch, R J

1992-01-01

A plasma lipopolysaccharide (LPS)-binding protein (LBP) has been shown to regulate the response of rabbit peritoneal macrophages and human blood monocytes to endotoxin (LPS). We investigated whether LBP is present in lung fluids and the effects of LBP on the response of lung macrophages to LPS. Immunoreactive LBP was detectable in the lavage fluids of patients with the adult respiratory distress syndrome by immunoprecipitation followed by Western blotting, and also by specific immunoassay. In rabbits, the LBP appeared to originate outside of the lungs, inasmuch as mRNA transcripts for LBP were identified in total cellular RNA from liver, but not from lung homogenates or alveolar macrophages. Purified LBP enhanced the response of human and rabbit alveolar macrophages to both smooth form LPS (Escherichia coli O111B:4) and rough form LPS (Salmonella minnesota Re595). In the presence of LBP and LPS, the onset of tumor necrosis factor-alpha (TNF alpha) production occurred earlier and at an LPS threshold dose that was as much as 1,000-fold lower for both types of LPS. In rabbit alveolar macrophages treated with LBP and LPS, TNF alpha mRNA appeared earlier, reached higher levels, and had a prolonged half-life as compared with LPS treatment alone. Neither LPS nor LPS and LBP affected pHi or [Cai++] in alveolar macrophages. Specific monoclonal antibodies to CD14, a receptor that binds LPS/LBP complexes, inhibited TNF alpha production by human alveolar macrophages stimulated with LPS alone or with LPS/LBP complexes, indicating the importance of CD14 in mediating the effects of LPS on alveolar macrophages. Thus, immunoreactive LBP accumulates in lung lavage fluids in patients with lung injury and enhances LPS-stimulated TNF alpha gene expression in alveolar macrophages by a pathway that depends on the CD14 receptor. LBP may play an important role in augmenting TNF alpha expression by alveolar macrophages within the lungs. Images PMID:1281827

Effects of normal saline and selenium-enriched hot spring water on experimentally induced rhinosinusitis in rats.

PubMed

Kim, Dong-Hyun; Yeo, Sang Won

2013-01-01

This prospective, randomized, and controlled study examined the effects of normal saline and selenium-enriched hot spring water on experimentally induced rhinosinusitis in rats. The study comprised two control groups (untreated and saline-treated) and three experimental groups of Sprague Dawley rats. The experimental groups received an instillation of lipopolysaccharide (LPS) only, LPS+normal saline (LPS/saline), or LPS+selenium-enriched hot spring water (LPS/selenium). Histopathological changes were identified using hematoxylin-eosin staining. Leakage of exudate was identified using fluorescence microscopy. Microvascular permeability was measured using the Evans blue dye technique. Expression of the Muc5ac gene was measured using reverse transcription-polymerase chain reaction. Mucosal edema and expression of the Muc5ac gene were significantly lower in the LPS/saline group than in the LPS group. Microvascular permeability, mucosal edema, and expression of the Muc5ac gene were significantly lower in the LPS/selenium group than in the LPS group. Mucosal edema was similar in the LPS/selenium group and LPS/saline group, but capillary permeability and Muc5ac expression were lower in the LPS/selenium group. This study shows that normal saline and selenium-enriched hot spring water reduce inflammatory activity and mucus hypersecretion in LPS-induced rhinosinusitis in rats. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Crosstalk between virulence loci: regulation of Salmonella enterica pathogenicity island 1 (SPI-1) by products of the std fimbrial operon.

PubMed

López-Garrido, Javier; Casadesús, Josep

2012-01-01

Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD, stdE, and stdF). We present evidence that two such loci (stdE and stdF) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam(-) background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam(+) background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam(-) mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (>60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed.

Genetic Locus for Streptolysin S Production by Group A Streptococcus

PubMed Central

Nizet, Victor; Beall, Bernard; Bast, Darrin J.; Datta, Vivekananda; Kilburn, Laurie; Low, Donald E.; De Azavedo, Joyce C. S.

2000-01-01

Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis and invasive infections, including necrotizing fasciitis. Streptolysin S (SLS) is the cytolytic factor that creates the zone of beta-hemolysis surrounding GAS colonies grown on blood agar. We recently reported the discovery of a potential genetic determinant involved in SLS production, sagA, encoding a small peptide of 53 amino acids (S. D. Betschel, S. M. Borgia, N. L. Barg, D. E. Low, and J. C. De Azavedo, Infect. Immun. 66:1671–1679, 1998). Using transposon mutagenesis, chromosomal walking steps, and data from the GAS genome sequencing project (www.genome.ou.edu/strep.html), we have now identified a contiguous nine-gene locus (sagA to sagI) involved in SLS production. The sag locus is conserved among GAS strains regardless of M protein type. Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype. Targeted integrations (i) upstream of the sagA promoter and (ii) downstream of a terminator sequence after sagI did not affect SLS production, establishing the functional boundaries of the operon. A rho-independent terminator sequence between sagA and sagB appears to regulate the amount of sagA transcript produced versus transcript for the entire operon. Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant. Finally, heterologous expression of the intact sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis. We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production. Sequence homologies of sag operon gene products suggest that SLS is related to the bacteriocin family of microbial toxins. PMID:10858242

Expression of the Arginine Deiminase Pathway Genes in Lactobacillus sakei Is Strain Dependent and Is Affected by the Environmental pH

PubMed Central

Rimaux, T.; Rivière, A.; Illeghems, K.; Weckx, S.; De Vuyst, L.

2012-01-01

The adaptation of Lactobacillus sakei to a meat environment is reflected in its metabolic potential. For instance, the ability to utilize arginine through the arginine deiminase (ADI) pathway, resulting in additional ATP, represents a competitive benefit. In L. sakei CTC 494, the arc operon (arcABCTDR) shows the same gene order and organization as that in L. sakei 23K, the genome sequence of which is known. However, differences in relative gene expression were found, and these seemed to be optimal in different growth phases, namely, the highest relative gene expression level was in the end exponential growth phase in the case of L. sakei CTC 494 and in the mid-exponential growth phase of L. sakei 23K. Also, the environmental pH influenced the relative expression level of the arc operon, as shown for L. sakei CTC 494, with the highest relative expression level occurring at the optimal pH for growth (pH 6.0). Deviations from this optimal pH (pH 5.0 and pH 7.0) resulted in an overall decline of the relative expression level of all genes of the arc operon. Furthermore, a differential relative expression of the individual genes of the arc operon was found, with the highest relative gene expression occurring for the first two genes of the arc operon (arcA and arcB). Finally, it was shown that some L. sakei strains were able to convert agmatine into putrescine, suggesting an operational agmatine deiminase pathway in these strains, a metabolic trait that is undesirable in meat fermentations. This study shows that this metabolic trait is most probably encoded by a previously erroneously annotated second putative arc operon. PMID:22544250

Sensitivity and Specificity of an Operon Immunochromatographic Test in Serum and Whole-Blood Samples for the Diagnosis of Trypanosoma cruzi Infection in Spain, an Area of Nonendemicity

PubMed Central

Flores-Chavez, María; Cruz, Israel; Nieto, Javier; Gárate, Teresa; Navarro, Miriam; Pérez-Ayala, Ana; López-Vélez, Rogelio

2012-01-01

Trypanosoma cruzi infection is an imported parasitic disease in Spain, and the majority of infected individuals are in the chronic phase of the disease. This study evaluated the sensitivity and specificity of the Operon immunochromatographic test (ICT-Operon; Simple Stick Chagas and Simple Chagas WB [whole blood]; Operon S.A., Spain) for different biological samples. Well-characterized serum samples were obtained from chagasic patients (n = 63), nonchagasic individuals (n = 95), visceral leishmaniasis patients (n = 38), and malaria patients (n = 55). Noncharacterized specimens were obtained from Latin American immigrants and individuals at risk with a clinical and/or epidemiological background: these specimens were recovered serum or plasma samples (n = 450), whole peripheral blood (n = 94), and capillary blood (n = 282). The concordance of the results by enzyme-linked immunosorbent assay and indirect immunofluorescence test was considered to be the “gold standard” for diagnosis. Serum and plasma samples were analyzed by Stick Chagas, and whole blood was analyzed by Simple Chagas WB. The sensitivity and specificity of the ICT-Operon in well-characterized samples were 100% and 97.9%, respectively. No cross-reactivity was found with samples obtained from visceral leishmaniasis patients. In contrast, a false-positive result was obtained in 27.3% of samples from malaria patients. The sensitivities of the rapid test in noncharacterized serum or plasma, peripheral blood, and capillary blood samples were 100%, 92.1%, and 86.4%, respectively, while the specificities were 91.6%, 93.6%, and 95% in each case. ICT-Operon showed variable sensitivity, depending on the kind of sample, performing better when serum or plasma samples were used. It could therefore be used for serological screening combined with any other conventional test. PMID:22761296

Two Paralogous Families of a Two-Gene Subtilisin Operon Are Widely Distributed in Oral Treponemes

PubMed Central

Correia, Frederick F.; Plummer, Alvin R.; Ellen, Richard P.; Wyss, Chris; Boches, Susan K.; Galvin, Jamie L.; Paster, Bruce J.; Dewhirst, Floyd E.

2003-01-01

Certain oral treponemes express a highly proteolytic phenotype and have been associated with periodontal diseases. The periodontal pathogen Treponema denticola produces dentilisin, a serine protease of the subtilisin family. The two-gene operon prcA-prtP is required for expression of active dentilisin (PrtP), a putative lipoprotein attached to the treponeme's outer membrane or sheath. The purpose of this study was to examine the diversity and structure of treponemal subtilisin-like proteases in order to better understand their distribution and function. The complete sequences of five prcA-prtP operons were determined for Treponema lecithinolyticum, “Treponema vincentii,” and two canine species. Partial operon sequences were obtained for T. socranskii subsp. 04 as well as 450- to 1,000-base fragments of prtP genes from four additional treponeme strains. Phylogenetic analysis demonstrated that the sequences fall into two paralogous families. The first family includes the sequence from T. denticola. Treponemes possessing this operon family express chymotrypsin-like protease activity and can cleave the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (SAAPFNA). Treponemes possessing the second paralog family do not possess chymotrypsin-like activity or cleave SAAPFNA. Despite examination of a range of protein and peptide substrates, the specificity of the second protease family remains unknown. Each of the fully sequenced prcA and prtP genes contains a 5′ hydrophobic leader sequence with a treponeme lipobox. The two paralogous families of treponeme subtilisins represent a new subgroup within the subtilisin family of proteases and are the only subtilisin lipoprotein family. The present study demonstrated that the subtilisin paralogs comprising a two-gene operon are widely distributed among treponemes. PMID:14617650

Intramammary infusion of Escherichia coli lipopolysaccharide negatively affects feed intake, chewing, and clinical variables, but some effects are stronger in cows experiencing subacute rumen acidosis.

PubMed

Aditya, S; Humer, E; Pourazad, P; Khiaosa-Ard, R; Huber, J; Zebeli, Q

2017-02-01

Feeding high-grain diets increases the risk of subacute rumen acidosis (SARA) and adversely affects rumen health. This condition might impair the responsiveness of cows when they are exposed to external infectious stimuli such as lipopolysaccharide (LPS). The main objective of this study was to evaluate various responses to intramammary LPS infusion in healthy dairy cows and those experimentally subjected to SARA. Eighteen early-lactating Simmental cows were subjected to SARA (n = 12) or control (CON; n = 6) feeding conditions. Cows of the control group received a diet containing 40% concentrates (DM basis) throughout the experiment. The intermittent SARA feeding regimen consisted in feeding the cows a ration with 60% concentrate (DM basis) for 32 d, consisting of a first SARA induction for 8 d, switched to the CON diet for 7 d, and re-induction during the last 17 d. On d 30 of the experiment, 6 SARA (SARA-LPS) and 6 CON (CON-LPS) cows were intramammary challenged once with a single dose of 50 μg of LPS from Escherichia coli (O26:B6), whereas the other 6 SARA cows (SARA-PLA) received 10 mL of sterile saline solution as placebo. To confirm the induction of SARA, the reticular pH was continuously monitored via wireless pH probes. The DMI remained unchanged between SARA and CON cows during the feeding experiment, but was reduced in both treatment groups receiving the LPS infusion compared with SARA-PLA, whereby a significant decline was observed for cows of the SARA-LPS treatment (-38%) compared with CON-LPS (-19%). The LPS infusion did not affect the reticuloruminal pH dynamics, but significantly enhanced ruminal temperature and negatively affected chewing behavior. The ruminal temperature increased after the LPS infusion and peaked about 1 h earlier in SARA-LPS cows compared with the cows of the CON-LPS treatment. Moreover, a significant decline in milk yield was found in SARA-LPS compared with CON-LPS following the LPS infusion. Cows receiving LPS had elevated somatic cell counts, protein, and fat contents in milk as well as decreased lactose contents and pH following the LPS infusion, whereby the changes in milk constituents were more pronounced in SARA-LPS than CON-LPS cows. Rectal temperature and pulse rate were highest 6 h after LPS infusion, but rumen contractions were not affected by the LPS infusion. The data suggest that a single intramammary LPS infusion induced fever and negatively affected feed intake, chewing activity, rectal temperature, and milk yield and composition, whereby these effects were more pronounced in SARA cows. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

PubMed Central

Mariano, F.S.; Campanelli, A.P.; Nociti, F.H.; Mattos-Graner, R.O.; Gonçalves, R.B.

2012-01-01

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens. PMID:22850872

Lipopolysaccharide stimulates HepG2 human hepatoma cells in the presence of lipopolysaccharide-binding protein via CD14.

PubMed

Nanbo, A; Nishimura, H; Muta, T; Nagasawa, S

1999-02-01

Lipopolysaccharide (LPS)-binding protein (LBP), an opsonin for activation of macrophages by bacterial LPS, is synthesized in hepatocytes and is known to be an acute phase protein. Recently, cytokine-induced production of LBP was reported to increase 10-fold in hepatocytes isolated from LPS-treated rats, compared with those from normal rats. However, the mechanism by which the LPS treatment enhances the effect of cytokines remains to be clarified. In the present study, we examined whether LPS alone or an LPS/LBP complex directly stimulates the hepatocytes, leading to acceleration of the cytokine-induced LBP production. HepG2 cells (a human hepatoma cell line) were shown to express CD14, a glycosylphosphatidylinositol-anchored LPS receptor, by both RT/PCR and flow cytometric analyses. An LPS/LBP complex was an effective stimulator for LBP and CD14 production in HepG2 cells, but stimulation of the cells with either LPS or LBP alone did not significantly accelerate the production of these proteins. The findings were confirmed by semiquantitative RT/PCR analysis of mRNA levels of LBP and CD14 in HepG2 cells after stimulation with LPS alone and an LPS/LBP complex. In addition, two monoclonal antibodies (mAbs) to CD14 (3C10 and MEM-18) inhibited LPS/LBP-induced cellular responses of HepG2 cells. Furthermore, prestimulation of HepG2 cells with LPS/LBP augmented cytokine-induced production and gene expression of LBP and CD14. All these findings suggest that an LPS/LBP complex, but not free LPS, stimulates HepG2 cells via CD14 leading to increased basal and cytokine-induced LBP and CD14 production.

Anti-LPS antibodies reduce endotoxemia in whole body Co-60 irradiated primates - A preliminary report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wells, M.T.; Gaffin, S.L.; Wessels, B.C.

1990-09-01

A previously established primate model was used to evaluate the role of lipopolysaccharide (LPS, endotoxin) in radiation sickness. Vervet monkeys were Co-60 irradiated with an LD100 exposure and had periodic blood samples taken for the determination of LPS and anti-LPS lgG antibodies and for bacteriological studies. On day 2 postirradiation, primates were treated with either sterile 0.9 percent saline, or equine anti-LPS hyperimmune plasma, or tripotassium-dicitrato-bismuthate (Denol). Results indicate that anti-LPS-treated animals survived significantly longer than both the other groups and, since LPS may cause nausea, vomiting, diarrhea, anorexia, and headaches, it is suggested that Anti-LPS administration may be ofmore » value in reducing plasma LPS concentration in humans and improving their performance and survivability. 24 refs.« less

Anti-Inflammatory and Osteoprotective Effects of Cannabinoid-2 Receptor Agonist HU-308 in a Rat Model of Lipopolysaccharide-Induced Periodontitis.

PubMed

Ossola, Cesar A; Surkin, Pablo N; Mohn, Claudia E; Elverdin, Juan C; Fernández-Solari, Javier

2016-06-01

Anti-inflammatory and immunologic properties of cannabinoids have been reported in several tissues. Expression of cannabinoid receptor Type 2 was reported in osteoblasts and osteoclasts, suggesting a key role in bone metabolism. The aim of this study is to assess the effect of treatment with cannabinoid-2 receptor agonist HU-308 in the oral health of rats subjected to lipopolysaccharide (LPS)-induced periodontitis. Twenty-four rats were distributed in four groups (six rats per group): 1) control rats; 2) sham rats; 3) rats submitted to experimental periodontitis (LPS); and 4) rats submitted to experimental periodontitis and treated with HU-308 (LPS+HU). In groups LPS and LPS+HU, periodontitis was induced by LPS (1 mg/mL) injected into the gingival tissue (GT) of maxillary and mandibular first molars and into the interdental space between the first and second molars, 3 days per week for 6 weeks. In group LPS+HU, HU-308 (500 ng/mL) was applied topically to the GT daily. Alveolar bone loss resulting from LPS-induced periodontitis was significantly attenuated with HU-308 treatment (LPS+HU), measured by macroscopic and histologic examination. Treatment also reduced gingival production of inflammatory mediators augmented in LPS-injected rats, such as: 1) inducible nitric oxide (iNOS) activity (LPS: 90.18 ± 36.51 pmol/minute/mg protein versus LPS+HU: 16.37 ± 4.73 pmol/minute/mg protein; P <0.05); 2) tumor necrosis factor alpha (LPS: 185.70 ± 25.63 pg/mg protein versus LPS+HU: 95.89 ± 17.47 pg/mg protein; P <0.05); and 3) prostaglandin E2 (PGE2) (LPS: 159.20 ± 38.70 pg/mg wet weight versus LPS+HU: 71.25 ± 17.75 pg/mg wet weight; P <0.05). Additionally, HU-308 treatment prevented the inhibitory effect of LPS-induced periodontitis on the salivary secretory response to pilocarpine. Moreover, iNOS activity and PGE2 content, which were increased by LPS-induced periodontitis in the submandibular gland, returned to control values after HU-308 treatment. This study demonstrates anti-inflammatory, osteoprotective, and prohomeostatic effects of HU-308 in oral tissues of rats with LPS-induced periodontitis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eckert, C. A.; Sullivan, R.; Johnson, C.

CO2 and H2 are promising feedstocks for production of valuable biocompounds. Ralstonia eutropha utilizes these feedstocks to generate energy (ATP) and reductant (NAD(P)H) via oxidation of H2 by a membrane-bound (MBH) and a soluble hydrogenase (SH) for CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle. Increased expression of the enzyme that fixes CO2 (RubisCO) resulted in 6-fold activity improvement in vitro, while increased expression of the MBH operon or the SH operon plus MBH operon maturation factors necessary for activity resulted in a 10-fold enhancement. Current research involves genetic manipulation of two endogenous cbb operons for increased expression, analysis of expressionmore » and activity of CBB/MBH/SH, cofactor ratios, and downstream products during autotrophic growth in control versus enhanced strains, and development of strategies for long-term, optimal overexpression. These studies will improve our understanding of autotrophic metabolism and provide a chassis strain for autotrophic production of biodiesel and other valuable carbon biocompounds.« less

Organization and post-transcriptional processing of the psb B operon from chloroplasts of Populus deltoides.

PubMed

Dixit, R; Trivedi, P K; Nath, P; Sane, P V

1999-09-01

Chloroplast genes are typically organized into polycistronic transcription units that give rise to complex sets of mono- and oligo-cistronic overlapping RNAs through a series of processing steps. The psbB operon contains genes for the PSII (psbB, psbT, psbH) and cytochrome b(6)f (petB and petD) complexes which are needed in different amounts during chloroplast biogenesis. The functional significance of gene organization in this polycistronic unit, containing information for two different complexes, is not known and is of interest. To determine the organization and expression of these complexes, studies have been carried out on crop plants by different groups, but not much information is known about trees. We present the nucleotide sequences of PSII genes and RNA profiles of the genes located in the psbB operon from Populus deltoides, a tree species. Although the gene organization of this operon in P. deltoides is similar to that in other species, a few variations have been observed in the processing scheme.

(S)-3-hydroxy-3-methylglutaryl coenzyme A reductase, a product of the mva operon of Pseudomonas mevalonii, is regulated at the transcriptional level.

PubMed Central

Wang, Y L; Beach, M J; Rodwell, V W

1989-01-01

We have cloned and sequenced a 505-base-pair (bp) segment of DNA situated upstream of mvaA, the structural gene for (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) of Pseudomonas mevalonii. The DNA segment that we characterized includes the promoter region for the mva operon. Nuclease S1 mapping and primer extension analysis showed that mvaA is the promoter-proximal gene of the mva operon. Transcription initiates at -56 bp relative to the first A (+1) of the translation start site. Transcription in vivo was induced by mevalonate. Structural features of the mva promoter region include an 80-bp A + T-rich region, and -12, -24 consensus sequences that resemble sequences of sigma 54 promoters in enteric organisms. The relative amplitudes of catalytic activity, enzyme protein, and mvaA mRNA are consistent with a model of regulation of this operon at the transcriptional level. Images PMID:2477360

Genomic analysis of a xylose operon and characterization of novel xylose isomerase and xylulokinase from Bacillus coagulans NL01.

PubMed

Zheng, Zhaojuan; Lin, Xi; Jiang, Ting; Ye, Weihua; Ouyang, Jia

2016-08-01

To investigate the xylose operon and properties of xylose isomerase and xylulokinase in Bacillus coagulans that can effectively ferment xylose to lactic acid. The xylose operon is widely present in B. coagulans. It is composed of four putative ORFs. Novel xylA and xylB from B. coagulans NL01 were cloned and expressed in Escherichia coli. Sequence of xylose isomerase was more conserved than that of xylulokinase. Both the enzymes exhibited maximum activities at pH 7-8 but with a high temperature maximum of 80-85 °C, divalent metal ion was prerequisite for their activation. Xylose isomerase and xylulokinase were most effectively activated by Ni(2+) and Co(2+), respectively. Genomic analysis of xylose operon has contributed to understanding xylose metabolism in B. coagulans and the novel xylose isomerase and xylulokinase might provide new alternatives for metabolic engineering of other strains to improve their fermentation performance on xylose.

The organization of the fuc regulon specifying L-fucose dissimilation in Escherichia coli K12 as determined by gene cloning.

PubMed

Chen, Y M; Zhu, Y; Lin, E C

1987-12-01

In Escherichia coli the six known genes specifying the utilization of L-fucose as carbon and energy source cluster at 60.2 min and constitute a regulon. These genes include fucP (encoding L-fucose permease), fucI (encoding L-fucose isomerase), fucK (encoding L-fuculose kinase), fucA (encoding L-fuculose 1-phosphate aldolase), fucO (encoding L-1,2-propanediol oxidoreductase), and fucR (encoding the regulatory protein). In this study the fuc genes were cloned and their positions on the chromosome were established by restriction endonuclease and complementation analyses. Clockwise, the gene order is: fucO-fucA-fucP-fucI-fucK-fucR. The operons comprising the structural genes and the direction of transcription were determined by complementation analysis and Southern blot hybridization. The fucPIK and fucA operons are transcribed clockwise. The fucO operon is transcribed counterclockwise. The fucR gene product activates the three structural operons in trans.

The rpoE operon regulates heat stress response in Burkholderia pseudomallei.

PubMed

Vanaporn, Muthita; Vattanaviboon, Paiboon; Thongboonkerd, Visith; Korbsrisate, Sunee

2008-07-01

Burkholderia pseudomallei is a gram-negative bacterium and the causative agent of melioidosis, one of the important lethal diseases in tropical regions. In this article, we demonstrate the crucial role of the B. pseudomallei rpoE locus in the response to heat stress. The rpoE operon knockout mutant exhibited growth retardation and reduced survival when exposed to a high temperature. Expression analysis using rpoH promoter-lacZ fusion revealed that heat stress induction of rpoH, which encodes heat shock sigma factor (sigma(H)), was abolished in the B. pseudomallei rpoE mutant. Analysis of the rpoH promoter region revealed sequences sharing high homology to the consensus sequence of sigma(E)-dependent promoters. Moreover, the putative heat-induced sigma(H)-regulated heat shock proteins (i.e. GroEL and HtpG) were also absent in the rpoE operon mutant. Altogether, our data suggest that the rpoE operon regulates B. pseudomallei heat stress response through the function of rpoH.

Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1-IKKϵ-IRF3 axis activation.

PubMed

Tsukamoto, Hiroki; Takeuchi, Shino; Kubota, Kanae; Kobayashi, Yohei; Kozakai, Sao; Ukai, Ippo; Shichiku, Ayumi; Okubo, Misaki; Numasaki, Muneo; Kanemitsu, Yoshitomi; Matsumoto, Yotaro; Nochi, Tomonori; Watanabe, Kouichi; Aso, Hisashi; Tomioka, Yoshihisa

2018-05-14

Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-β expression. However, LPS-stimulated late activation of NFκB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-β pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

Effect of molecular hydrogen on uterine inflammation during preterm labour

PubMed Central

Nakano, Tomoko; Kotani, Tomomi; Imai, Kenji; Iitani, Yukako; Ushida, Takafumi; Tsuda, Hiroyuki; Li, Hua; Iwase, Akira; Toyokuni, Shinya; Kikkawa, Fumitaka

2018-01-01

Intrauterine inflammation causes preterm birth and is associated with complications in preterm neonates. Thus, strategies aimed at suppressing inflammation are expected to be effective for reducing the risk of preterm birth and associated complications. Our previous studies demonstrated that molecular hydrogen (H2), an anti-inflammatory agent, prevented inflammation-induced impairment in foetal brain and lung tissues in lipopolysaccharide (LPS)-induced rodent models. However, it remains unclear whether H2 is capable of inhibiting preterm labour. The aim of the current study was therefore to investigate the effect of H2 on inflammation-induced preterm labour. Pregnant ICR (CD-1) mice were divided into three groups: Control, LPS and H2 water (HW) + LPS. In the control and LPS groups, vehicle and LPS, respectively, were intraperitoneally injected on embryonic day 15.5. In the HW + LPS group, HW was administered 24 h prior to LPS injection. The time from LPS administration to parturition was compared between the LPS and HW + LPS groups. Maternal uterus was collected 6 h after LPS injection and the transcript levels of pro-inflammatory cytokines, contractile-associated proteins (CAPs), matrix metalloproteinase-3 (Mmp3) and endothelin-1 (Et1) were assessed by reverse transcription-quantitative polymerase chain reaction. The protein levels of cyclooxygenase-2 (Cox2) were also evaluated by immunohistochemistry. The time from LPS administration to parturition in the HW + LPS group was significantly increased compared with that in the LPS group (33.5±3.4 vs. 18.3±8.8 h, respectively, P=0.020). H2 administration also resulted in significantly higher progesterone levels compared with LPS treatment alone (P=0.002). The transcript levels of pro-inflammatory cytokines, CAPs, Mmp3 and Et1 in the uteri of the LPS group were significantly higher than those in the control group (all P<0.05). In turn, all these levels with the exception of interleukin-8 and Mmp3 were significantly lower in the HW + LPS group compared with those in the LPS group (all P<0.05). The protein levels of Cox2 in the LPS group were also significantly increased compared with those in the control (P<0.001) and HW + LPS (P=0.003) groups. These results suggest that inflammation-induced changes in the uterus may be ameliorated through maternal H2 administration. Preventive H2 administration may therefore represent an effective strategy for the suppression of inflammation during preterm labour. PMID:29732148

Phenotypical Analysis of the Lactobacillus rhamnosus GG Fimbrial spaFED Operon: Surface Expression and Functional Characterization of Recombinant SpaFED Pili in Lactococcus lactis

PubMed Central

Kant, Ravi; Palva, Airi; von Ossowski, Ingemar

2014-01-01

A noticeable genomic feature of many piliated Gram-positive bacterial species is the presence of more than one pilus-encoding operon. Paradigmatically, the gut-adapted Lactobacillus rhamnosus GG strain contains two different fimbrial operons in its genome. However, whereas one of these operons (called spaCBA) is encoding for the functionally mucus-/collagen-binding SpaCBA pilus, for the other operon (called spaFED) any native expression of the SpaFED-called pili is still the subject of some uncertainty. Irrespective of such considerations, we decided it would be of relevance or interest to decipher the gross structure of this pilus type, and as well assess its functional capabilities for cellular adhesion and immunostimulation. For this, and by following the approach we had used previously to explicate the immuno-properties of SpaCBA pili, we constructed nisin-inducible expression clones producing either wild-type or SpaF pilin-deleted surface-assembled L. rhamnosus GG SpaFED pili on Lactococcus lactis cells. Using these piliated lactococcal constructs, we found that the pilin-polymerized architecture of a recombinant-produced SpaFED pilus coincides with sequence-based functional predictions of the related pilins, and in fact is prototypical of those other sortase-dependent pilus-like structures thus far characterized for piliated Gram-positive bacteria. Moreover, we confirmed that among the different pilin subunits encompassing spaFED operon-encoded pili, the SpaF pilin is a main adhesion determinant, and when present in the assembled structure can mediate pilus binding to mucus, certain extracellular matrix proteins, and different gut epithelial cell lines. However, somewhat unexpectedly, when recombinant SpaFED pili are surface-attached, we found that they could not potentiate the existing lactococcal cell-induced immune responses so elicited from intestinal- and immune-related cells, but rather instead, they could dampen them. Accordingly, we have now provided the first phenotypical description of a spaFED pilus operon, and with that furthered the functional understanding of surface piliation for a particular gut-commensalic genre of piliated Gram-positive bacteria. PMID:25415357

Phenotypical analysis of the Lactobacillus rhamnosus GG fimbrial spaFED operon: surface expression and functional characterization of recombinant SpaFED pili in Lactococcus lactis.

PubMed

Rintahaka, Johanna; Yu, Xia; Kant, Ravi; Palva, Airi; von Ossowski, Ingemar

2014-01-01

A noticeable genomic feature of many piliated Gram-positive bacterial species is the presence of more than one pilus-encoding operon. Paradigmatically, the gut-adapted Lactobacillus rhamnosus GG strain contains two different fimbrial operons in its genome. However, whereas one of these operons (called spaCBA) is encoding for the functionally mucus-/collagen-binding SpaCBA pilus, for the other operon (called spaFED) any native expression of the SpaFED-called pili is still the subject of some uncertainty. Irrespective of such considerations, we decided it would be of relevance or interest to decipher the gross structure of this pilus type, and as well assess its functional capabilities for cellular adhesion and immunostimulation. For this, and by following the approach we had used previously to explicate the immuno-properties of SpaCBA pili, we constructed nisin-inducible expression clones producing either wild-type or SpaF pilin-deleted surface-assembled L. rhamnosus GG SpaFED pili on Lactococcus lactis cells. Using these piliated lactococcal constructs, we found that the pilin-polymerized architecture of a recombinant-produced SpaFED pilus coincides with sequence-based functional predictions of the related pilins, and in fact is prototypical of those other sortase-dependent pilus-like structures thus far characterized for piliated Gram-positive bacteria. Moreover, we confirmed that among the different pilin subunits encompassing spaFED operon-encoded pili, the SpaF pilin is a main adhesion determinant, and when present in the assembled structure can mediate pilus binding to mucus, certain extracellular matrix proteins, and different gut epithelial cell lines. However, somewhat unexpectedly, when recombinant SpaFED pili are surface-attached, we found that they could not potentiate the existing lactococcal cell-induced immune responses so elicited from intestinal- and immune-related cells, but rather instead, they could dampen them. Accordingly, we have now provided the first phenotypical description of a spaFED pilus operon, and with that furthered the functional understanding of surface piliation for a particular gut-commensalic genre of piliated Gram-positive bacteria.

Identification of Surprisingly Diverse Type IV Pili, across a Broad Range of Gram-Positive Bacteria

PubMed Central

Roos, David S.; Pohlschröder, Mechthild

2011-01-01

Background In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available. Results To assess TFP diversity in Gram-positive bacteria and facilitate pilin identification, we compiled a comprehensive list of putative Gram-positive pilins encoded by operons containing highly conserved pilus biosynthetic genes (pilB, pilC). A surprisingly large number of species were found to contain multiple TFP operons (pil, com and/or tad). The N-terminal sequences of predicted pilins were exploited to develop PilFind, a rule-based algorithm for genome-wide identification of otherwise poorly conserved type IV pilins in any species, regardless of their association with TFP biosynthetic operons (http://signalfind.org). Using PilFind to scan 53 Gram-positive genomes (encoding >187,000 proteins), we identified 286 candidate pilins, including 214 in operons containing TFP biosynthetic genes (TBG+ operons). Although trained on Gram-positive pilins, PilFind identified 55 of 58 manually curated Gram-negative pilins in TBG+ operons, as well as 53 additional pilin candidates in operons lacking biosynthetic genes in ten species (>38,000 proteins), including 27 of 29 experimentally verified pilins. False positive rates appear to be low, as PilFind predicted only four pilin candidates in eleven bacterial species (>13,000 proteins) lacking TFP biosynthetic genes. Conclusions We have shown that Gram-positive bacteria contain a highly diverse set of type IV pili. PilFind can be an invaluable tool to study bacterial cellular processes known to involve type IV pilus-like structures. Its use in combination with other currently available computational tools should improve the accuracy of predicting the subcellular localization of bacterial proteins. PMID:22216142

Effect of chromium on bioenergetics and leukocyte dynamics following immunoactivation in lactating Holstein cows.

PubMed

Horst, E A; Kvidera, S K; Mayorga, E J; Shouse, C S; Al-Qaisi, M; Dickson, M J; Ydstie, J; Ramirez Ramirez, H A; Keating, A F; Dickson, D J; Griswold, K E; Baumgard, L H

2018-06-01

Activated immune cells are insulin sensitive and utilize copious amounts of glucose. Because chromium (Cr) increases insulin sensitivity and may be immunomodulatory, our objective was to evaluate the effect of supplemental Cr (KemTrace Cr propionate, 20 g/d; Kemin Industries Inc., Des Moines, IA) on immune system glucose utilization and immune system dynamics following an intravenous endotoxin challenge in lactating Holstein cows. Twenty cows (320 ± 18 d in milk) were randomly assigned to 1 of 4 treatments: (1) pair-fed (PF) control (PF-CON; 5 mL of saline; n = 5), (2) PF and Cr supplemented (PF-Cr; 5 mL of saline; n = 5), (3) lipopolysaccharide (LPS)-euglycemic clamp and control supplemented (LPS-CON; 0.375 µg/kg of body weight LPS; n = 5), and (4) LPS-euglycemic clamp and Cr supplemented (LPS-Cr; 0.375 µg/kg of body weight LPS; n = 5). The experiment was conducted serially in 3 periods (P). During P1 (3 d), cows received their respective dietary treatments and baseline values were obtained. At the initiation of P2 (2 d), either a 12-h LPS-euglycemic clamp was conducted or cows were PF to their respective dietary counterparts. During P3 (3 d), cows consumed feed ad libitum and continued to receive their respective dietary treatment. During P2, LPS administration decreased dry matter intake (DMI; 40%) similarly among diets, and by experimental design the pattern and magnitude of reduced DMI were similar in the PF cohorts. During P3, LPS-Cr cows tended to have decreased DMI (6%) relative to LPS-CON cows. Relative to controls, milk yield from LPS-challenged cows decreased (58%) during P2 and LPS-Cr cows produced less (16%) milk than LPS-CON cows. During P3, milk yield progressively increased similarly in LPS-administered cows, but overall milk yield remained decreased (24%) compared with PF controls. There were no dietary treatment differences in milk yield during P3. Circulating insulin increased 9- and 15-fold in LPS-administered cows at 6 and 12 h postbolus, respectively, compared with PF controls. Compared with LPS-CON cows, circulating insulin in LPS-Cr cows was decreased (48%) at 6 h postbolus. Relative to PF cows, circulating LPS binding protein and serum amyloid A from LPS-administered cows increased 2- and 5-fold, respectively. Compared with PF cows, blood neutrophil counts in LPS-infused cows initially decreased, then gradually increased 163%. Between 18 and 48 h postbolus, the number of neutrophils was increased (12%) in LPS-Cr versus LPS-CON cows. The 12-h total glucose deficit was 220 and 1,777 g for the PF and LPS treatments, respectively, but glucose utilization following immune activation was not influenced by Cr. In summary, supplemental Cr reduced the insulin response and increased circulating neutrophils following an LPS challenge but did not appear to alter the immune system's glucose requirement following acute and intense activation. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Naringenin degradation by the endophytic diazotroph Herbaspirillum seropedicae SmR1.

PubMed

Marin, A M; Souza, E M; Pedrosa, F O; Souza, L M; Sassaki, G L; Baura, V A; Yates, M G; Wassem, R; Monteiro, R A

2013-01-01

Several bacteria are able to degrade flavonoids either to use them as carbon sources or as a detoxification mechanism. Degradation pathways have been proposed for several bacteria, but the genes responsible are not known. We identified in the genome of the endophyte Herbaspirillum seropedicae SmR1 an operon potentially associated with the degradation of aromatic compounds. We show that this operon is involved in naringenin degradation and that its expression is induced by naringenin and chrysin, two closely related flavonoids. Mutation of fdeA, the first gene of the operon, and fdeR, its transcriptional activator, abolished the ability of H. seropedicae to degrade naringenin.

The excludon: a new concept in bacterial antisense RNA-mediated gene regulation.

PubMed

Sesto, Nina; Wurtzel, Omri; Archambaud, Cristel; Sorek, Rotem; Cossart, Pascale

2013-02-01

In recent years, non-coding RNAs have emerged as key regulators of gene expression. Among these RNAs, the antisense RNAs (asRNAs) are particularly abundant, but in most cases the function and mechanism of action for a particular asRNA remains elusive. Here, we highlight a recently discovered paradigm termed the excludon, which defines a genomic locus encoding an unusually long asRNA that spans divergent genes or operons with related or opposing functions. Because these asRNAs can inhibit the expression of one operon while functioning as an mRNA for the adjacent operon, they act as fine-tuning regulatory switches in bacteria.

Modulation of lipopolysaccharide-induced chorioamnionitis in fetal sheep by maternal betamethasone.

PubMed

Wolfe, Katherine B; Snyder, Candice C; Gisslen, Tate; Kemp, Matthew W; Newnham, John P; Kramer, Boris W; Jobe, Alan H; Kallapur, Suhas

2013-12-01

We tested the hypothesis that the order of exposure to maternal betamethasone and intra-amniotic (IA) lipopolysaccharide (LPS) will differentially modulate inflammation in the chorioamnion. Time-mated Merino ewes with singleton fetuses received saline alone, IA LPS alone, maternal betamethasone before LPS, or betamethasone after LPS. We assessed inflammatory markers in the chorioamnion and the amniotic fluid. Inflammatory cell infiltration, expression of myeloperoxidase, serum amyloid A3 (acute phase reactant) in the chorioamnion, and levels of interleukin (IL)-8 in the amniotic fluid increased 7 days after LPS exposure. Betamethasone prior to LPS decreased infiltration of the inflammatory cells, CD3+ T cells, and decreased the levels of IL-1β and IL-8 in the amniotic fluid. Betamethasone 7 days prior to LPS exposure suppressed LPS-induced inflammation. The markers of inflammation largely had returned to the baseline 14 days after LPS exposure.

Lipopolysaccharide-reactive immunoglobulin E is associated with lower mortality and organ failure in traumatically injured patients.

PubMed Central

DiPiro, J T; Hamilton, R G; Howdieshell, T R; Adkinson, N F; Mansberger, A R

1994-01-01

Antilipopolysaccharide (anti-LPS) immunoglobulin G (IgG) and IgM have been associated with protection from LPS effects in vivo. We investigated the presence of IgE and anti-LPS in 32 patients that had experienced severe traumatic injury and in 35 healthy volunteers; we also investigated whether IgE anti-LPS was associated with important clinical events. Plasma samples were collected daily from patients in the intensive care unit and on one occasion from volunteers; the samples were assayed for IgE anti-LPS. IgE anti-LPS was assayed by enzyme-linked immunosorbent assay with monoclonal anti-human IgE as the capture antibody. Detection was accomplished with biotin-labeled LPS (Escherichia coli J5 mutant) followed by streptavidin-peroxidase with 2,2'-azino(3-ethylbenzthiazoline)sulfonic acid as the substrate. The assay was demonstrated to be specific for IgE and LPS-biotin by nonreactivity of control sera with high-titer anti-LPS IgG and IgM and by inhibition with unlabeled LPS. IgE anti-LPS was detected in 1 of 35 healthy controls (2.9%) and 25 of 32 traumatically injured patients (78%) (P < 0.001). The presence of IgE anti-LPS was associated with a lower incidence of death (P = 0.026) and of renal failure (P = 0.0012). There was no apparent temporal relationship between detection of IgE anti-LPS and clinical events. IgG anti-LPS was detected more frequently in patients that were positive for IgE anti-LPS (P = 0.06) but was not associated with clinical events. The inability to detect IgE anti-LPS may be related to adverse clinical events through depletion of specific IgE due to LPS exposure after trauma or through saturation of the assay by IgE with other specificities. We have reported increased total IgE concentrations in these patients. (J.T. DiPiro, R.G. Hamilton, T. R. Howdieshell, N. F. Adkinson, and A. R. Mansberger, Ann. Surg. 215:460-466, 1992). PMID:7496965

The rapidly emerging ESBL-producing Escherichia coli O25-ST131 clone carries LPS core synthesis genes of the K-12 type.

PubMed

Szijártó, Valéria; Pal, Tibor; Nagy, Gabor; Nagy, Eszter; Ghazawi, Akela; al-Haj, Mohammed; El Kurdi, Sylvia; Sonnevend, Agnes

2012-07-01

The clone Escherichia coli O25 ST131, typically producing extended-spectrum beta-lactamases (ESBLs), has spread globally and became the dominant type among extraintestinal isolates at many parts of the world. However, the reasons behind the emergence and success of this clone are only partially understood. We compared the core type genes by PCR of ESBL-producing and ESBL-nonproducing strains isolated from urinary tract infections in the United Arab Emirates and found a surprisingly high frequency of the K-12 core type (44.6%) among members of the former group, while in the latter one, it was as low (3.7%), as reported earlier. The high figure was almost entirely attributable to the presence of members of the clone O25 ST131 among ESBL producers. Strains from the same clone isolated in Europe also carried the K-12 core type genes. Sequencing the entire core operon of an O25 ST131 isolate revealed a high level of similarity to known K-12 core gene sequences and an almost complete identity with a recently sequenced non-O25 ST131 fecal isolate. The exact chemical structure and whether and how this unusual core type contributed to the sudden emergence of ST131 require further investigations. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems ▿†‡

PubMed Central

Makarova, Kira S.; Wolf, Yuri I.; Snir, Sagi; Koonin, Eugene V.

2011-01-01

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic “sinks” that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands. PMID:21908672

Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.

PubMed

Makarova, Kira S; Wolf, Yuri I; Snir, Sagi; Koonin, Eugene V

2011-11-01

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic "sinks" that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.

The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.

PubMed

Luebke, Justin L; Shen, Jiangchuan; Bruce, Kevin E; Kehl-Fie, Thomas E; Peng, Hui; Skaar, Eric P; Giedroc, David P

2014-12-01

How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR [Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor] represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high-resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60' interprotomer cross-links, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR. © 2014 John Wiley & Sons Ltd.

The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus

PubMed Central

Luebke, Justin L.; Shen, Jiangchuan; Bruce, Kevin E.; Kehl-Fie, Thomas E.; Peng, Hui; Skaar, Eric P.; Giedroc, David P.

2014-01-01

How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR (Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor) represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60’ interprotomer crosslinks, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR. PMID:25318663

Improved vectors for transcriptional/translational signal screening in corynebacteria using the melC operon from Streptomyces glaucescens as reporter.

PubMed

Adham, Sirin A I; Rodríguez, Sonia; Ramos, Angelina; Santamaría, Ramón I; Gil, José A

2003-07-01

The tyrosinase operon ( melC) from Streptomyces glaucescens was cloned and functionally expressed in Brevibacterium lactofermentum and Corynebacterium glutamicum under the control of the promoter of the kan gene from Tn 5. Recombinant corynebacterial cells containing the tyrosinase operon produced melanin on agar plates and in liquid culture when supplemented with copper and tyrosine. A conjugative bifunctional replacement vector for transcriptional/translational signal screening (pEMel-1) was constructed using expression of the melC operon from S. glaucescens, which can be used for cloning promoter sequences as EcoRI- NdeI fragments. When the DNA fragments with promoter activity such as cspBp or trpp were inserted into pEMel-1, B. lactofermentum harboring the chimeric plasmids produced melanin at different stages of growth, allowing temporal detection of promoter activity. The vector was also used to detect the activity of a Streptomyces promoter ( xysAp), which was inactive in B. lactofermentum, after PCR mutagenesis. The melC operon can be used for the visual, inexpensive (compared to the high price of starch azure for amylase detection), and non-selective (in contrast to the kan or cat genes) screening of several thousand clones at high colony density without killing of the transformants due to the presence of iodine (as in the case of amylase assay).

Identification of new members of the Escherichia coli K-12 MG1655 SlyA regulon

PubMed Central

Curran, Thomas D; Abacha, Fatima; Hibberd, Stephen P; Green, Jeffrey

2017-01-01

SlyA is a member of the MarR family of bacterial transcriptional regulators. Previously, SlyA has been shown to directly regulate only two operons in Escherichia coli K-12 MG1655, fimB and hlyE (clyA). In both cases, SlyA activates gene expression by antagonizing repression by the nucleoid-associated protein H-NS. Here, the transcript profiles of aerobic glucose-limited steady-state chemostat cultures of E. coli K-12 MG1655, slyA mutant and slyA over-expression strains are reported. The transcript profile of the slyA mutant was not significantly different from that of the parent; however, that of the slyA expression strain was significantly different from that of the vector control. Transcripts representing 27 operons were increased in abundance, whereas 3 were decreased. Of the 30 differentially regulated operons, 24 have previously been associated with sites of H-NS binding, suggesting that antagonism of H-NS repression is a common feature of SlyA-mediated transcription regulation. Direct binding of SlyA to DNA located upstream of a selection of these targets permitted the identification of new operons likely to be directly regulated by SlyA. Transcripts of four operons coding for cryptic adhesins exhibited enhanced expression, and this was consistent with enhanced biofilm formation associated with the SlyA over-producing strain. PMID:28073397

Identification of new members of the Escherichia coli K-12 MG1655 SlyA regulon.

PubMed

Curran, Thomas D; Abacha, Fatima; Hibberd, Stephen P; Rolfe, Matthew D; Lacey, Melissa M; Green, Jeffrey

2017-03-01

SlyA is a member of the MarR family of bacterial transcriptional regulators. Previously, SlyA has been shown to directly regulate only two operons in Escherichia coli K-12 MG1655, fimB and hlyE (clyA). In both cases, SlyA activates gene expression by antagonizing repression by the nucleoid-associated protein H-NS. Here, the transcript profiles of aerobic glucose-limited steady-state chemostat cultures of E. coli K-12 MG1655, slyA mutant and slyA over-expression strains are reported. The transcript profile of the slyA mutant was not significantly different from that of the parent; however, that of the slyA expression strain was significantly different from that of the vector control. Transcripts representing 27 operons were increased in abundance, whereas 3 were decreased. Of the 30 differentially regulated operons, 24 have previously been associated with sites of H-NS binding, suggesting that antagonism of H-NS repression is a common feature of SlyA-mediated transcription regulation. Direct binding of SlyA to DNA located upstream of a selection of these targets permitted the identification of new operons likely to be directly regulated by SlyA. Transcripts of four operons coding for cryptic adhesins exhibited enhanced expression, and this was consistent with enhanced biofilm formation associated with the SlyA over-producing strain.

Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic.

PubMed

Mirkovic, Nemanja; Polovic, Natalija; Vukotic, Goran; Jovcic, Branko; Miljkovic, Marija; Radulovic, Zorica; Diep, Dzung B; Kojic, Milan

2016-04-01

Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins.Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes,lmgA ,lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes,lmgF,lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization-time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility.

PubMed

Huertas, Mónica G; Zárate, Lina; Acosta, Iván C; Posada, Leonardo; Cruz, Diana P; Lozano, Marcela; Zambrano, María M

2014-12-01

Klebsiella pneumoniae is an opportunistic pathogen important in hospital-acquired infections, which are complicated by the rise of drug-resistant strains and the capacity of cells to adhere to surfaces and form biofilms. In this work, we carried out an analysis of the genes in the K. pneumoniae yfiRNB operon, previously implicated in biofilm formation. The results indicated that in addition to the previously reported effect on type 3 fimbriae expression, this operon also affected biofilm formation due to changes in cellulose as part of the extracellular matrix. Deletion of yfiR resulted in enhanced biofilm formation and an altered colony phenotype indicative of cellulose overproduction when grown on solid indicator media. Extraction of polysaccharides and treatment with cellulase were consistent with the presence of cellulose in biofilms. The enhanced cellulose production did not, however, correlate with virulence as assessed using a Caenorhabditis elegans assay. In addition, cells bearing mutations in genes of the yfiRNB operon varied with respect to the WT control in terms of susceptibility to the antibiotics amikacin, ciprofloxacin, imipenem and meropenem. These results indicated that the yfiRNB operon is implicated in the production of exopolysaccharides that alter cell surface characteristics and the capacity to form biofilms--a phenotype that does not necessarily correlate with properties related with survival, such as resistance to antibiotics. © 2014 The Authors.

Genome analysis of E. coli isolated from Crohn's disease patients.

PubMed

Rakitina, Daria V; Manolov, Alexander I; Kanygina, Alexandra V; Garushyants, Sofya K; Baikova, Julia P; Alexeev, Dmitry G; Ladygina, Valentina G; Kostryukova, Elena S; Larin, Andrei K; Semashko, Tatiana A; Karpova, Irina Y; Babenko, Vladislav V; Ismagilova, Ruzilya K; Malanin, Sergei Y; Gelfand, Mikhail S; Ilina, Elena N; Gorodnichev, Roman B; Lisitsyna, Eugenia S; Aleshkin, Gennady I; Scherbakov, Petr L; Khalif, Igor L; Shapina, Marina V; Maev, Igor V; Andreev, Dmitry N; Govorun, Vadim M

2017-07-19

Escherichia coli (E. coli) has been increasingly implicated in the pathogenesis of Crohn's disease (CD). The phylogeny of E. coli isolated from Crohn's disease patients (CDEC) was controversial, and while genotyping results suggested heterogeneity, the sequenced strains of E. coli from CD patients were closely related. We performed the shotgun genome sequencing of 28 E. coli isolates from ten CD patients and compared genomes from these isolates with already published genomes of CD strains and other pathogenic and non-pathogenic strains. CDEC was shown to belong to A, B1, B2 and D phylogenetic groups. The plasmid and several operons from the reference CD-associated E. coli strain LF82 were demonstrated to be more often present in CDEC genomes belonging to different phylogenetic groups than in genomes of commensal strains. The operons include carbon-source induced invasion GimA island, prophage I, iron uptake operons I and II, capsular assembly pathogenetic island IV and propanediol and galactitol utilization operons. Our findings suggest that CDEC are phylogenetically diverse. However, some strains isolated from independent sources possess highly similar chromosome or plasmids. Though no CD-specific genes or functional domains were present in all CD-associated strains, some genes and operons are more often found in the genomes of CDEC than in commensal E. coli. They are principally linked to gut colonization and utilization of propanediol and other sugar alcohols.

Cyanobacterial lipopolysaccharides and human health – a review

PubMed Central

Stewart, Ian; Schluter, Philip J; Shaw, Glen R

2006-01-01

Cyanobacterial lipopolysaccharide/s (LPS) are frequently cited in the cyanobacteria literature as toxins responsible for a variety of heath effects in humans, from skin rashes to gastrointestinal, respiratory and allergic reactions. The attribution of toxic properties to cyanobacterial LPS dates from the 1970s, when it was thought that lipid A, the toxic moiety of LPS, was structurally and functionally conserved across all Gram-negative bacteria. However, more recent research has shown that this is not the case, and lipid A structures are now known to be very different, expressing properties ranging from LPS agonists, through weak endotoxicity to LPS antagonists. Although cyanobacterial LPS is widely cited as a putative toxin, most of the small number of formal research reports describe cyanobacterial LPS as weakly toxic compared to LPS from the Enterobacteriaceae. We systematically reviewed the literature on cyanobacterial LPS, and also examined the much lager body of literature relating to heterotrophic bacterial LPS and the atypical lipid A structures of some photosynthetic bacteria. While the literature on the biological activity of heterotrophic bacterial LPS is overwhelmingly large and therefore difficult to review for the purposes of exclusion, we were unable to find a convincing body of evidence to suggest that heterotrophic bacterial LPS, in the absence of other virulence factors, is responsible for acute gastrointestinal, dermatological or allergic reactions via natural exposure routes in humans. There is a danger that initial speculation about cyanobacterial LPS may evolve into orthodoxy without basis in research findings. No cyanobacterial lipid A structures have been described and published to date, so a recommendation is made that cyanobacteriologists should not continue to attribute such a diverse range of clinical symptoms to cyanobacterial LPS without research confirmation. PMID:16563160

Increase in hypothalamic AMPK phosphorylation induced by prolonged exposure to LPS involves ghrelin and CB1R signaling.

PubMed

Rivas, Priscila M S; Vechiato, Fernanda M V; Borges, Beatriz C; Rorato, Rodrigo; Antunes-Rodrigues, Jose; Elias, Lucila L K

2017-07-01

Acute administration of lipopolysaccharide (LPS) from Gram-negative bacteria induces hypophagia. However, the repeated administration of LPS leads to desensitization of hypophagia, which is associated with increased hypothalamic p-AMPK expression. Because ghrelin and endocannabinoids modulate AMPK activity in the hypothalamus, we hypothesized that these neuromodulators play a role in the reversal of tolerance to hypophagia in rats under long-term exposure to LPS. Male Wistar rats were treated with single (1 LPS, 100μg/kg body weight, ip) or repeated injections of LPS over 6days (6 LPS). Food intake was reduced in the 1 LPS, but not in the 6 LPS group. 6 LPS rats showed an increased serum concentration of acylated ghrelin and reduced ghrelin receptor mRNA expression in the hypothalamus. Ghrelin injection (40μg/kg body weight, ip) increased food intake, body weight gain, p-AMPK hypothalamic expression, neuropeptide Y (NPY) and Agouti related peptide (AgRP) mRNA expression in control animals (Saline). However, in 6 LPS rats, ghrelin did not alter these parameters. Central administration of a CB1R antagonist (AM251, 200ng/μl in 5μl/rat) induced hypophagia in 6 LPS animals, suggesting that the endocannabinoid system contributes to preserved food intake during LPS tolerance. In the presence of AM251, the ability of ghrelin to phosphorylate AMPK in the hypothalamus of 6 LPS group was restored, but not its orexigenic effect. Our data highlight that the orexigenic effects of ghrelin require CB1R signaling downstream of AMPK activation. Moreover, CB1R-mediated pathways contribute to the absence of hypophagia during repeated exposure to endotoxin. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of the First Molluscicidal Lipopolysaccharide from Moraxella osloensis

PubMed Central

Tan, Li; Grewal, Parwinder S.

2003-01-01

Moraxella osloensis is a bacterium that is mutualistically associated with Phasmarhabditis hermaphrodita, a nematode that has potential for the biocontrol of mollusk pests, especially the slug Deroceras reticulatum. We discovered that purified M. osloensis lipopolysaccharide (LPS) possesses a lethal toxicity to D. reticulatum when administered by injection but no contact or oral toxicity to this slug. The toxicity of the LPS resides in the lipid A moiety. M. osloensis LPS was semiquantitated at 6 × 107 endotoxin units per mg. The LPS is a rough-type LPS with an estimated molecular weight of 5,300. Coinjection of galactosamine with the LPS increased the LPS's toxicity to the slug two- to four-fold. The galactosamine-induced sensitization of the slug to the LPS was reversed completely by uridine. PMID:12788774

Characterization of the first molluscicidal lipopolysaccharide from Moraxella osloensis.

PubMed

Tan, Li; Grewal, Parwinder S

2003-06-01

Moraxella osloensis is a bacterium that is mutualistically associated with Phasmarhabditis hermaphrodita, a nematode that has potential for the biocontrol of mollusk pests, especially the slug Deroceras reticulatum. We discovered that purified M. osloensis lipopolysaccharide (LPS) possesses a lethal toxicity to D. reticulatum when administered by injection but no contact or oral toxicity to this slug. The toxicity of the LPS resides in the lipid A moiety. M. osloensis LPS was semiquantitated at 6 x 10(7) endotoxin units per mg. The LPS is a rough-type LPS with an estimated molecular weight of 5,300. Coinjection of galactosamine with the LPS increased the LPS's toxicity to the slug two- to four-fold. The galactosamine-induced sensitization of the slug to the LPS was reversed completely by uridine.

The Sinorhizobium fredii HH103 Lipopolysaccharide Is Not Only Relevant at Early Soybean Nodulation Stages but Also for Symbiosome Stability in Mature Nodules

PubMed Central

Margaret, Isabel; Lucas, M. Mercedes; Acosta-Jurado, Sebastián; Buendía-Clavería, Ana M.; Fedorova, Elena; Hidalgo, Ángeles; Rodríguez-Carvajal, Miguel A.; Rodriguez-Navarro, Dulce N.; Ruiz-Sainz, José E.; Vinardell, José M.

2013-01-01

In this work we have characterised the Sinorhizobium fredii HH103 greA lpsB lpsCDE genetic region and analysed for the first time the symbiotic performance of Sinorhizobium fredii lps mutants on soybean. The organization of the S. fredii HH103 greA, lpsB, and lpsCDE genes was equal to that of Sinorhizobium meliloti 1021. S. fredii HH103 greA, lpsB, and lpsE mutant derivatives produced altered LPS profiles that were characteristic of the gene mutated. In addition, S. fredii HH103 greA mutants showed a reduction in bacterial mobility and an increase of auto-agglutination in liquid cultures. RT-PCR and qPCR experiments demonstrated that the HH103 greA gene has a positive effect on the transcription of lpsB. Soybean plants inoculated with HH103 greA, lpsB or lpsE mutants formed numerous ineffective pseudonodules and showed severe symptoms of nitrogen starvation. However, HH103 greA and lps mutants were also able to induce the formation of a reduced number of soybean nodules of normal external morphology, allowing the possibility of studying the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis. The infected cells of these nodules showed signs of early termination of symbiosis and lytical clearance of bacteroids. These cells also had very thick walls and accumulation of phenolic-like compounds, pointing to induced defense reactions. Our results show the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis and their role in preventing host cell defense reactions. S. fredii HH103 lpsB mutants also showed reduced nodulation with Vigna unguiculata, although the symbiotic impairment was less pronounced than in soybean. PMID:24098345

Transcriptional regulation of bone sialoprotein gene by Porphyromonas gingivalis lipopolysaccharide.

PubMed

Li, Xinyue; Kato, Naoko; Mezawa, Masaru; Li, Zhengyang; Wang, Zhitao; Yang, Li; Sasaki, Yoko; Kaneko, Takashi; Takai, Hideki; Yoshimura, Atsutoshi; Ogata, Yorimasa

2010-07-01

Lipopolysaccharide (LPS) is a major mediator of inflammatory response. Periodontopathic bacterium Porphyromonas gingivalis LPS has quite different character from Escherichia coli LPS. E. coli LPS is agonist for Toll-like receptor 4 (TLR4), whereas P. gingivalis LPS worked as antagonist for TLR4. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of P. gingivalis LPS on BSP transcription, we used rat osteoblast-like ROS17/2.8 cells. BSP mRNA levels were decreased by 0.1 microg/ml and increased by 0.01 microg/ml P. gingivalis LPS at 12 h. Results of luciferase assays showed that 0.1 microg/ml decreased and 0.01 microg/ml P. gingivalis LPS increased BSP transcription in -116 to +60 BSP construct. The effects of P. gingivalis LPS were abrogated by double mutations in cAMP response element (CRE) and FGF2 response element (FRE). Tyrosine kinase inhibitor herbimycin A, ERK1/2 inhibitor and antioxidant N-acetylcystein inhibited effects of P. gingivalis LPS. Protein kinase A inhibitor and PI3-kinase/Akt inhibitor only abolished the effect of 0.01 microg/ml P. gingivalis LPS. Furthermore, 0.1 microg/ml LPS decreased the CRE- and FRE-protein complexes formation, whereas 0.01 microg/ml P. gingivalis LPS increased the nuclear protein binding to CRE and FRE. ChIP assays revealed increased binding of CREB1, JunD, Fra2, Runx2, Dlx5, and Smad1 to a chromatin fragment containing the CRE and FRE by 0.01 microg/ml P. gingivalis LPS. These studies therefore indicated that 0.1 microg/ml suppressed, and 0.01 microg/ml P. gingivalis LPS increased BSP gene transcription mediated through CRE and FRE elements in the rat BSP gene promoter. J. Cell. Biochem. 110: 823-833, 2010. (c) 2010 Wiley-Liss, Inc.

EphA2 Receptor Signaling Mediates Inflammatory Responses in Lipopolysaccharide-Induced Lung Injury.

PubMed

Hong, Ji Young; Shin, Mi Hwa; Chung, Kyung Soo; Kim, Eun Young; Jung, Ji Ye; Kang, Young Ae; Kim, Young Sam; Kim, Se Kyu; Chang, Joon; Park, Moo Suk

2015-07-01

Eph receptors and ephrin ligands have several functions including angiogenesis, cell migration, axon guidance, fluid homeostasis, oncogenesis, inflammation and injury repair. The EphA2 receptor potentially mediates the regulation of vascular permeability and inflammation in response to lung injury. Mice were divided into 3 experimental groups to study the role of EphA2 signaling in the lipopolysaccharide (LPS)-induced lung injury model i.e., IgG+phosphate-buffered saline (PBS) group (IgG instillation before PBS exposure), IgG+LPS group (IgG instillation before LPS exposure) and EphA2 monoclonal antibody (mAb)+LPS group (EphA2 mAb pretreatment before LPS exposure). EphA2 and ephrinA1 were upregulated in LPS-induced lung injury. The lung injury score of the EphA2 mAb+LPS group was lower than that of the IgG+LPS group (4.30±2.93 vs. 11.45±1.20, respectively; p=0.004). Cell counts (EphA2 mAb+LPS: 11.33×10(4)±8.84×10(4) vs. IgG+LPS: 208.0×10(4)±122.6×10(4); p=0.018) and total protein concentrations (EphA2 mAb+LPS: 0.52±0.41 mg/mL vs. IgG+LPS: 1.38±1.08 mg/mL; p=0.192) were decreased in EphA2 mAb+LPS group, as compared to the IgG+LPS group. In addition, EphA2 antagonism reduced the expression of phospho-p85, phosphoinositide 3-kinase 110γ, phospho-Akt, nuclear factor κB, and proinflammatory cytokines. This results of the study indicated a role for EphA2-ephrinA1 signaling in the pathogenesis of LPS-induced lung injury. Furthermore, EphA2 antagonism inhibits the phosphoinositide 3-kinase-Akt pathway and attenuates inflammation.

Accumulation mode particles and LPS exposure induce TLR-4 dependent and independent inflammatory responses in the lung.

PubMed

Fonceca, Angela M; Zosky, Graeme R; Bozanich, Elizabeth M; Sutanto, Erika N; Kicic, Anthony; McNamara, Paul S; Knight, Darryl A; Sly, Peter D; Turner, Debra J; Stick, Stephen M

2018-01-22

Accumulation mode particles (AMP) are formed from engine combustion and make up the inhalable vapour cloud of ambient particulate matter pollution. Their small size facilitates dispersal and subsequent exposure far from their original source, as well as the ability to penetrate alveolar spaces and capillary walls of the lung when inhaled. A significant immuno-stimulatory component of AMP is lipopolysaccharide (LPS), a product of Gram negative bacteria breakdown. As LPS is implicated in the onset and exacerbation of asthma, the presence or absence of LPS in ambient particulate matter (PM) may explain the onset of asthmatic exacerbations to PM exposure. This study aimed to delineate the effects of LPS and AMP on airway inflammation, and potential contribution to airways disease by measuring airway inflammatory responses induced via activation of the LPS cellular receptor, Toll-like receptor 4 (TLR-4). The effects of nebulized AMP, LPS and AMP administered with LPS on lung function, cellular inflammatory infiltrate and cytokine responses were compared between wildtype mice and mice not expressing TLR-4. The presence of LPS administered with AMP appeared to drive elevated airway resistance and sensitivity via TLR-4. Augmented TLR4 driven eosinophilia and greater TNF-α responses observed in AMP-LPS treated mice independent of TLR-4 expression, suggests activation of allergic responses by TLR4 and non-TLR4 pathways larger than those induced by LPS administered alone. Treatment with AMP induced macrophage recruitment independent of TLR-4 expression. These findings suggest AMP-LPS as a stronger stimulus for allergic inflammation in the airways then LPS alone.

Lead effects on development and function of bone marrow-derived dendritic cells promote Th2 immune responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao Donghong; Mondal, Tapan K.; Lawrence, David A.

2007-07-01

Although lead (Pb) has significant effects on the development and function of macrophages, B cells, and T cells and has been suggested to promote allergic asthma in mice and humans, Pb modulation of bone marrow (BM)-derived dendritic cells (DCs) and the resultant DC effects on Th1 and Th2 development have not been examined. Accordingly, we cultured BM cells with murine granulocyte macrophage-colony stimulating factor (mGM-CSF) {+-} PbCl{sub 2}. At day 10, culture supernatant (SN) and non-adherent cells were harvested for analysis. Additionally, day 10 non-adherent BM-DCs were harvested and recultured with mGM-CSF + LPS {+-} Pb for 2 days. Themore » day 10 Pb exposure significantly inhibited BM-DC generation, based on CD11c expression. Although fewer DCs were generated with Pb, the existing Pb-exposed DCs had significantly greater MHC-II expression than did the non-Pb-exposed DCs. However, these differences diminished upon LPS stimulation. After LPS stimulation, CD80, CD86, CD40, CD54, and MHC-II were all up-regulated on both Pb-DCs and DCs, but Pb-DCs expressed significantly less CD80 than did DCs. The CD86:CD80 ratio suggests a Pb-DC potential for Th2 cell development. After LPS stimulation, IL-6, IL-10, IL-12 (p70), and TNF-{alpha} levels significantly increased with both Pb-DCs and DCs, but Pb-DCs produced significantly less cytokines than did DCs, except for IL-10, which further supports Pb-DC preferential skewing toward type-2 immunity. In vitro studies confirm that Pb-DCs have the ability to polarize antigen-specific T cells to Th2 cells. Pb-DCs also enhanced allogeneic and autologous T cell proliferation in vitro, and in vivo studies suggested that Pb-DCs inhibited Th1 effects on humoral and cell-mediated immunity. The Pb effect was mainly on DCs, rather than on T cells, and Pb's modification of DC function appears to be the main cause of Pb's promotion of type-2-related immunity, which may relate to Pb's enhanced activation of the Erk/MAP kinase pathway.« less

The binding capability of plasma phospholipid transfer protein, but not HDL pool size, is critical to repress LPS induced inflammation.

PubMed

Yu, Yang; Cui, Yingjie; Zhao, Yanan; Liu, Shuai; Song, Guohua; Jiao, Peng; Li, Bin; Luo, Tian; Guo, Shoudong; Zhang, Xiangjian; Wang, Hao; Jiang, Xian-Cheng; Qin, Shucun

2016-02-09

Phospholipid transfer protein (PLTP) participates in high density lipoprotein (HDL) metabolism. Increased plasma PLTP activity was observed in lipopolysaccharide (LPS) triggered acute inflammatory diseases. This study aimed to determine the exact role of PLTP in LPS induced inflammation. HDL pool size was shrunk both in PLTP deficient mice (PLTP-/-) and PLTP transgenic mice (PLTP-Tg). PLTP displayed a strong protective effect on lethal endotoxemia in mice survival study. Furthermore, after LPS stimulation, the expression of pro-inflammatory cytokines were increased in bone marrow derived macrophage (BMDM) from PLTP-/-, while decreased in BMDM from PLTP-Tg compared with BMDM from wild-type mice (WT). Moreover, LPS induced nuclear factor kappa-B (NFκB) activation was enhanced in PLTP-/- BMDM or PLTP knockdown RAW264.7. Conversely, PLTP overexpression countered the NFκB activation in LPS challenged BMDM. Additionally, the activation of toll like receptor 4 (TLR4) induced by LPS showed no alteration in PLTP-/- BMDM. Finally, PLTP could bind to LPS, attenuate the pro-inflammatory effects of LPS, and improve the cell viability in vitro. To sum up, these findings elucidated that PLTP repressed LPS induced inflammation due to extracellular LPS binding capability, and the protective effects were not related to HDL pool size in mice.

Effects of BCG infection on the susceptibility of mouse macrophages to endotoxin.

PubMed Central

Peavy, D L; Baughn, R E; Musher, D M; Musher, D M

1979-01-01

Mice infected intravenously with Mycobacterium bovis (BCG) are 100 to 1,000 times more sensitive to the lethal effects of bacterial lipopolysaccharides (LPS). Since BCG infection results in macrophage activation and LPS may cause pathophysiological effects through interaction with this cell type, it was of interest to determine whether macrophages from BCG-infected animals were more susceptible to the toxic effects of LPS in vitro. When LPS-susceptible, C57BL/6 mice were infected with BCG, a significant reduction in the 50% lethal dose of LPS was first observed after 7 days and persisted for several weeks. Macrophages from these animals had greatly increased susceptibility to LPS in vitro, which correlated with the development of acquired cellular resistance as determined by their ability to inhibit the growth of Listeria monocytogenes. In contrast, BCG infection of C3H/HeJ mice, a strain resistant to LPS, did not alter the 50% lethal dose of LPS for these animals or increase the sensitivity of their peritoneal macrophages to LPS in vitro. These results indicate that susceptibility of BCG-infected mice to the lethal effects of LPS parallels the susceptibility of their macrophages in vitro; release of vasoactive substances from LPS-susceptible activated macrophages in vivo may be, in part, responsible for lethality. PMID:378847

Alkaline Phosphatase Protects Lipopolysaccharide-Induced Early Pregnancy Defects in Mice

PubMed Central

Lei, Wei; Ni, Hua; Herington, Jennifer; Reese, Jeff; Paria, Bibhash C.

2015-01-01

Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women. PMID:25910276

Alkaline phosphatase protects lipopolysaccharide-induced early pregnancy defects in mice.

PubMed

Lei, Wei; Ni, Hua; Herington, Jennifer; Reese, Jeff; Paria, Bibhash C

2015-01-01

Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women.

Chondroitin Sulfate-Rich Extract of Skate Cartilage Attenuates Lipopolysaccharide-Induced Liver Damage in Mice.

PubMed

Song, Yeong Ok; Kim, Mijeong; Woo, Minji; Baek, Jang-Mi; Kang, Keon-Hee; Kim, Sang-Ho; Roh, Seong-Soo; Park, Chan Hum; Jeong, Kap-Seop; Noh, Jeong-Sook

2017-06-15

The protective effects of a chondroitin sulfate-rich extract (CSE) from skate cartilage against lipopolysaccharide (LPS)-induced hepatic damage were investigated, and its mechanism of action was compared with that of chondroitin sulfate (CS) from shark cartilage. ICR mice were orally administrated 200 mg/kg body weight (BW) of CS or 400 mg/kg BW of CSE for 3 consecutive days, followed by a one-time intraperitoneal injection of LPS (20 mg/kg BW). The experimental groups were vehicle treatment without LPS injection (NC group), vehicle treatment with LPS injection (LPS group), CS pretreatment with LPS injection (CS group), and CSE pretreatment with LPS injection (CSE group). Hepatic antioxidant enzyme expression levels in the CS and CSE groups were increased relative to those in the LPS group. In LPS-insulted hepatic tissue, inflammatory factors were augmented relative to those in the NC group, but were significantly suppressed by pretreatment with CS or CSE. Moreover, CS and CSE alleviated the LPS-induced apoptotic factors and mitogen-activated protein kinase (MAPK). In addition, CS and CSE effectively decreased the serum lipid concentrations and downregulated hepatic sterol regulatory element-binding proteins expression. In conclusion, the skate CSE could protect against LPS-induced hepatic dyslipidemia, oxidative stress, inflammation, and apoptosis, probably through the regulation of MAPK signaling.

Chondroitin Sulfate-Rich Extract of Skate Cartilage Attenuates Lipopolysaccharide-Induced Liver Damage in Mice

PubMed Central

Song, Yeong Ok; Kim, Mijeong; Woo, Minji; Baek, Jang-Mi; Kang, Keon-Hee; Kim, Sang-Ho; Roh, Seong-Soo; Park, Chan Hum; Jeong, Kap-Seop; Noh, Jeong-Sook

2017-01-01

The protective effects of a chondroitin sulfate-rich extract (CSE) from skate cartilage against lipopolysaccharide (LPS)-induced hepatic damage were investigated, and its mechanism of action was compared with that of chondroitin sulfate (CS) from shark cartilage. ICR mice were orally administrated 200 mg/kg body weight (BW) of CS or 400 mg/kg BW of CSE for 3 consecutive days, followed by a one-time intraperitoneal injection of LPS (20 mg/kg BW). The experimental groups were vehicle treatment without LPS injection (NC group), vehicle treatment with LPS injection (LPS group), CS pretreatment with LPS injection (CS group), and CSE pretreatment with LPS injection (CSE group). Hepatic antioxidant enzyme expression levels in the CS and CSE groups were increased relative to those in the LPS group. In LPS-insulted hepatic tissue, inflammatory factors were augmented relative to those in the NC group, but were significantly suppressed by pretreatment with CS or CSE. Moreover, CS and CSE alleviated the LPS-induced apoptotic factors and mitogen-activated protein kinase (MAPK). In addition, CS and CSE effectively decreased the serum lipid concentrations and downregulated hepatic sterol regulatory element-binding proteins expression. In conclusion, the skate CSE could protect against LPS-induced hepatic dyslipidemia, oxidative stress, inflammation, and apoptosis, probably through the regulation of MAPK signaling. PMID:28617322

Relationship between luminous fish and symbiosis. I. Comparative studies of lipopolysaccharides isolated from symbiotic luminous bacteria of the luminous marine fish, Physiculus japonicus.

PubMed

Kuwae, T; Andoh, M; Fukasawa, S; Kurata, M

1983-01-01

In order to investigate the relationship between host and symbiosis in the luminous marine fish, Physiculus japonicus, the bacterial lipopolysaccharides (LPS) of symbiotic luminous bacteria were compared serologically and electrophoretically. Five symbiotic luminous bacteria (PJ strains) were separately isolated from five individuals of this fish species caught at three points, off the coasts of Chiba, Nakaminato, and Oharai. LPS preparations were made from these bacteria by Westphal's phenol-water method and highly purified by repeated ultracentrifugation. These LPSs contained little or no 2-keto-3-deoxyoctonate and had powerful mitogenic activity. In sodium dodecylsulfate polyacrylamide gel electrophoresis, these PJ-1 to -5 LPSs were separated by their electrophoretic patterns into three groups; the first group included PJ-1 and PJ-4, the second group PJ-2 and PJ-3, and the third group PJ-5 alone. The results agreed with those of the double immunodiffusion test; precipitin lines completely coalesced within each group but not with other groups. In immunoelectrophoresis, one precipitin line was observed between anti PJ-2 LPS serum and PJ-5 LPS but the electrophoretic mobility of PJ-5 LPS was clearly different from that of the PJ-2 LPS group. Furthermore, in a 50% inhibition test with PJ-2 LPS by the passive hemolysis system, the doses of PJ-2 LPS, PJ-3 LPS, and PJ-5 LPS required for 50% inhibition (ID50) in this system were 0.25, 0.25, and 21.6 micrograms/ml for each alkali-treated LPS, respectively, and the ID50's of both PJ-1 LPS and PJ-4 LPS were above 1,000 micrograms/ml. These results indicate that PJ-5 LPS has an antigenic determinant partially in common with LPS from the PJ-2 group but not with LPS from the PJ-1 group and that the symbiotic luminous bacterium PJ-5 is more closely related to the PJ-2 group than to the PJ-1 group. These results show that the species Physiculus japonicus is symbiotically associated with at least three immunologically different strains of luminous marine bacteria in its specialized light organ.

Effect of gamma irradiation on the change of solubility and anti-inflammation activity of chrysin in macrophage cells and LPS-injected endotoxemic mice

NASA Astrophysics Data System (ADS)

Byun, Eui-Baek; Jang, Beom-Su; Byun, Eui-Hong; Sung, Nak-Yun

2016-10-01

This study evaluated the changes of solubility and anti-inflammatory properties of structurally modified gamma-irradiated chrysin. Chrysin was irradiated at various doses for a physical analysis and determining any structural changes and solubility. As shown through the physical analysis, the main peak of the chrysin was decreased as the irradiation dose increased, and it was concomitant with the appearance of several new peaks, which were highly increased in 50 kGy gamma-irradiated chrysin. The solubility was markedly increased in the gamma-irradiated groups. As shown through a physiological analysis, both gamma-irradiated- (15-50 kGy) and intact-chrysin (0 kGy) did not exert cytotoxicity to bone-marrow derived macrophages. The treatment of LPS-stimulated macrophages with 50 kGy gamma-irradiated chrysin resulted in a dose-dependent decrease in pro-inflammatory mediators, such as iNOS-mediated NO, PGE2, COX-2, and cell surface marker (CD80 and CD86), as well as pro-inflammatory cytokines (TNF-α and IL-6), when compared to the intact-chrysin treated group. Mechanically, we found that the inhibition of these pro-inflammatory mediators induced by gamma-irradiated chrysin occurred through an inhibition of MAPKs (ERK1/2 and p38) and the NF-κB signaling pathways. Furthermore, the anti-inflammatory activity remained in the LPS-injected animal model. In this model, gamma-irradiated chrysin treatment highly increased the mouse survival, and significantly decreased the serum cytokine (TNF-α, IL-6 and IL-1β) levels. From these findings, the anti-inflammatory action by gamma-irradiated chrysin may be closely mediated with structural modification. It seems likely that gamma irradiation can be an effective tool for improvement of the physical and physiological properties of polyphenols.

Identification of Proteus mirabilis Mutants with Increased Sensitivity to Antimicrobial Peptides

PubMed Central

McCoy, Andrea J.; Liu, Hongjian; Falla, Timothy J.; Gunn, John S.

2001-01-01

Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example, Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilis mutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility. PMID:11408219

Peptidylarginine deiminases: novel drug targets for prevention of neuronal damage following hypoxic ischemic insult (HI) in neonates.

PubMed

Lange, Sigrun; Rocha-Ferreira, Eridan; Thei, Laura; Mawjee, Priyanka; Bennett, Kate; Thompson, Paul R; Subramanian, Venkataraman; Nicholas, Anthony P; Peebles, Donald; Hristova, Mariya; Raivich, Gennadij

2014-08-01

Neonatal hypoxic ischaemic (HI) injury frequently causes neural impairment in surviving infants. Our knowledge of the underlying molecular mechanisms is still limited. Protein deimination is a post-translational modification caused by Ca(+2) -regulated peptidylarginine deiminases (PADs), a group of five isozymes that display tissue-specific expression and different preference for target proteins. Protein deimination results in altered protein conformation and function of target proteins, and is associated with neurodegenerative diseases, gene regulation and autoimmunity. In this study, we used the neonatal HI and HI/infection [lipopolysaccharide (LPS) stimulation] murine models to investigate changes in protein deimination. Brains showed increases in deiminated proteins, cell death, activated microglia and neuronal loss in affected brain areas at 48 h after hypoxic ischaemic insult. Upon treatment with the pan-PAD inhibitor Cl-amidine, a significant reduction was seen in microglial activation, cell death and infarct size compared with control saline or LPS-treated animals. Deimination of histone 3, a target protein of the PAD4 isozyme, was increased in hippocampus and cortex specifically upon LPS stimulation and markedly reduced following Cl-amidine treatment. Here, we demonstrate a novel role for PAD enzymes in neural impairment in neonatal HI Encephalopathy, highlighting their role as promising new candidates for drug-directed intervention in neurotrauma. Hypoxic Ischaemic Insult (HI) results in activation of peptidylarginine deiminases (PADs) because of calcium dysregulation. Target proteins undergo irreversible changes of protein bound arginine to citrulline, resulting in protein misfolding. Infection in synergy with HI causes up-regulation of TNFα, nuclear translocation of PAD4 and change in gene regulation as a result of histone deimination. Pharmacological PAD inhibition significantly reduced HI brain damage. © 2014 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.

LPS from Escherichia coli protects against indomethacin-induced gastropathy in rats--role of ATP-sensitive potassium channels.

PubMed

Gomes, Antoniella S; Lima, Lívia M F; Santos, Camila L; Cunha, Fernando Q; Ribeiro, Ronaldo A; Souza, Marcellus H L P

2006-10-10

The effect of lipopolysaccharide (LPS) in gastric protection has not been elucidated, but ATP-sensitive potassium (K(ATP)) channels are known to be involved in gastric defense. We evaluated the effect of LPS administration on indomethacin-induced gastropathy, and the role of K(ATP) channels in this event. Rats received intravenous (i.v.) LPS administration. After 1/2, 6, 24 or 48 h, indomethacin was injected. 3H later, gastric damage and myeloperoxidase activity were determined. Another group received LPS and 5 h later, glibenclamide, diazoxide or glibenclamide plus diazoxide. After 1 h, the rats received indomethacin and 3 h later, gastric damage and myeloperoxidase activity were evaluated. LPS reduced dose dependently gastric damage and myeloperoxidase activity induced by indomethacin. Glibenclamide reversed this LPS effect on indomethacin-induced gastropathy. Glibenclamide plus diazoxide administration did not change this LPS effect. Thus LPS has a protective effect against indomethacin-induced gastropathy, probably through activation of K(ATP) channels.

Designed β-Boomerang Antiendotoxic and Antimicrobial Peptides

PubMed Central

Bhunia, Anirban; Mohanram, Harini; Domadia, Prerna N.; Torres, Jaume; Bhattacharjya, Surajit

2009-01-01

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like β-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nm concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the β-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate β-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane. PMID:19520860

Pulmonary exposure to diesel exhaust particles enhances coagulatory disturbance with endothelial damage and systemic inflammation related to lung inflammation.

PubMed

Inoue, Ken-Ichiro; Takano, Hirohisa; Sakurai, Miho; Oda, Toshio; Tamura, Hiroshi; Yanagisawa, Rie; Shimada, Akinori; Yoshikawa, Toshikazu

2006-11-01

Pulmonary exposure to diesel exhaust particles (DEP) enhances lung inflammation related to bacterial endotoxin (lipopolysaccharide [LPS]) in mice. Severe lung inflammation can reportedly induce coagulatory abnormalities and systemic inflammation. This study examined the effects of components of DEP on lung inflammation, pulmonary permeability, coagulatory changes, systemic inflammatory response, and lung-to-systemic translocation of LPS in a murine model of lung inflammation. ICR mice were divided into six experimental groups that intratracheally received vehicle, LPS (2.5 mg/kg), organic chemicals in DEP (DEP-OC; 4 mg/kg) extracted with dicloromethane), residual carbonaceous nuclei of DEP (washed DEP: 4 mg/kg), DEP-OC + LPS, or washed DEP + LPS. Both DEP components exacerbated lung inflammation, vascular permeability, and the increased fibrinogen and E-selectin levels induced by LPS. With overall trends, the exacerbation was more prominent with washed DEP than with DEP-OC. Washed DEP + LPS significantly decreased activated protein C and antithrombin-III and elevated circulatory levels of interleukin (IL)-6, keratinocyte chemoattractant (KC), and LPS as compared with LPS alone, whereas DEP-OC + LPS elevated IL-6, KC, and LPS without significance. These results show that DEP components, especially washed DEP, amplify the effects if LPS on the respiratory system and suggest that they contribute to the adverse health effects of particulate air pollution on the sensitive populations with predisposing vascular and/or pulmonary diseases, including ischemic vascular diseases and respiratory infection.

Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1beta, TNF-alpha and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS.

PubMed

Diya Zhang; Lili Chen; Shenglai Li; Zhiyuan Gu; Jie Yan

2008-04-01

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been shown to differ from enterobacterial LPS in structure and function; therefore, the Toll-like receptors (TLRs) and the intracellular inflammatory signaling pathways are accordingly different. To elucidate the signal transduction pathway of P. gingivalis, LPS-induced pro-inflammatory cytokine production in the human monocytic cell line THP-1 was measured by ELISA, and the TLRs were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors as well as Phospho-ELISA kits were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. In this study, P. gingivalis LPS showed the ability to induce cytokine production in THP-1 cells and its induction was significantly (P < 0.05) suppressed by anti-TLR2 antibody or JNK inhibitor, and the phosphorylation level of JNK was significantly increased (P < 0.05). These results indicate that TLR2-JNK is the main signaling pathway of P. gingivalis LPS-induced cytokine production, while the cytokine induction by E. coli LPS was mainly via TLR4-NF-kappaB and TLR4-p38MAPK. This suggests that P. gingivalis LPS differs from E. coli LPS in its signaling pathway in THP-1 cells, and that the TLR2-JNK pathway might play a significant role in P. gingivalis LPS-induced chronic inflammatory periodontal disease.

Endogenous pleiotrophin and midkine regulate LPS-induced glial responses.

PubMed

Fernández-Calle, Rosalía; Vicente-Rodríguez, Marta; Gramage, Esther; de la Torre-Ortiz, Carlos; Pérez-García, Carmen; Ramos, María P; Herradón, Gonzalo

2018-01-01

Pleiotrophin (PTN) and Midkine (MK) are two growth factors that modulate neuroinflammation. PTN overexpression in the brain prevents LPS-induced astrocytosis in mice but potentiates microglial activation. The modest astrocytic response caused by a low dose of LPS (0.5mg/kg) is blocked in the striatum of MK-/- mice whereas microglial response is unaffected. We have now tested the effects of an intermediate dose of LPS (7.5mg/kg) in glial response in PTN-/- and MK-/- mice. We found that LPS-induced astrocytosis is prevented in prefrontal cortex and striatum of both PTN-/- and MK-/- mice. Some of the morphological changes of microglia induced by LPS tended to increase in both genotypes, particularly in PTN-/- mice. Since we previously showed that PTN potentiates LPS-induced activation of BV2 microglial cells, we tested the activation of FYN kinase, a substrate of the PTN receptor RPTPβ/ζ, and the subsequent ERK1/2 phosphorylation on LPS and PTN-treated BV2 cells. LPS effects on BV2 cells were not affected by the addition of PTN, suggesting that PTN does not recruit the FYN-MAP kinase signaling pathway in order to modulate LPS effects on microglial cells. Taking together, evidences demonstrate that regulation of astroglial responses to LPS administration are highly dependent on the levels of expression of PTN and MK. Further studies are needed to clarify the possible roles of endogenous expression of PTN and MK in LPS-induced microglial responses. Copyright © 2017 Elsevier B.V. All rights reserved.

Zinc supplementation during pregnancy protects against lipopolysaccharide-induced fetal growth restriction and demise through its anti-inflammatory effect.

PubMed

Chen, Yuan-Hua; Zhao, Mei; Chen, Xue; Zhang, Ying; Wang, Hua; Huang, Ying-Ying; Wang, Zhen; Zhang, Zhi-Hui; Zhang, Cheng; Xu, De-Xiang

2012-07-01

LPS is associated with adverse developmental outcomes, including preterm delivery, fetal death, teratogenicity, and intrauterine growth restriction (IUGR). Previous reports showed that zinc protected against LPS-induced teratogenicity. In the current study, we investigated the effects of zinc supplementation during pregnancy on LPS-induced preterm delivery, fetal death and IUGR. All pregnant mice except controls were i.p. injected with LPS (75 μg/kg) daily from gestational day (GD) 15 to GD17. Some pregnant mice were administered zinc sulfate through drinking water (75 mg elemental Zn per liter) throughout the pregnancy. As expected, an i.p. injection with LPS daily from GD15 to GD17 resulted in 36.4% (4/11) of dams delivered before GD18. In dams that completed the pregnancy, 63.2% of fetuses were dead. Moreover, LPS significantly reduced fetal weight and crown-rump length. Of interest, zinc supplementation during pregnancy protected mice from LPS-induced preterm delivery and fetal death. In addition, zinc supplementation significantly alleviated LPS-induced IUGR and skeletal development retardation. Further experiments showed that zinc supplementation significantly attenuated LPS-induced expression of placental inflammatory cytokines and cyclooxygenase-2. Zinc supplementation also significantly attenuated LPS-induced activation of NF-κB and MAPK signaling in mononuclear sinusoidal trophoblast giant cells of the labyrinth zone. It inhibited LPS-induced placental AKT phosphorylation as well. In conclusion, zinc supplementation during pregnancy protects against LPS-induced fetal growth restriction and demise through its anti-inflammatory effect.

Protection of BALB/C mice against Brucella abortus 544 challenge by vaccination with combination of recombinant human serum albumin-l7/l12 (Brucella abortus ribosomal protein) and lipopolysaccharide.

PubMed

Pakzad, Iraj; Rezaee, Abbas; Rasaee, Mohammad J; Hossieni, Ahmad Zavaran; Tabbaraee, Bahman; Kazemnejad, Anoshirvan

2010-01-01

The immunogenic Brucella abortus ribosomal protein L7/L12 and Lipopolysaccharide (LPS) are promising candidate antigens for the development of subunit vaccines against brucellosis. This study was aimed to evaluate the protection of combination of recombinant HSA-L7/L12 fusion protein with LPS in Balb/c mouse. The recombinant HSA-L7/L12 fusion protein in Saccharomyces cerevisiae was expressed and purified by affinity chromatography column. LPS was extracted by n-butanol, purified by ultracentrifugation. BALB/c mouses were immunized in 9 groups with PBS, HSA, tHSA-L7/L12, L7/L12, LPS, LPS+ HSA, LPS+ tHSA-L7/L12, LPS+ L7/L12, B. abortus S19. ELISA, LTT tests and challenging two weeks after last injection were carried out. Bacterial count of spleen of immunized BALB/c mouse was done four weeks after challenging with virulent strain B. abortus 544. In ELISA test the specific antibodies of tHSA-L7/L12 exhibited a dominance of immunoglobulin IgG1 over IgG2a. LPS-HSA and tHSA-L7/L12 + LPS produced a significantly higher antibody titer than LPS alone and L7/L12+LPS (P < 0.05). The predominant IgG subtype for LPS and L7/L12+LPS were IgG3. However, tHSA-L7/L12+ LPS and LPS+ HAS elicited predominantly IgG1 and IgG3 subtypes. In addition, the tHSA-L7/L12 fusion protein and L7/L12 elicited a strong T-cell proliferative response upon restimulation in vitro with recombinant tHSA-L7/L12 and L7/L12, suggesting the induction of a cellular immunity response in vivo. However, there was no significant difference proliferative response in L7/L12 and tHSA-L7/L12 fusion protein (P > 0.05). The combination of tHSA-L7/L12 fusion protein with LPS and B. abortus S19 induce higher level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice than other groups (P = 0.005). The combination of tHSA-L7/L12 fusion protein with LPS had higher protective ability than LPS and fusion protein distinctly.

6-Mercaptopurine attenuates tumor necrosis factor-α production in microglia through Nur77-mediated transrepression and PI3K/Akt/mTOR signaling-mediated translational regulation.

PubMed

Huang, Hsin-Yi; Chang, Hui-Fen; Tsai, Ming-Jen; Chen, Jhih-Si; Wang, Mei-Jen

2016-04-13

The pathogenesis of several neurodegenerative diseases often involves the microglial activation and associated inflammatory processes. Activated microglia release pro-inflammatory factors that may be neurotoxic. 6-Mercaptopurine (6-MP) is a well-established immunosuppressive drug. Common understanding of their immunosuppressive properties is largely limited to peripheral immune cells. However, the effect of 6-MP in the central nervous system, especially in microglia in the context of neuroinflammation is, as yet, unclear. Tumor necrosis factor-α (TNF-α) is a key cytokine of the immune system that initiates and promotes neuroinflammation. The present study aimed to investigate the effect of 6-MP on TNF-α production by microglia to discern the molecular mechanisms of this modulation. Lipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Released TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression was determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Signaling molecules were analyzed by western blotting, and activation of NF-κB was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Chromatin immunoprecipitation (ChIP) analysis was performed to examine NF-κB p65 and coactivator p300 enrichments and histone modifications at the endogenous TNF-α promoter. Treatment of LPS-activated microglia with 6-MP significantly attenuated TNF-α production. In 6-MP pretreated microglia, LPS-induced MAPK signaling, IκB-α degradation, NF-κB p65 nuclear translocation, and in vitro p65 DNA binding activity were not impaired. However, 6-MP suppressed transactivation activity of NF-κB and TNF-α promoter by inhibiting phosphorylation and acetylation of p65 on Ser276 and Lys310, respectively. ChIP analyses revealed that 6-MP dampened LPS-induced histone H3 acetylation of chromatin surrounding the TNF-α promoter, ultimately leading to a decrease in p65/coactivator-mediated transcription of TNF-α gene. Furthermore, 6-MP enhanced orphan nuclear receptor Nur77 expression. Using RNA interference approach, we further demonstrated that Nur77 upregulation contribute to 6-MP-mediated inhibitory effect on TNF-α production. Additionally, 6-MP also impeded TNF-α mRNA translation through prevention of LPS-activated PI3K/Akt/mTOR signaling cascades. These results suggest that 6-MP might have a therapeutic potential in neuroinflammation-related neurodegenerative disorders through downregulation of microglia-mediated inflammatory processes.

Histological changes and some in vitro biological activities induced by lipopolysaccharide from Bacteroides gingivalis.

PubMed

Isogai, H; Isogai, E; Fujii, N; Oguma, K; Kagota, W; Takano, K

1988-07-01

The biological activities of lipopolysaccharide from Bacteroides gingivalis 381 (B-LPS) were examined in vivo and in vitro. Intra-oral mucosal injection of B-LPS induced an acute inflammation at the injection site. Intravenous injection of B-LPS induced necrotic lesions with many thrombi in the liver and lymphocytic reduction in the spleen. By immunohistochemical examination, B-LPS was detected in macrophages in the liver, spleen and lymph nodes. In vitro analysis showed that B-LPS was a potent activator of both neutrophils and macrophages in luminol-dependent response and IL-1 secretion from macrophages and was mitogenic to the spleen cells not only from BALB/c mice but also from LPS-non-responder C3H/HeJ mice. Interferon production from human peripheral mononuclear leucocytes was induced, in vitro, by stimulation with B-LPS but not with the other enterobacterial LPS. These findings clarified the various biological activities of B-LPS affecting various cells and tissues, especially neutrophils, macrophages and lymphocytes. The potent inflammability of B-LPS shown in the present study indicates that it is one of the effective agents to induce periodontitis.

Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate.

PubMed

Oyler, Benjamin L; Khan, Mohd M; Smith, Donald F; Harberts, Erin M; Kilgour, David P A; Ernst, Robert K; Cross, Alan S; Goodlett, David R

2018-06-01

Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS 3 analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts. Graphical Abstract ᅟ.

Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro

PubMed Central

Reisinger, Nicole; Schaumberger, Simone; Nagl, Veronika; Hessenberger, Sabine; Schatzmayr, Gerd

2014-01-01

The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application. PMID:25290524

Which one is more effective for the treatment of rat sepsis model: thalidomide or etanercept?

PubMed

Ilhan, N; Susam, S; Gul, H F; Bardas, R; Ilhan, N

2017-01-01

We aimed to investigate the protective effect of selected treatment agents on liver injury in lipopolysaccharide (LPS)-induced rat sepsis model. The sepsis includes complex inflammatory responses between a microbial pathogen and the host immune system, and leads to organ failure and also death. This study was performed with 29 male Wistar Albino rats. Rats were divided randomly into five groups: Sham group, LPS-treated sepsis group, LPS+thalidomide treated group, LPS+etanercept treated group and LPS+thalidomide+etanercept treated group, respectively. Liver tissue tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) levels were determined by enzyme-linked immuno-sorbent assay (ELISA) method. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was performed using western blot analysis. The levels of tissue TNF-α, IL-1β and IL-6 were found statistically significantly higher in sepsis group than in the sham group. TNF-α levels were found statistically significantly decreased in LPS+etanercept and LPS+thalidomide+etanercept treated groups when compared with LPS group (p < 0.05). For IL-1β and IL-6 levels a statistically significant decline was observed in the LPS+thalidomide and LPS+etanercept treated groups compared to the LPS group (p < 0.05). Expression of NF-κB protein in liver tissue was significantly elevated in the LPS group compared to sham group (p < 0.001). In treatment groups, a marked decrease was observed in NF-κB protein expression. The results of this investigation suggested that etanercept and thalidomide administration may have a beneficial effect on LPS-induced sepsis. So, the present study may have significant clinical relevance, but clinical trials are needed to confirm these results (Tab. 1, Fig. 1, Ref. 36).

Role of T Cells and Gamma Interferon during Induction of Hypersensitivity to Lipopolysaccharide by Toxic Shock Syndrome Toxin 1 in Mice

PubMed Central

Dinges, Martin M.; Schlievert, Patrick M.

2001-01-01

The superantigenic function of toxic shock syndrome toxin 1 (TSST-1) is generally regarded as an important determinant of its lethal effects in humans or experimental animals. This study examined the role of superantigenicity in a BALB/c mouse model of lethal TSST-1-induced hypersensitivity to lipopolysaccharide (LPS). In this model, TSST-1 greatly potentiated both LPS-induced lethality, as well as LPS-induced serum tumor necrosis factor alpha (TNF-α) activity. Although BALB/c-SCID mice were resistant to these LPS enhancement effects of TSST-1, BALB/c-SCID mice reconstituted with T cells were completely susceptible to the enhancement effect of TSST-1 on LPS-induced serum TNF-α. Mice pretreated with cyclosporine (Cs) or neutralizing antibodies against gamma interferon (IFN-γ) did not develop lethal LPS hypersensitivity when injected with TSST-1, and these agents reduced the enhancement effect of TSST-1 on LPS-induced serum TNF-α by 99 and 85%, respectively. Cs pretreatment also completely inhibited the known capacity of TSST-1 to amplify LPS-induced levels of IFN-γ in serum. In contrast, mice given Cs after a priming injection of TSST-1, but before LPS, still exhibited lethal hypersensitivity to LPS. Cs given after TSST-1 also did not inhibit enhancement of LPS-induced serum TNF-α by TSST-1 but inhibited the enhancement effect of TSST-1 on LPS-induced serum IFN-γ by 50%. These experiments support the theory that TSST-1-induced hypersensitivity to LPS is mediated primarily by IFN-γ derived from superantigen-activated T cells. PMID:11179286

Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate

NASA Astrophysics Data System (ADS)

Oyler, Benjamin L.; Khan, Mohd M.; Smith, Donald F.; Harberts, Erin M.; Kilgour, David P. A.; Ernst, Robert K.; Cross, Alan S.; Goodlett, David R.

2018-02-01

Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS3 analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts. [Figure not available: see fulltext.

Milk thistle extract and silymarin inhibit lipopolysaccharide induced lamellar separation of hoof explants in vitro.

PubMed

Reisinger, Nicole; Schaumberger, Simone; Nagl, Veronika; Hessenberger, Sabine; Schatzmayr, Gerd

2014-10-06

The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application.

Nonhemolytic Streptococcus pyogenes Isolates That Lack Large Regions of the sag Operon Mediating Streptolysin S Production▿

PubMed Central

Yoshino, Miho; Murayama, Somay Y.; Sunaoshi, Katsuhiko; Wajima, Takeaki; Takahashi, Miki; Masaki, Junko; Kurokawa, Iku; Ubukata, Kimiko

2010-01-01

Among nonhemolytic Streptococcus pyogenes (group A streptococcus) strains (n = 9) isolated from patients with pharyngitis or acute otitis media, we identified three deletions in the region from the epf gene, encoding the extracellular matrix binding protein, to the sag operon, mediating streptolysin S production. PMID:20018818

A Modified Consumer Inkjet for Spatiotemporal Control of Gene Expression

PubMed Central

Cohen, Daniel J.; Morfino, Roberto C.; Maharbiz, Michel M.

2009-01-01

This paper presents a low-cost inkjet dosing system capable of continuous, two-dimensional spatiotemporal regulation of gene expression via delivery of diffusible regulators to a custom-mounted gel culture of E. coli. A consumer-grade, inkjet printer was adapted for chemical printing; E. coli cultures were grown on 750 µm thick agar embedded in micro-wells machined into commercial compact discs. Spatio-temporal regulation of the lac operon was demonstrated via the printing of patterns of lactose and glucose directly into the cultures; X-Gal blue patterns were used for visual feedback. We demonstrate how the bistable nature of the lac operon's feedback, when perturbed by patterning lactose (inducer) and glucose (inhibitor), can lead to coordination of cell expression patterns across a field in ways that mimic motifs seen in developmental biology. Examples of this include sharp boundaries and the generation of traveling waves of mRNA expression. To our knowledge, this is the first demonstration of reaction-diffusion effects in the well-studied lac operon. A finite element reaction-diffusion model of the lac operon is also presented which predicts pattern formation with good fidelity. PMID:19763256

Prevalence of transcription promoters within archaeal operons and coding sequences

PubMed Central

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements. PMID:19536208

Prevalence of transcription promoters within archaeal operons and coding sequences.

PubMed

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of approximately 64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes-events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

The Bacillus subtilis GntR family repressor YtrA responds to cell wall antibiotics.

PubMed

Salzberg, Letal I; Luo, Yun; Hachmann, Anna-Barbara; Mascher, Thorsten; Helmann, John D

2011-10-01

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σ(M). Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB. These results suggest that YtrA is an additional regulator of cell envelope stress responses.

The Bacillus subtilis GntR Family Repressor YtrA Responds to Cell Wall Antibiotics▿§

PubMed Central

Salzberg, Letal I.; Luo, Yun; Hachmann, Anna-Barbara; Mascher, Thorsten; Helmann, John D.

2011-01-01

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σM. Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB. These results suggest that YtrA is an additional regulator of cell envelope stress responses. PMID:21856850

Cellulose production in Pseudomonas syringae pv. syringae: a compromise between epiphytic and pathogenic lifestyles.

PubMed

Arrebola, Eva; Carrión, Víctor J; Gutiérrez-Barranquero, José Antonio; Pérez-García, Alejandro; Rodríguez-Palenzuela, Pablo; Cazorla, Francisco M; de Vicente, Antonio

2015-07-01

Genome sequencing and annotation have revealed a putative cellulose biosynthetic operon in the strain Pseudomonas syringae pv. syringae UMAF0158, the causal agent of bacterial apical necrosis. Bioinformatics analyses and experimental methods were used to confirm the functionality of the cellulose biosynthetic operon. In addition, the results showed the contribution of the cellulose operon to important aspects of P. syringae pv. syringae biology, such as the formation of biofilms and adhesion to the leaf surface of mango, suggesting that this operon increases epiphytic fitness. However, based on the incidence and severity of the symptoms observed in tomato leaflets, cellulose expression reduces virulence, as cellulose-deficient mutants increased the area of necrosis, whereas the cellulose-overproducing strain decreased the area of necrosis compared with the wild type. In conclusion, the results of this study show that the epiphytic and pathogenic stages of the P. syringae pv. syringae UMAF0158 lifestyle are intimately affected by cellulose production. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

rrndb: the Ribosomal RNA Operon Copy Number Database

PubMed Central

Klappenbach, Joel A.; Saxman, Paul R.; Cole, James R.; Schmidt, Thomas M.

2001-01-01

The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary from one to as many as 15 copies. Despite the widespread use of 16S rRNA gene sequences for identification of prokaryotes, information on the number and sequence of individual rRNA genes in a genome is not readily accessible. In an attempt to understand the evolutionary implications of rRNA operon redundancy, we have created a phylogenetically arranged report on rRNA gene copy number for a diverse collection of prokaryotic microorganisms. Each entry (organism) in the rrndb contains detailed information linked directly to external websites including the Ribosomal Database Project, GenBank, PubMed and several culture collections. Data contained in the rrndb will be valuable to researchers investigating microbial ecology and evolution using 16S rRNA gene sequences. The rrndb web site is directly accessible on the WWW at http://rrndb.cme.msu.edu. PMID:11125085

A distinct alleles and genetic recombination of pmrCAB operon in species of Acinetobacter baumannii complex isolates.

PubMed

Kim, Dae Hun; Ko, Kwan Soo

2015-07-01

To investigate pmrCAB sequence divergence in 5 species of Acinetobacter baumannii complex, a total of 80 isolates from a Korean hospital were explored. We evaluated nucleotide and amino acid polymorphisms of pmrCAB operon, and phylogenetic trees were constructed for each gene of prmCAB operon. Colistin and polymyxin B susceptibility was determined for all isolates, and multilocus sequence typing was also performed for A. baumannii isolates. Our results showed that each species of A. baumannii complex has divergent pmrCAB operon sequences. We identified a distinct pmrCAB allele allied with Acinetobacter nosocomialis in gene trees. Different grouping in each gene tree suggests sporadic recombination or emergence of pmrCAB genes among Acinetobacter species. Sequence polymorphisms among Acinetobacter species might not be associated with colistin resistance. We revealed that a distinct pmrCAB allele may be widespread across the continents such as North America and Asia and that sporadic genetic recombination or emergence of pmrCAB genes might occur. Copyright © 2015 Elsevier Inc. All rights reserved.

Membrane insertion for the detection of lipopolysaccharides: Exploring the dynamics of amphiphile-in-lipid assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stromberg, Loreen R.; Hengartner, Nicolas W.; Swingle, Kirstie L.

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assaysmore » to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. In addition, we also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.« less

Membrane insertion for the detection of lipopolysaccharides: Exploring the dynamics of amphiphile-in-lipid assays

DOE PAGES

Stromberg, Loreen R.; Hengartner, Nicolas W.; Swingle, Kirstie L.; ...

2016-05-26

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assaysmore » to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. In addition, we also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.« less

The effects of dietary chromium(III) picolinate on growth performance, vital signs, and blood measurements of pigs during immune stress.

PubMed

Kim, Beob G; Lindemann, Merlin D; Cromwell, Gary L

2010-06-01

This experiment used 24 pigs (26.0 kg) to investigate the effects of dietary chromium (Cr) on pigs challenged with lipopolysaccharide (LPS). Following 35 days of diet exposure, the immune stress treatments were: (1) phosphate-buffered saline (PBS) injection and no Cr, (2) LPS injection and no Cr, (3) LPS injection and Cr 1,000 ppb, and (4) LPS injection and Cr 2,000 ppb. At 0 h, PBS or LPS was injected intraperitoneally in each pig. During the first 12 h post-injection, pigs challenged with LPS lost 951 g, while the PBS group gained 170 g (p < 0.001). Compared with the PBS group, LPS-challenged pigs consumed less feed (p < 0.01) during the first 24 h. The LPS group had higher rectal temperature at 2 and 4 h and higher respiratory rate at 1.3 and 8.5 h than the PBS group (p < 0.05). Plasma collected at 3 h had higher cortisol (p < 0.001) and lower glucose (p < 0.05) concentrations in the LPS group than the PBS group. However, supplemental Cr did not affect the response variables. Overall, the LPS challenge affects growth performance, vital signs, and plasma variables, but dietary Cr is unable to moderate stress-related effects associated with an LPS challenge.

Long-term alterations in neuroimmune responses after neonatal exposure to lipopolysaccharide.

PubMed

Boissé, Lysa; Mouihate, Abdeslam; Ellis, Shaun; Pittman, Quentin J

2004-05-26

Fever is an integral part of the host's defense to infection that is orchestrated by the brain. A reduced febrile response is associated with reduced survival. Consequently, we have asked if early life immune exposure will alter febrile and neurochemical responses to immune stress in adulthood. Fourteen-day-old neonatal male rats were given Escherichia coli lipopolysaccharide (LPS) that caused either fever or hypothermia depending on ambient temperature. Control rats were given pyrogen-free saline. Regardless of the presence of neonatal fever, adult animals that had been neonatally exposed to LPS displayed attenuated fevers in response to intraperitoneal LPS but unaltered responses to intraperitoneal interleukin 1beta or intracerebroventricular prostaglandin E(2). The characteristic reduction in activity that accompanies fever was unaltered, however, as a function of neonatal LPS exposure. Treatment of neonates with an antigenically dissimilar LPS (Salmonella enteritidis) was equally effective in reducing adult responses to E. coli LPS, indicating an alteration in the innate immune response. In adults treated as neonates with LPS, basal levels of hypothalamic cyclooxygenase 2 (COX-2), determined by semiquantitative Western blot analysis, were significantly elevated compared with controls. In addition, whereas adult controls responded to LPS with the expected induction of COX-2, adults pretreated neonatally with LPS responded to LPS with a reduction in COX-2. Thus, neonatal LPS can alter CNS-mediated inflammatory responses in adult rats.

FLICE-like inhibitory protein (FLIP) protects against apoptosis and suppresses NF-kappaB activation induced by bacterial lipopolysaccharide.

PubMed

Bannerman, Douglas D; Eiting, Kristine T; Winn, Robert K; Harlan, John M

2004-10-01

Bacterial lipopolysaccharide (LPS) via its activation of Toll-like receptor-4 contributes to much of the vascular injury/dysfunction associated with gram-negative sepsis. Inhibition of de novo gene expression has been shown to sensitize endothelial cells (EC) to LPS-induced apoptosis, the onset of which correlates with decreased expression of FLICE-like inhibitory protein (FLIP). We now have data that conclusively establish a role for FLIP in protecting EC against LPS-induced apoptosis. Overexpression of FLIP protected against LPS-induced apoptosis, whereas down-regulation of FLIP using antisense oligonucleotides sensitized EC to direct LPS killing. Interestingly, FLIP overexpression suppressed NF-kappaB activation induced by LPS, but not by phorbol ester, suggesting a specific role for FLIP in mediating LPS activation. Conversely, mouse embryo fibroblasts (MEF) obtained from FLIP -/- mice showed enhanced LPS-induced NF-kappaB activation relative to those obtained from wild-type mice. Reconstitution of FLIP-/- MEF with full-length FLIP reversed the enhanced NF-kappaB activity elicited by LPS in the FLIP -/- cells. Changes in the expression of FLIP had no demonstrable effect on other known LPS/Tlr-4-activated signaling pathways including the p38, Akt, and Jnk pathways. Together, these data support a dual role for FLIP in mediating LPS-induced apoptosis and NF-kappaB activation.

Sulfate-Dependent Repression of Genes That Function in Organosulfur Metabolism in Bacillus subtilis Requires Spx

PubMed Central

Erwin, Kyle N.; Nakano, Shunji; Zuber, Peter

2005-01-01

Oxidative stress in Bacillus subtilis results in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase α subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of the ytmI, yxeI, and ssu operons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZ fusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfate-dependent repression. Both the ytmI operon and the divergently transcribed ytlI, encoding a LysR-type regulator that positively controls ytmI operon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. The yxeL-lacZ and ssu-lacZ fusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions. PMID:15937167

A NodD-like protein activates transcription of genes involved with naringenin degradation in a flavonoid-dependent manner in Herbaspirillum seropedicae.

PubMed

Wassem, R; Marin, A M; Daddaoua, A; Monteiro, R A; Chubatsu, L S; Ramos, J L; Deakin, W J; Broughton, W J; Pedrosa, F O; Souza, E M

2017-03-01

Herbaspirillum seropedicae is an associative, endophytic non-nodulating diazotrophic bacterium that colonises several grasses. An ORF encoding a LysR-type transcriptional regulator, very similar to NodD proteins of rhizobia, was identified in its genome. This nodD-like gene, named fdeR, is divergently transcribed from an operon encoding enzymes involved in flavonoid degradation (fde operon). Apigenin, chrysin, luteolin and naringenin strongly induce transcription of the fde operon, but not that of the fdeR, in an FdeR-dependent manner. The intergenic region between fdeR and fdeA contains several generic LysR consensus sequences (T-N 11 -A) and we propose a binding site for FdeR, which is conserved in other bacteria. DNase I foot-printing revealed that the interaction with the FdeR binding site is modified by the four flavonoids that stimulate transcription of the fde operon. Moreover, FdeR binds naringenin and chrysin as shown by isothermal titration calorimetry. Interestingly, FdeR also binds in vitro to the nod-box from the nodABC operon of Rhizobium sp. NGR234 and is able to activate its transcription in vivo. These results show that FdeR exhibits two features of rhizobial NodD proteins: nod-box recognition and flavonoid-dependent transcription activation, but its role in H. seropedicae and related organisms seems to have evolved to control flavonoid metabolism. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Origin and Evolution of the Sodium -Pumping NADH: Ubiquinone Oxidoreductase

PubMed Central

Reyes-Prieto, Adrian; Barquera, Blanca; Juárez, Oscar

2014-01-01

The sodium -pumping NADH: ubiquinone oxidoreductase (Na+-NQR) is the main ion pump and the primary entry site for electrons into the respiratory chain of many different types of pathogenic bacteria. This enzymatic complex creates a transmembrane gradient of sodium that is used by the cell to sustain ionic homeostasis, nutrient transport, ATP synthesis, flagellum rotation and other essential processes. Comparative genomics data demonstrate that the nqr operon, which encodes all Na+-NQR subunits, is found in a large variety of bacterial lineages with different habitats and metabolic strategies. Here we studied the distribution, origin and evolution of this enzymatic complex. The molecular phylogenetic analyses and the organizations of the nqr operon indicate that Na+-NQR evolved within the Chlorobi/Bacteroidetes group, after the duplication and subsequent neofunctionalization of the operon that encodes the homolog RNF complex. Subsequently, the nqr operon dispersed through multiple horizontal transfer events to other bacterial lineages such as Chlamydiae, Planctomyces and α, β, γ and δ -proteobacteria. Considering the biochemical properties of the Na+-NQR complex and its physiological role in different bacteria, we propose a detailed scenario to explain the molecular mechanisms that gave rise to its novel redox- dependent sodium -pumping activity. Our model postulates that the evolution of the Na+-NQR complex involved a functional divergence from its RNF homolog, following the duplication of the rnf operon, the loss of the rnfB gene and the recruitment of the reductase subunit of an aromatic monooxygenase. PMID:24809444

ThrR, a DNA-binding transcription factor involved in controlling threonine biosynthesis in Bacillus subtilis.

PubMed

Rosenberg, Jonathan; Müller, Peter; Lentes, Sabine; Thiele, Martin J; Zeigler, Daniel R; Tödter, Dominik; Paulus, Henry; Brantl, Sabine; Stülke, Jörg; Commichau, Fabian M

2016-09-01

The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria. © 2016 John Wiley & Sons Ltd.

Immunization of cows with novel core glycolipid vaccine induces anti-endotoxin antibodies in bovine colostrum.

PubMed

Cross, Alan S; Karreman, Hubert J; Zhang, Lei; Rosenberg, Zeil; Opal, Steven M; Lees, Andrew

2014-10-21

Translocation of gut-derived Gram-negative bacterial (GNB) lipopolysaccharide (LPS, or endotoxin) is a source of systemic inflammation that exacerbates HIV, cardiovascular and gastrointestinal diseases and malnutrition. The oral administration of bovine colostrum (BC) reduces endotoxemia in patients with impaired gut barrier function. Consequently, BC enriched in antibodies to LPS may ameliorate endotoxemia-related morbidities. We developed a detoxified J5 LPS/group B meningococcal outer membrane protein (J5dLPS/OMP) vaccine that induces antibodies against a highly conserved core region of LPS and protects against heterologous GNB infection. We now examine the ability of this vaccine to elicit anti-core endotoxin antibodies in BC. Two cohorts of pregnant cows were immunized with this vaccine in combination with FICA (Cohort 1) or Emulsigen-D (Cohort 2) adjuvants. Antibody responses to the J5 core LPS antigen were measured in both serum and colostrum and compared to antibody levels elicited by a commercially available veterinary vaccine (J5 Bacterin) comprised of heat-killed Escherichia coli O111, J5 mutant bacteria, from which the J5 LPS was purified. The J5dLPS/OMP vaccine induced high titers of serum IgG antibody to J5 LPS in all seven cows. Both IgG and to a lesser extent IgA anti-J5 LPS antibodies were generated in the colostrum. The J5dLPS/OMP vaccine was significantly more immunogenic in mice than was the J5 Bacterin. BC enriched in anti-J5 LPS antibody reduced circulating endotoxin levels in neutropenic rats, a model of "leaky gut". The J5dLPS/OMP vaccine elicits high titers of serum anti-endotoxin antibodies in cows that is passed to the colostrum. This BC enriched in anti-core LPS antibodies has the potential to reduce endotoxemia and ameliorate endotoxin-related systemic inflammation in patients with impaired gut barrier function. Since this vaccine is significantly more immunogenic than the J5 Bacterin vaccine, this J5dLPS/OMP vaccine might prove to be more useful for veterinary indications as well. Copyright © 2014 Elsevier Ltd. All rights reserved.

Designed beta-boomerang antiendotoxic and antimicrobial peptides: structures and activities in lipopolysaccharide.

PubMed

Bhunia, Anirban; Mohanram, Harini; Domadia, Prerna N; Torres, Jaume; Bhattacharjya, Surajit

2009-08-14

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like beta-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nM concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the beta-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate beta-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.

The Use of Amino Sugars by Bacillus subtilis: Presence of a Unique Operon for the Catabolism of Glucosamine

PubMed Central

Gaugué, Isabelle; Oberto, Jacques; Putzer, Harald; Plumbridge, Jacqueline

2013-01-01

B. subtilis grows more rapidly using the amino sugar glucosamine as carbon source, than with N-acetylglucosamine. Genes for the transport and metabolism of N-acetylglucosamine (nagP and nagAB) are found in all the sequenced Bacilli (except Anoxybacillus flavithermus). In B. subtilis there is an additional operon (gamAP) encoding second copies of genes for the transport and catabolism of glucosamine. We have developed a method to make multiple deletion mutations in B. subtilis employing an excisable spectinomycin resistance cassette. Using this method we have analysed the contribution of the different genes of the nag and gam operons for their role in utilization of glucosamine and N-acetylglucosamine. Faster growth on glucosamine is due to the presence of the gamAP operon, which is strongly induced by glucosamine. Although the gamA and nagB genes encode isozymes of GlcN6P deaminase, catabolism of N-acetylglucosamine relies mostly upon the gamA gene product. The genes for use of N-acetylglucosamine, nagAB and nagP, are repressed by YvoA (NagR), a GntR family regulator, whose gene is part of the nagAB yvoA(nagR) operon. The gamAP operon is repressed by YbgA, another GntR family repressor, whose gene is expressed divergently from gamAP. The nagAB yvoA synton is found throughout the Bacilli and most firmicutes. On the other hand the ybgA-gamAP synton, which includes the ybgB gene for a small protein of unknown provenance, is only found in B. subtilis (and a few very close relatives). The origin of ybgBA-gamAP grouping is unknown but synteny analysis suggests lateral transfer from an unidentified donor. The presence of gamAP has enabled B. subtilis to efficiently use glucosamine as carbon source. PMID:23667565

Molecular Diversity of Plasmids Bearing Genes That Encode Toluene and Xylene Metabolism in Pseudomonas Strains Isolated from Different Contaminated Sites in Belarus

PubMed Central

Sentchilo, Vladimir S.; Perebituk, Alexander N.; Zehnder, Alexander J. B.; van der Meer, Jan Roelof

2000-01-01

Twenty different Pseudomonas strains utilizing m-toluate were isolated from oil-contaminated soil samples near Minsk, Belarus. Seventeen of these isolates carried plasmids ranging in size from 78 to about 200 kb (assigned pSVS plasmids) and encoding the meta cleavage pathway for toluene metabolism. Most plasmids were conjugative but of unknown incompatibility groups, except for one, which belonged to the IncP9 group. The organization of the genes for toluene catabolism was determined by restriction analysis and hybridization with xyl gene probes of pWW0. The majority of the plasmids carried xyl-type genes highly homologous to those of pWW53 and organized in a similar manner (M. T. Gallegos, P. A. Williams, and J. L. Ramos, J. Bacteriol. 179:5024–5029, 1997), with two distinguishable meta pathway operons, one upper pathway operon, and three xylS-homologous regions. All of these plasmids also possessed large areas of homologous DNA outside the catabolic genes, suggesting a common ancestry. Two other pSVS plasmids carried only one meta pathway operon, one upper pathway operon, and one copy each of xylS and xylR. The backbones of these two plasmids differed greatly from those of the others. Whereas these parts of the plasmids, carrying the xyl genes, were mostly conserved between plasmids of each group, the noncatabolic parts had undergone intensive DNA rearrangements. DNA sequencing of specific regions near and within the xylTE and xylA genes of the pSVS plasmids confirmed the strong homologies to the xyl genes of pWW53 and pWW0. However, several recombinations were discovered within the upper pathway operons of the pSVS plasmids and pWW0. The main genetic mechanisms which are thought to have resulted in the present-day configuration of the xyl operons are discussed in light of the diversity analysis carried out on the pSVS plasmids. PMID:10877777

A Coarse-Grained Biophysical Model of E. coli and Its Application to Perturbation of the rRNA Operon Copy Number

PubMed Central

Tadmor, Arbel D.; Tlusty, Tsvi

2008-01-01

We propose a biophysical model of Escherichia coli that predicts growth rate and an effective cellular composition from an effective, coarse-grained representation of its genome. We assume that E. coli is in a state of balanced exponential steady-state growth, growing in a temporally and spatially constant environment, rich in resources. We apply this model to a series of past measurements, where the growth rate and rRNA-to-protein ratio have been measured for seven E. coli strains with an rRNA operon copy number ranging from one to seven (the wild-type copy number). These experiments show that growth rate markedly decreases for strains with fewer than six copies. Using the model, we were able to reproduce these measurements. We show that the model that best fits these data suggests that the volume fraction of macromolecules inside E. coli is not fixed when the rRNA operon copy number is varied. Moreover, the model predicts that increasing the copy number beyond seven results in a cytoplasm densely packed with ribosomes and proteins. Assuming that under such overcrowded conditions prolonged diffusion times tend to weaken binding affinities, the model predicts that growth rate will not increase substantially beyond the wild-type growth rate, as indicated by other experiments. Our model therefore suggests that changing the rRNA operon copy number of wild-type E. coli cells growing in a constant rich environment does not substantially increase their growth rate. Other observations regarding strains with an altered rRNA operon copy number, such as nucleoid compaction and the rRNA operon feedback response, appear to be qualitatively consistent with this model. In addition, we discuss possible design principles suggested by the model and propose further experiments to test its validity. PMID:18437222

Awakening sleeping beauty: production of propionic acid in Escherichia coli through the sbm operon requires the activity of a methylmalonyl-CoA epimerase.

PubMed

Gonzalez-Garcia, Ricardo Axayacatl; McCubbin, Tim; Wille, Annalena; Plan, Manuel; Nielsen, Lars Keld; Marcellin, Esteban

2017-07-17

Propionic acid is used primarily as a food preservative with smaller applications as a chemical building block for the production of many products including fabrics, cosmetics, drugs, and plastics. Biological production using propionibacteria would be competitive against chemical production through hydrocarboxylation of ethylene if native producers could be engineered to reach near-theoretical yield and good productivity. Unfortunately, engineering propionibacteria has proven very challenging. It has been suggested that activation of the sleeping beauty operon in Escherichia coli is sufficient to achieve propionic acid production. Optimising E. coli production should be much easier than engineering propionibacteria if tolerance issues can be addressed. Propionic acid is produced in E. coli via the sleeping beauty mutase operon under anaerobic conditions in rich medium via amino acid degradation. We observed that the sbm operon enhances amino acids degradation to propionic acid and allows E. coli to degrade isoleucine. However, we show here that the operon lacks an epimerase reaction that enables propionic acid production in minimal medium containing glucose as the sole carbon source. Production from glucose can be restored by engineering the system with a methylmalonyl-CoA epimerase from Propionibacterium acidipropionici (0.23 ± 0.02 mM). 1-Propanol production was also detected from the promiscuous activity of the native alcohol dehydrogenase (AdhE). We also show that aerobic conditions are favourable for propionic acid production. Finally, we increase titre 65 times using a combination of promoter engineering and process optimisation. The native sbm operon encodes an incomplete pathway. Production of propionic acid from glucose as sole carbon source is possible when the pathway is complemented with a methylmalonyl-CoA epimerase. Although propionic acid via the restored succinate dissimilation pathway is considered a fermentative process, the engineered pathway was shown to be functional under anaerobic and aerobic conditions.

Transcription attenuation is the major mechanism by which the leu operon of Salmonella typhimurium is controlled.

PubMed

Searles, L L; Wessler, S R; Calvo, J M

1983-01-25

Three mutations, each causing constitutive expression of the Salmonella typhimurium leu operon, were cloned into phage vector lambda gt4 on EcoRI DNA fragments carrying all of that operon except for part of the promoter-distal last gene. Sequence analysis of DNA from these phage demonstrated that each contains a single base change in the leu attenuator. Transcription of mutant DNA in vitro resulted in transcription beyond the usual site of termination. The level of beta-IPM dehydrogenase, the leuB enzyme, was elevated 40-fold in a strain carrying one of these mutations, and starvation of this strain for leucine had little effect on the amount of activity expressed. Using a strain with a wild-type promoter-leader region of the leu operon, the rates of synthesis and degradation of leu leader RNA and readthrough RNA (leu mRNA) were measured by DNA-RNA hybridizations with specific DNA probes. The rate of synthesis of the leu leader was about the same in cells grown with excess or with limiting leucine. On the other hand, the rate of synthesis of leu mRNA was 12-fold higher for cells grown in limiting leucine as opposed to excess leucine. The rate of degradation of these RNA species was the same under both conditions of growth. Thus, the variation in expression of the leu operon observed for cells grown in minimal medium is, for the most part, not caused by control over the frequency of initiation or by the differential stability of these RNA species. Rather, the variation is a direct result of the frequency of transcription termination at an attenuator site. These results taken together suggest that transcription attenuation is the major mechanism by which leucine regulates expression of the leu operon of S. typhimurium for cells growing in a minimal medium.

Anti-inflammatory and Anti-oxidative Effects of Dexpanthenol on Lipopolysaccharide Induced Acute Lung Injury in Mice.

PubMed

Li-Mei, Wan; Jie, Tan; Shan-He, Wan; Dong-Mei, Meng; Peng-Jiu, Yu

2016-10-01

The aim of this study is to investigate the effects of dexpanthenol in a model of acute lung injury (ALI) induced by lipopolysaccharides (LPS). Lung injury was induced by exposure to atomized LPS. Mice were randomly divided into four groups: control group; Dxp (500 mg/kg) group; LPS group; LPS + Dxp (500 mg/kg) group. The effects of dexpanthenol on LPS-induced neutrophil recruitment, cytokine levels, total protein concentration, myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) contents were examined. Additionally, lung tissue was examined by histology to investigate the changes in pathology in the presence and absence of dexpanthenol. In LPS-challenged mice, dexpanthenol significantly improved lung edema. Dexpanthenol also markedly inhibited the LPS-induced neutrophiles influx, protein leakage, and release of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF). Furthermore, dexpanthenol attenuated MPO activity and MDA contents and increased SOD and GSH activity in the LPS-challenged lung tissue. These data suggest that dexpanthenol protects mice from LPS-induced acute lung injury by its anti-inflammatory and anti-oxidative activities.

Visualization and analysis of lipopolysaccharide distribution in binary phospholipid bilayers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henning, Maria Florencia; Sanchez, Susana; Bakas, Laura, E-mail: lbakas@biol.unlp.edu.ar

2009-05-22

Lipopolysaccharide (LPS) is an endotoxin released from the outer membrane of Gram-negative bacteria during infections. It have been reported that LPS may play a role in the outer membrane of bacteria similar to that of cholesterol in eukaryotic plasma membranes. In this article we compare the effect of introducing LPS or cholesterol in liposomes made of dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine on the solubilization process by Triton X-100. The results show that liposomes containing LPS or cholesterol are more resistant to solubilization by Triton X-100 than the binary phospholipid mixtures at 4 {sup o}C. The LPS distribution was analyzed on GUVs of DPPC:DOPC usingmore » FITC-LPS. Solid and liquid-crystalline domains were visualized labeling the GUVs with LAURDAN and GP images were acquired using a two-photon microscope. The images show a selective distribution of LPS in gel domains. Our results support the hypothesis that LPS could aggregate and concentrate selectively in biological membranes providing a mechanism to bring together several components of the LPS-sensing machinery.« less

Light Spectral Quality Effects on the Growth of Potato (Solanum Tuberosum L.) Nodal Cuttings in Vitro

NASA Technical Reports Server (NTRS)

Wilson, Deborah A.; Weigel, Russell C.; Wheeler, Raymond M.; Sager, John C.

1993-01-01

The effects of light spectral quality on the growth of in vitro nodal cuttings of potato (Solanum tuberosum L.) cultivars Norland, Superior, Kennebec, and Denali were examined. The different light spectra were provided by Vita-Lite fluorescent (VF) (a white light control), blue fluorescent (BF), red fluorescent (RF), low-pressure sodium (LPS), and a combination of low-pressure sodium plus cool-white fluorescent lamps (LPS/CWF). For cultivars, stem lengths after 4 wks were longest under LPS, follow by RF, LPS/CWF, VF, and BF (in descending order). Microscopic studies revealed that cells were shortest when cultured in BF or VF environments, and were longest in RF or LPS lamp environments. The highest number axillary branches occurred on plantlets grown with LPS or LPS/CWF, whereas the lowest number occurred with BF. No leaf or stem edema (callus or gall-like growths) occurred iwth LPS or LPS/cwf lighting, and no edema occurred on cv. Norland plantlets, regardless of lighting. Results suggest that shoot morphologic development of in vitro grown potato plants can be controlled by controlling irradiant spectral quality.

Endothelial expression of selectins during endotoxin preconditioning.

PubMed

Bauer, P; Welbourne, T; Shigematsu, T; Russell, J; Granger, D N

2000-12-01

Although bacterial endotoxins [lipopolysaccharide (LPS)] can confer tissue resistance to subsequent inflammatory insults, the mechanisms that underlie this LPS-preconditioning (LPS-PC) response remain poorly defined. The dual-radiolabeled monoclonal antibody technique was used to examine whether LPS-PC alters the upregulation (protein) of E- and P-selectins after subsequent LPS challenge. In the gut of wild-type (C57BL/6J) mice, LPS-PC was associated with a reduction in E- (66%) and P-selectin (33%) expression. A similar reduction in E-selectin expression was observed in mutant mice that were genetically deficient in either the endothelial or inducible isoform of nitric oxide synthase or that overexpressed the human gene for Cu/Zn superoxide dismutase. Severe combined immunodeficient mice, genetically devoid of lymphocytes, did exhibit partial inhibition of the LPS-PC response. We conclude that 1) LPS-PC can be demonstrated for E- and P-selectins in some vascular beds (e.g., gut), 2) the mechanism(s) underlying this blunted selectin response does not include a major role for either nitric oxide and superoxide, and 3) circulating lymphocytes may contribute to the LPS-PC response.

The Role of RaxST, a Prokaryotic Sulfotransferase, and RaxABC, a Putative Type I Secretion System, in Activation of the Rice XA21-Mediated Immune Response

PubMed Central

Ronald, Pamela C.

2014-01-01

Tyrosine sulfation is an important posttranslational modification that determines the outcome of serious diseases in plants and animals. We have recently demonstrated that the plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) carries a functional sulfotransferase (RaxST). raxST is required for activation of rice Xa21-mediated immunity indicating the critical, but unknown, function of raxST in mediating the Xoo/rice interaction. The raxST gene resides in the same operon (raxSTAB) as components of a predicted type I secretion and processing system (RaxA and RaxB). These observations suggest a model where RaxST sulfates a molecule that contains a leader peptide, which is cleaved by the peptidase domain of the RaxB protein and secreted outside the bacterial cell by the RaxABC T1SS. PMID:25386383

The Role of RaxST, a Prokaryotic Sulfotransferase, and RaxABC, a Putative Type I Secretion System, in Activation of the Rice XA21-Mediated Immune Response.

PubMed

Ronald, Pamela C

2014-01-01

Tyrosine sulfation is an important posttranslational modification that determines the outcome of serious diseases in plants and animals. We have recently demonstrated that the plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) carries a functional sulfotransferase (RaxST). raxST is required for activation of rice Xa21-mediated immunity indicating the critical, but unknown, function of raxST in mediating the Xoo/rice interaction. The raxST gene resides in the same operon (raxSTAB) as components of a predicted type I secretion and processing system (RaxA and RaxB). These observations suggest a model where RaxST sulfates a molecule that contains a leader peptide, which is cleaved by the peptidase domain of the RaxB protein and secreted outside the bacterial cell by the RaxABC T1SS.

LPS Increases 5-LO Expression on Monocytes via an Activation of Akt-Sp1/NF-κB Pathways.

PubMed

Lee, Seung Jin; Seo, Kyo Won; Kim, Chi Dae

2015-05-01

5-Lipoxygenase (5-LO) plays a pivotal role in the progression of atherosclerosis. Therefore, this study investigated the molecular mechanisms involved in 5-LO expression on monocytes induced by LPS. Stimulation of THP-1 monocytes with LPS (0~3 µg/ml) increased 5-LO promoter activity and 5-LO protein expression in a concentration-dependent manner. LPS-induced 5-LO expression was blocked by pharmacological inhibition of the Akt pathway, but not by inhibitors of MAPK pathways including the ERK, JNK, and p38 MAPK pathways. In line with these results, LPS increased the phosphorylation of Akt, suggesting a role for the Akt pathway in LPS-induced 5-LO expression. In a promoter activity assay conducted to identify transcription factors, both Sp1 and NF-κB were found to play central roles in 5-LO expression in LPS-treated monocytes. The LPS-enhanced activities of Sp1 and NF-κB were attenuated by an Akt inhibitor. Moreover, the LPS-enhanced phosphorylation of Akt was significantly attenuated in cells pretreated with an anti-TLR4 antibody. Taken together, 5-LO expression in LPS-stimulated monocytes is regulated at the transcriptional level via TLR4/Akt-mediated activations of Sp1 and NF-κB pathways in monocytes.

Doxycycline Attenuates Lipopolysaccharide-Induced Microvascular Endothelial Cell Derangements.

PubMed

Wiggins-Dohlvik, Katie; Stagg, Hayden W; Han, Min Suk; Alluri, Himakarnika; Oakley, Ryan P; Anasooya Shaji, Chinchusha; Davis, Matthew L; Tharakan, Binu

2016-06-01

Lipopolysaccharide (LPS) is known to induce vascular derangements. The pathophysiology involved therein is unknown, but matrix metalloproteinases (MMPs) may be an important mediator. We hypothesized that in vitro LPS provokes vascular permeability, damages endothelial structural proteins, and increases MMP activity; that in vivo LPS increases permeability and fluid requirements; and that the MMP inhibitor doxycycline mitigates such changes. Rat lung microvascular endothelial cells were divided into four groups: control, LPS, LPS plus doxycycline, and doxycycline. Permeability, structural proteins β-catenin and Filamentous-actin, and MMP-9 activity were examined. Sprauge Dawley rats were divided into sham, IV LPS, and IV LPS plus IV doxycycline groups. Mesenteric postcapillary venules were observed. Blood pressure was measured as animals were resuscitated and fluid requirements were compared. Statistical analysis was conducted using Student's t-test and ANOVA. In vitro LPS increased permeability, damaged adherens junctions, induced actin stress fiber formation, and increased MMP-9 enzyme activity. In vivo, IV LPS administration induced vascular permeability. During resuscitation, significantly more fluid was necessary to maintain normotension in the IV LPS group. Doxycycline mitigated all derangements observed. We conclude that LPS increases permeability, damages structural proteins, and increases MMP-9 activity in endothelial cells. Additionally, endotoxemia induces hyperpermeability and increases the amount of IV fluid required to maintain normotension in vivo. Doxycycline mitigates such changes both in vitro and in vivo. Our findings illuminate the possible role of matrix metalloproteinases in the pathophysiology of lipopolysaccharide-induced microvascular hyperpermeability and pave the way for better understanding and treatment of this process.

Inhibition of LPS toxicity by hepatic argininosuccinate synthase (ASS): novel roles for ASS in innate immune responses to bacterial infection.

PubMed

Prima, Victor; Wang, Alvin; Molina, Gabriel; Wang, Kevin K W; Svetlov, Stanislav I

2011-09-01

Lipopolysaccharide (LPS), a structural component of Gram-negative bacteria, is implicated in the pathogenesis of endotoxemia/sepsis and multi-organ injury, including liver damage. We have shown that argininosuccinate synthase (ASS), a hepatic enzyme of the urea cycle, accumulates in circulation within 1h after treatment with both LPS alone and hepatotoxic combination of LPS and D-Galactosamine. These findings indicate ASS as a sensitive biomarker of liver responses to bacterial endotoxin. Furthermore, we suggest that the ASS release represents a potential counteracting liver reaction to LPS, and demonstrates anti-LPS activity of recombinant ASS (rASS) in vitro and in rodent models of endotoxemia in vivo. rASS physically bound to LPS, as indicated by a gel shift assay, and suppressed Escherichia coli growth in cultures consistent with direct antimicrobial properties of ASS. rASS reduced LPS cytotoxicity, TNF-α production, and increased cell viability in cultured mouse macrophages, even when added one hour following LPS challenge. Intraperitoneal injection of rASS (5 mg/kg) after treatment with a high dose of LPS remarkably increased survival of rodents, with a concomitant decrease of sepsis markers TNF-α, C-reactive protein (CRP), and lactate dehydrogenase (LDH) levels in blood. These results suggest that the endogenous ASS constitutes a novel liver-derived component of the innate immune response to bacterial LPS, and that recombinant ASS could mitigate the lethal effects of bacterial endotoxins during sepsis. Copyright © 2011 Elsevier B.V. All rights reserved.

A central role for the mammalian target of rapamycin in LPS-induced anorexia in mice.

PubMed

Yue, Yunshuang; Wang, Yi; Li, Dan; Song, Zhigang; Jiao, Hongchao; Lin, Hai

2015-01-01

Bacterial lipopolysaccharide (LPS), also known as endotoxin, induces profound anorexia. However, the LPS-provoked pro-inflammatory signaling cascades and the neural mechanisms underlying the development of anorexia are not clear. Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. This study aimed to determine whether the mTOR pathway is involved in LPS-induced anorexia. Effects of LPS on hypothalamic gene/protein expression in mice were measured by RT-PCR or western blotting analysis. To determine whether inhibition of mTOR signaling could attenuate LPS-induced anorexia, we administered an i.c.v. injection of rapamycin, an mTOR inhibitor, on LPS-treated male mice. In this study, we showed that LPS stimulates the mTOR signaling pathway through the enhanced phosphorylation of mTOR(Ser2448) and p70S6K(Thr389). We also showed that LPS administration increased the phosphorylation of FOXO1(Ser256), the p65 subunit of nuclear factor kappa B (P<0.05), and FOXO1/3a(Thr) (24) (/) (32) (P<0.01). Blocking the mTOR pathway significantly attenuated the LPS-induced anorexia by decreasing the phosphorylation of p70S6K(Thr389), FOXO1(Ser256), and FOXO1/3a(Thr) (24) (/) (32). These results suggest promising approaches for the prevention and treatment of LPS-induced anorexia. © 2015 Society for Endocrinology.

The civRT operon is important for Campylobacter jejuni strain 81-176 host cell interactions through regulation of the formate dehydrogenase operon

USDA-ARS?s Scientific Manuscript database

C. jejuni colonizes the intestinal mucosa, and the severity of disease in different strains is correlated with host cell interaction and invasion. A microarray screen to identify genes differentially regulated during C. jejuni interaction with tissue culture cells revealed the up-regulation of a two...

Heterologous expression of the Desulfovibrio gigas [NiFe] hydrogenase in Desulfovibrio fructosovorans MR400.

PubMed

Rousset, M; Magro, V; Forget, N; Guigliarelli, B; Belaich, J P; Hatchikian, E C

1998-09-01

The ability of Desulfovibrio fructosovorans MR400 DeltahynABC to express the heterologous cloned [NiFe] hydrogenase of Desulfovibrio gigas was investigated. The [NiFe] hydrogenase operon from D. gigas, hynABCD, was cloned, sequenced, and introduced into D. fructosovorans MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native D. gigas protein. A chimeric operon containing hynAB from D. gigas and hynC from D. fructosovorans placed under the control of the D. fructosovorans hynAp promoter was constructed and expressed in D. fructosovorans MR400. Under these conditions, the same level of activity was obtained as with the D. gigas hydrogenase operon.

Participation of S. Typhimurium cysJIH Operon in the H2S-mediated Ciprofloxacin Resistance in Presence of Sulfate as Sulfur Source

PubMed Central

Álvarez, Ricardo; Frávega, Jorge; Rodas, Paula I.; Fuentes, Juan A.; Paredes-Sabja, Daniel; Calderón, Iván L.; Gil, Fernando

2015-01-01

H2S production has been proposed as a mechanism to explain bacterial resistance to antibiotics. In this work, we present evidence for the role of the cysJIH operon in resistance to ciprofloxacin mediated by H2S production with different sulfate as the only sulfur source. We found that the products of the cysJIH operon are involved in ciprofloxacin resistance by increasing both, the levels of H2S and reduced thiols apparently counteracting antimicrobial-induced reactive oxygen species (ROS). This protective effect was observed only when bacteria were cultured in the presence of sulfate, but not with cysteine, as the sole sulfur source.

Effects of betaine on lipopolysaccharide-induced memory impairment in mice and the involvement of GABA transporter 2

PubMed Central

2011-01-01

Background Betaine (glycine betaine or trimethylglycine) plays important roles as an osmolyte and a methyl donor in animals. While betaine is reported to suppress expression of proinflammatory molecules and reduce oxidative stress in aged rat kidney, the effects of betaine on the central nervous system are not well known. In this study, we investigated the effects of betaine on lipopolysaccharide (LPS)-induced memory impairment and on mRNA expression levels of proinflammatory molecules, glial markers, and GABA transporter 2 (GAT2), a betaine/GABA transporter. Methods Mice were continuously treated with betaine for 13 days starting 1 day before they were injected with LPS, or received subacute or acute administration of betaine shortly before or after LPS injection. Then, their memory function was evaluated using Y-maze and novel object recognition tests 7 and 10-12 days after LPS injection (30 μg/mouse, i.c.v.), respectively. In addition, mRNA expression levels in hippocampus were measured by real-time RT-PCR at different time points. Results Repeated administration of betaine (0.163 mmol/kg, s.c.) prevented LPS-induced memory impairment. GAT2 mRNA levels were significantly increased in hippocampus 24 hr after LPS injection, and administration of betaine blocked this increase. However, betaine did not affect LPS-induced increases in levels of mRNA related to inflammatory responses. Both subacute administration (1 hr before, and 1 and 24 hr after LPS injection) and acute administration (1 hr after LPS injection) of betaine also prevented LPS-induced memory impairment in the Y-maze test. Conclusions These data suggest that betaine has protective effects against LPS-induced memory impairment and that prevention of LPS-induced changes in GAT2 mRNA expression is crucial to this ameliorating effect. PMID:22053950

Usefulness of ELISA Methods for Assessing LPS Interactions with Proteins and Peptides

PubMed Central

Martínez-Sernández, Victoria; Orbegozo-Medina, Ricardo A.; Romarís, Fernanda; Paniagua, Esperanza; Ubeira, Florencio M.

2016-01-01

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, can trigger severe inflammatory responses during bacterial infections, possibly leading to septic shock. One approach to combatting endotoxic shock is to neutralize the most conserved part and major mediator of LPS activity (lipid A) with LPS-binding proteins or peptides. Although several available assays evaluate the biological activity of these molecules on LPS (e.g. inhibition of LPS-induced TNF-α production in macrophages), the development of simple and cost-effective methods that would enable preliminary screening of large numbers of potential candidate molecules is of great interest. Moreover, it would be also desirable that such methods could provide information about the possible biological relevance of the interactions between proteins and LPS, which may enhance or neutralize LPS-induced inflammatory responses. In this study, we designed and evaluated different types of ELISA that could be used to study possible interactions between LPS and any protein or peptide. We also analysed the usefulness and limitations of the different ELISAs. Specifically, we tested the capacity of several proteins and peptides to bind FITC-labeled LPSs from Escherichia coli serotypes O111:B4 and O55:B5 in an indirect ELISA and in two competitive ELISAs including casein hydrolysate (hCAS) and biotinylated polymyxin B (captured by deglycosylated avidin; PMX) as LPS-binding agents in the solid phase. We also examined the influence of pH, detergents and different blocking agents on LPS binding. Our results showed that the competitive hCAS-ELISA performed under mildly acidic conditions can be used as a general method for studying LPS interactions, while the more restrictive PMX-ELISA may help to identify proteins/peptides that are likely to have neutralizing properties in vitro or in vivo. PMID:27249227

Contribution of sensory nerves to LPS-induced hyperresponsiveness of human isolated bronchi.

PubMed

Calzetta, Luigino; Luongo, Livio; Cazzola, Mario; Page, Clive; Rogliani, Paola; Facciolo, Francesco; Maione, Sabatino; Capuano, Annalisa; Rinaldi, Barbara; Matera, Maria Gabriella

2015-06-15

Bacterial lipopolysaccharide (LPS) can induce bronchial hyperresponsiveness (BHR), but the underlying mechanisms remain to be determined. Here, the possible contribution of sensory nerves to LPS-induced BHR was examined in human isolated bronchi to pharmacologically identify the mechanisms underlying this phenomenon. Human isolated bronchial tone was induced by electrical field stimulation (EFS). The responses of airways to LPS, with or without capsaicin desensitization or thiorphan treatment were studied and the transient receptor potential vanilloid type 1 (TRPV1) expression was assessed. We performed similar experiments in the presence of a TRPV1 or a neurokinin (NK) 2 receptor antagonist using SB366791 and GR159897, respectively. LPS increased (≃2.3-fold, P<0.001) the contraction induced by EFS, compared to control tissues. Acute administration of capsaicin enhanced (≃2.3-fold, P<0.001) the EFS-mediated contraction, but did not potentiate the effect of LPS. Thiorphan increased (≃1.3-fold, P<0.05) the contractile response of LPS treated tissues and, at lower frequencies, it enhanced (≃1.7-fold, P<0.001) the capsaicin-induced contraction. In capsaicin-desensitized bronchi, LPS did not modify (P>0.05) the EFS contractile response, nor after treatment with thiorphan. Capsaicin desensitization reduced (≃0.4-fold, P<0.001) the LPS-induced BHR. SB366791 and GR159897 prevented the LPS-induced BHR and the release of NKA. LPS increased (+85.3±9.5%, P<0.01) the surface membrane expression of TRPV1 in parasympathetic ganglia. Our results demonstrate the involvement of capsaicin-sensitive sensory nerves and neutral endopeptidases in LPS-induced BHR of the human bronchi, associated with an upregulation of TRPV1 and release of NKA. Copyright © 2015. Published by Elsevier Inc.

Prenatal exposure to lipopolysaccharide combined with pre- and postnatal high-fat diet result in lowered blood pressure and insulin resistance in offspring rats.

PubMed

Hao, Xue-Qin; Du, Jing-Xia; Li, Yan; Li, Meng; Zhang, Shou-Yan

2014-01-01

Adult metabolic syndrome may in part have origins in fetal or early life. This study was designed to explore the effect of prenatal exposure to lipopolysaccharide and high-fat diet on metabolic syndrome in offspring rats. 32 pregnant rats were randomly divided into four groups, including Control group; LPS group (pregnant rats were injected with LPS 0.4 mg/kg intraperitoneally on the 8(th), 10(th) and 12(th) day of pregnancy); High-fat group (maternal rats had high-fat diet during pregnancy and lactation period, and their pups also had high-fat diet up to the third month of life); LPS + High-fat group (rats were exposed to the identical experimental scheme with LPS group and High-fat group). Blood pressure elevated in LPS group and High-fat group, reduced in LPS+High-fat group, accompanied by the increase of serum leptin level in LPS and High-fat group and increase of serum IL-6, TNF-a in High-fat group; both serum insulin and cholesterol increased in High-fat and LPS+High-fat group, as well as insulin in LPS group. HOMA-IR value increased in LPS, High-fat and LPS+High-fat group, and QUICKI decreased in these groups; H-E staining showed morphologically pathological changes in thoracic aorta and liver tissue in the three groups. Increased serum alanine and aspartate aminotransferase suggest impaired liver function in LPS+High-fat group. Prenatal exposure to lipopolysaccharide combined with pre- and postnatal high-fat diet result in lowered blood pressure, insulin resistance and impaired liver function in three-month old offspring rats. The lowered blood pressure might benefit from the predictive adaptive response to prenatal inflammation.

The critical role played by endotoxin-induced liver autophagy in the maintenance of lipid metabolism during sepsis.

PubMed

Chung, Ki Wung; Kim, Kyung Mok; Choi, Yeon Ja; An, Hye Jin; Lee, Bonggi; Kim, Dae Hyun; Lee, Eun Kyeong; Im, Eunok; Lee, Jaewon; Im, Dong Soon; Yu, Byung Pal; Chung, Hae Young

2017-07-03

Macroautophagy/autophagy is a central mechanism by which cells maintain integrity and homeostasis, and endotoxin-induced autophagy plays important roles in innate immunity. Although TLR4 stimulation mediated by lipopolysaccharide (LPS) also upregulates autophagy in hepatocytes and liver, its physiological role remains elusive. The objective of this study was to determine the role of LPS-induced autophagy in the regulation of liver lipid metabolism. LPS treatment (5 mg/kg) increased autophagy, as detected by LC3 conversion and transmission electron microscopy (TEM) analysis in C57BL6 mouse livers. AC2F hepatocytes also showed increased autophagic flux after LPS treatment (1 μg/ml). To investigate the role of LPS-induced autophagy further, liver lipid metabolism changes in LPS-treated mice and fasted controls were compared. Interestingly, LPS-treated mice showed less lipid accumulation in liver than fasted mice despite increased fatty acid uptake and lipid synthesis-associated genes. In vitro analysis using AC2F hepatocytes demonstrated LPS-induced autophagy influenced the degradation of lipid droplets. Inhibition of LPS-induced autophagy using bafilomycin A 1 or Atg7 knockdown significantly increased lipid accumulation in AC2F hepatocytes. In addition, pretreatment with chloroquine aggravated LPS-induced lipid accumulation and inflammation in C57BL6 mouse livers. The physiological importance of autophagy was verified in LPS-treated young and aged rats. Autophagic response was diminished in LPS-treated aged rats and lipid metabolism was impaired during sepsis, indicating autophagy response is important for regulating lipid metabolism after endotoxin challenge. Our findings demonstrate endotoxin-induced autophagy is important for the regulation of lipid metabolism, and suggest that autophagy helps maintain lipid metabolism homeostasis during sepsis.

CD14 is a key mediator of both lysophosphatidic acid and lipopolysaccharide induction of foam cell formation.

PubMed

An, Dong; Hao, Feng; Zhang, Fuqiang; Kong, Wei; Chun, Jerold; Xu, Xuemin; Cui, Mei-Zhen

2017-09-01

Macrophage uptake of oxidized low-density lipoprotein (oxLDL) plays an important role in foam cell formation and the pathogenesis of atherosclerosis. We report here that lysophosphatidic acid (LPA) enhances lipopolysaccharide (LPS)-induced oxLDL uptake in macrophages. Our data revealed that both LPA and LPS highly induce the CD14 expression at messenger RNA and protein levels in macrophages. The role of CD14, one component of the LPS receptor cluster, in LPA-induced biological functions has been unknown. We took several steps to examine the role of CD14 in LPA signaling pathways. Knockdown of CD14 expression nearly completely blocked LPA/LPS-induced oxLDL uptake in macrophages, demonstrating for the first time that CD14 is a key mediator responsible for both LPA- and LPS-induced oxLDL uptake/foam cell formation. To determine the molecular mechanism mediating CD14 function, we demonstrated that both LPA and LPS significantly induce the expression of scavenger receptor class A type I (SR-AI), which has been implicated in lipid uptake process, and depletion of CD14 levels blocked LPA/LPS-induced SR-AI expression. We further showed that the SR-AI-specific antibody, which quenches SR-AI function, blocked LPA- and LPS-induced foam cell formation. Thus, SR-AI is the downstream mediator of CD14 in regulating LPA-, LPS-, and LPA/LPS-induced foam cell formation. Taken together, our results provide the first experimental evidence that CD14 is a novel connecting molecule linking both LPA and LPS pathways and is a key mediator responsible for LPA/LPS-induced foam cell formation. The LPA/LPS-CD14-SR-AI nexus might be the new convergent pathway, contributing to the worsening of atherosclerosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Duration of in vivo endotoxin tolerance in horses.

PubMed

Holcombe, Susan J; Jacobs, Carrie C; Cook, Vanessa L; Gandy, Jeffery C; Hauptman, Joseph G; Sordillo, Lorraine M

2016-05-01

Endotoxemia models are used to study mechanisms and treatments of early sepsis. Repeated endotoxin exposures induce periods of endotoxin tolerance, characterized by diminished proinflammatory responses to lipopolysaccharide (LPS) and modulated production of proinflammatory cytokines. Repeated measure designs using equine endotoxemia models are rarely performed, despite the advantages associated with reduced variability, because the altered responsiveness would confound study results and because the duration of equine endotoxin tolerance is unknown. We determined the interval of endotoxin tolerance, in vivo, in horses based on physical, clinicopathologic, and proinflammatory gene expression responses to repeated endotoxin exposures. Six horses received 30 ng/kg LPS in saline infused over 30 min. Behavior pain scores, physical examination parameters, and blood for complete blood count and proinflammatory gene expression were obtained at predetermined intervals for 24h. Horses received a total of 3 endotoxin exposures. The first exposure was LPS 1, followed 7 days later by LPS 7 or 14-21 days later by LPS 14-21. Lipopolysaccharide exposures were allocated in a randomized, crossover design. Lipopolysaccharide produced clinical and clinicopathologic signs of endotoxemia and increased expression of tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and IL-8, P<0.001. Horses exhibited evidence of endotoxin tolerance following LPS 7 but not following LPS 14-21. Horses had significantly lower pain scores, heart rates, respiratory rates and duration of fever, after LPS 7 compared to LPS 1 and LPS 14-21, P<0.001, and expression of TNFα was lower in the whole blood of horses after LPS 7, P=0.05. Clinical parameters and TNFα gene expression were similar or slightly increased in horses following LPS 14-21 compared to measurements made in horses following LPS 1, suggesting that endotoxin tolerance had subsided. A minimum of 3 weeks between experiments is warranted if repeated measures designs are used to assess in vivo response to endotoxin in horses. Copyright © 2016 Elsevier B.V. All rights reserved.

Magnesium sulfate differentially modulates fetal membrane inflammation in a time-dependent manner.

PubMed

Cross, Sarah N; Nelson, Rachel A; Potter, Julie A; Norwitz, Errol R; Abrahams, Vikki M

2018-04-30

Chorioamnionitis and infection-associated inflammation are major causes of preterm birth. Magnesium sulfate (MgSO 4 ) is widely used in obstetrics as a tocolytic; however, its mechanism of action is unclear. This study sought to investigate how MgSO 4 modulates infection-associated inflammation in fetal membranes (FMs), and whether the response was time dependent. Human FM explants were treated with or without bacterial lipopolysaccharide (LPS); with or without MgSO 4 added either: 1 hour before LPS; at the same time as LPS; 1 hour post-LPS; or 2 hours post-LPS. Explants were also treated with or without viral dsRNA and LPS, alone or in combination; and MgSO 4 added 1 hour post-LPS After 24 hours, supernatants were measured for cytokines/chemokines; and tissue lysates measured for caspase-1 activity. Lipopolysaccharide-induced FM inflammation by upregulating the secretion of a number of inflammatory cytokines/chemokines. Magnesium sulfate administered 1-hour post-LPS inhibited FM secretion of IL-1β, IL-6, G-CSF, RANTES, and TNFα. Magnesium sulfate administered 2 hours post-LPS augmented FM secretion of these factors as well as IL-8, IFNγ, VEGF, GROα and IP-10. Magnesium sulfate delivered 1- hour post-LPS inhibited LPS-induced caspase-1 activity, and inhibited the augmented IL-1β response triggered by combination viral dsRNA and LPS. Magnesium sulfate differentially modulates LPS-induced FM inflammation in a time-dependent manner, in part through its modulation of caspase-1 activity. Thus, the timing of MgSO 4 administration may be critical in optimizing its anti-inflammatory effects in the clinical setting. MgSO 4 might also be useful at preventing FM inflammation triggered by a polymicrobial viral-bacterial infection. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bacillus amyloliquefaciens supplementation alleviates immunological stress in lipopolysaccharide-challenged broilers at early age.

PubMed

Li, Y; Zhang, H; Chen, Y P; Yang, M X; Zhang, L L; Lu, Z X; Zhou, Y M; Wang, T

2015-07-01

This study was conducted to investigate the effect of Bacillus amyloliquefaciens ( BA: ) on the immune function of broilers challenged with lipopolysaccharide ( LPS: ). 192 one-day-old male Arbor Acre broiler chickens were randomly distributed into four treatments: 1) broilers fed a basal diet; 2) broilers fed a basal diet supplemented with BA; 3) LPS-challenged broilers fed a basal diet; and 4) LPS-challenged broilers fed a basal diet supplemented with BA. Each treatment consisted of six replicates with eight broilers per replicate. Broilers were intraperitoneally injected with either 500 μg LPS per kg body weight or sterile saline at 16, 18 and 20 d of age. LPS decreased the average daily gain ( ADG: , P = 0.001) and average daily feed intake (P = 0.001). The decreased ADG (P = 0.009) and increased feed conversion ratio (P = 0.047) in LPS-challenged broilers were alleviated by BA. LPS increased the relative spleen weight (P = 0.001). Relative spleen (P = 0.014) and bursa (P = 0.024) weights in the LPS-challenged broilers were reduced by BA. LPS increased white blood cell ( WBC: ) numbers (P = 0.001). However, the WBC numbers (P = 0.042) and the ratio of lymphocytes to WBC (P = 0.020) in LPS-challenged broilers were decreased with BA treatment. LPS decreased plasma lysozyme activity (P = 0.001), but increased concentrations of plasma corticosterone (P = 0.012) and IL-2 (P = 0.020). In contrast, BA increased lysozyme activity in plasma (P = 0.040). LPS increased mRNA abundances of splenic toll-like receptor 4 (P = 0.046), interferon γ (P = 0.008), IL-1β (P = 0.045) and IL-6, (P = 0.006). IL-2 (P = 0.014) and IL-6 (P = 0.074) mRNA abundances in LPS-challenged broilers were reduced by BA, although BA had an opposite effect for IL-10 mRNA expression in those broilers (P = 0.004). In conclusion, BA supplementation could partially alleviate the compromised growth performance and immune status of broilers under immune stress induced by LPS challenge at early age. © 2015 Poultry Science Association Inc.

Peripheral and central mediators of lipopolysaccharide induced suppression of defensive rage behavior in the cat.

PubMed

Bhatt, S; Bhatt, R S; Zalcman, S S; Siegel, A

2009-11-10

Based upon recent findings in our laboratory that cytokines microinjected into the medial hypothalamus or periaqueductal gray (PAG) powerfully modulate defensive rage behavior in cat, the present study determined the effects of peripherally released cytokines following lipopolysaccharide (LPS) challenge upon defensive rage. The study involved initial identification of the effects of peripheral administration of LPS upon defensive rage by electrical stimulation from PAG and subsequent determination of the peripheral and central mechanisms governing this process. The results revealed significant elevation in response latencies for defensive rage from 60 to 300 min, post LPS injection, with no detectable signs of sickness behavior present at 60 min. In contrast, head turning behavior elicited by stimulation of adjoining midbrain sites was not affected by LPS administration, suggesting a specificity of the effects of LPS upon defensive rage. Direct administration of LPS into the medial hypothalamus had no effect on defensive rage, suggesting that the effects of LPS were mediated by peripheral cytokines rather than by any direct actions upon hypothalamic neurons. Complete blockade of the suppressive effects of LPS by peripheral pretreatment with an Anti-tumor necrosis factor-alpha (TNFalpha) antibody but not with an anti- interleukin-1 (IL-1) antibody demonstrated that the effects of LPS were mediated through TNF-alpha rather than through an IL-1 mechanism. A determination of the central mechanisms governing LPS suppression revealed that pretreatment of the medial hypothalamus with PGE(2) or 5-HT(1A) receptor antagonists each completely blocked the suppressive effects of LPS, while microinjections of a TNF-alpha antibody into the medial hypothalamus were ineffective. Microinjections of -Iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) benzamide monohydrochloride (p-MPPI) into lateral hypothalamus (to test for anatomical specificity) had no effect upon LPS induced suppression of defensive rage. The results demonstrate that LPS suppresses defensive rage by acting through peripheral TNF-alpha in periphery and that central effects of LPS suppression of defensive rage are mediated through PGE(2) and 5-HT(1A) receptors in the medial hypothalamus.

Control of lipopolysaccharide biosynthesis and release by Escherichia coli and Salmonella typhimurium.

PubMed Central

Ishiguro, E E; Vanderwel, D; Kusser, W

1986-01-01

The influence of the relA gene on lipopolysaccharide (LPS) biosynthesis and release by Escherichia coli and Salmonella typhimurium was investigated. Similar results were obtained with both species. The incorporation of [3H]galactose into LPS by galE mutants was inhibited by at least 50% (as compared with normal growing controls) during amino acid deprivation of relA+ strains. This inhibition could be prevented by the treatment of the amino acid-deprived relA+ bacteria with chloramphenicol, a known antagonist of the stringent control mechanism. Furthermore, LPS biosynthesis was not inhibited during amino acid deprivation of isogenic relA mutant strains. These results indicate that LPS synthesis is regulated by the stringent control mechanism. Normal growing cells of both relA+ and relA strains released LPS into the culture fluid at low rates. Amino acid deprivation stimulated the rate of LPS release by relA mutants but not by relA+ bacteria. Chloramphenicol treatment markedly stimulated the release of cell-bound LPS by amino acid-deprived relA+ cells. Thus, a low rate of LPS release was characteristic of normal growth and could be increased in nongrowing cells by relaxing the control of LPS synthesis. Images PMID:3531174

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

PubMed Central

Arunmanee, Wanatchaporn; Pathania, Monisha; Solovyova, Alexandra S.; Le Brun, Anton P.; Ridley, Helen; Baslé, Arnaud; van den Berg, Bert; Lakey, Jeremy H.

2016-01-01

The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin–LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin–LPS interactions and a bridging calcium ion. PMID:27493217

Lycorine inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in RAW264.7 cells through suppressing P38 and STATs activation and increases the survival rate of mice after LPS challenge.

PubMed

Kang, Jingjing; Zhang, Yushun; Cao, Xiang; Fan, Jie; Li, Guilan; Wang, Qi; Diao, Ying; Zhao, Zhihui; Luo, Lan; Yin, Zhimin

2012-01-01

As a natural alkaloid extracted from Amaryllidaceae, lycorine shows various biological effects on tumor cells. Here we show that lycorine dose-dependently inhibited the LPS-induced up-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein level in RAW264.7 cells. Besides, it also inhibited NO, PGE(2), TNF-α and IL-6 release from LPS-treated RAW264.7 cells. RT-PCR experiments showed that lycorine suppressed LPS-induced iNOS but not COX-2 gene expression. Moreover, lycorine decreased LPS-induced mortality in mice. Mechanistically, LPS-induced activation of P38 and STATs pathways was suppressed significantly by lycorine. In addition, lycorine did not interfere with the phosphorylation of ERK1/2, JNK1/2 and NF-κB pathways. In conclusion, lycorine inhibits LPS-induced production of pro-inflammatory mediators and increases the survival rate of mice after LPS challenge, suggesting that lycorine could play an anti-inflammatory role in response to LPS. Copyright © 2011 Elsevier B.V. All rights reserved.

The role of lipopolysaccharide on the electrochemical behavior of titanium.

PubMed

Barão, V A; Mathew, M T; Assunção, W G; Yuan, J C; Wimmer, M A; Sukotjo, C

2011-05-01

Lipopolysaccharide (LPS) may induce peri-implantitis and implant failure. However, the role of LPS in titanium (Ti) electrochemical behavior remains unknown. We hypothesized that LPS in saliva with different pHs affects Ti corrosion properties. Thirty-six Ti discs (15 mm × 3 mm) were divided into 12 groups according to saliva pH (3, 6.5, and 9) and Escherichia coli LPS concentration (0, 0.15, 15, and 150 µg/mL). Electrochemical tests, such as open circuit potential, potentiodynamic, and electrochemical impedance spectroscopy, were conducted in a controlled environment. Data were evaluated by Pearson correlation and regression analysis (α = 0.05). LPS and pH affected Ti corrosive behavior. In general, lower pH and higher LPS concentration accelerated Ti corrosion. In the control group, the increase of pH significantly reduced the corrosion rate and increased the capacitance of the double layer. In LPS groups, the decrease of pH significantly increased the corrosion rate of Ti. LPS negatively influenced Ti corrosion behavior. C(dl), capacitance of double layer; E(corr), corrosion potential; EIS, electrochemical impedance spectroscopy; I(corr), corrosion current density; I(pass), passivation current density; LPS, lipopolysaccharide; OCP, open circuit potential; R(p), polarization resistance; Ti, titanium.

Lipopolysaccharide interactions of C-terminal peptides from human thrombin.

PubMed

Singh, Shalini; Kalle, Martina; Papareddy, Praveen; Schmidtchen, Artur; Malmsten, Martin

2013-05-13

Interactions with bacterial lipopolysaccharide (LPS), both in aqueous solution and in lipid membranes, were investigated for a series of amphiphilic peptides derived from the C-terminal region of human thrombin, using ellipsometry, dual polarization interferometry, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering, and z-potential measurements. The ability of these peptides to block endotoxic effects caused by LPS, monitored through NO production in macrophages, was compared to peptide binding to LPS and its endotoxic component lipid A, and to size, charge, and secondary structure of peptide/LPS complexes. While the antiendotoxic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) displayed significant binding to both LPS and lipid A, so did two control peptides with either selected D-amino acid substitutions or with maintained composition but scrambled sequence, both displaying strongly attenuated antiendotoxic effects. Hence, the extent of LPS or lipid A binding is not the sole discriminant for the antiendotoxic effect of these peptides. In contrast, helix formation in peptide/LPS complexes correlates to the antiendotoxic effect of these peptides and is potentially linked to this functionality. Preferential binding to LPS over lipid membrane was furthermore demonstrated for these peptides and preferential binding to the lipid A moiety within LPS inferred.

Protective effect of vitamin E in an animal model of LPS-induced inflammation.

PubMed

Mayorga, M; Iborra, A; Estany, S; Martínez, P

2004-12-01

Many sterility outcomes may be associated to the presence of an inflammatory response that would lead to an inability of the endometrium to support implantation and maintain viable embryos. We have established an animal model of inflammation in which the systemic administration of lipopolysaccharyde (LPS) results in a low embryo implantation rate. The purpose of this work was to investigate the effect of the inflammatory agent LPS on embryo viability and to verify the ability of vitamin E to modulate the inflammatory effect of LPS on embryo viability. For pre-implantation studies B6CBAF1 mice, which were intraperitoneally inoculated with LPS (4-10 mg/kg), were used. Mice were also treated with vitamin E (4-10 mg/kg) before or after LPS injection. Embryos were obtained from the oviduct after each treatment. The LPS produces a decrease in the number of pre-implantational embryos in a concentration dependent manner. The LPS effect can be partially reversed or prevented by vitamin E. Preliminary results show that inflammatory cytokines are secreted by intraperitoneal macrophages in LPS treated mice. Our results demonstrate the ability of vitamin E to avoid an inflammatory environment and to allow viability of embryos. (c) Blackwell Munksgaard, 2004

In situ longitudinal pre-stretch in the human femoropopliteal artery.

PubMed

Kamenskiy, Alexey; Seas, Andreas; Bowen, Grant; Deegan, Paul; Desyatova, Anastasia; Bohlim, Nick; Poulson, William; MacTaggart, Jason

2016-03-01

In situ longitudinal (axial) pre-stretch (LPS) plays a fundamental role in the mechanics of the femoropopliteal artery (FPA). It conserves energy during pulsation and prevents buckling of the artery during limb movement. We investigated how LPS is affected by demographics and risk factors, and how these patient characteristics associate with the structural and physiologic features of the FPA. LPS was measured in n=148 fresh human FPAs (14-80 years old). Mechanical properties were characterized with biaxial extension and histopathological characteristics were quantified with Verhoeff-Van Gieson Staining. Constitutive modeling was used to calculate physiological stresses and stretches which were then analyzed in the context of demographics, risk factors and structural characteristics. Age had the strongest negative effect (r=-0.812, p<0.01) on LPS and could alone explain 66% of LPS variability. Male gender, higher body mass index, hypertension, diabetes, coronary artery disease, dyslipidemia and tobacco use had negative effects on LPS, but only the effect of tobacco was not associated with aging. FPAs with less pre-stretch had thicker medial layers, but thinner intramural elastic fibers with less dense and more fragmented external elastic laminae. Elastin degradation was associated with decreased physiological tethering force and longitudinal stress, while circumferential stress remained constant. FPA wall pathology was negatively associated with LPS (r=-0.553, p<0.01), but the effect was due primarily to aging. LPS in the FPA may serve as an energy reserve for adaptive remodeling. Reduction of LPS due to degradation and fragmentation of intramural longitudinal elastin during aging can be accelerated in tobacco users. This work studies in situ longitudinal pre-stretch (LPS) in the human femoropopliteal artery. LPS has a fundamental role in arterial mechanics, but is rather poorly studied due to lack of direct in vivo measurement method. We have investigated LPS in the n=148 human femoropopliteal arteries in the context of subject demographics and risk factors, and structural and physiologic characteristics of the artery. Our results demonstrate that LPS reduces with age due to degradation and fragmentation of intramural elastin. LPS may serve as an energy reserve for adaptive remodeling, and reduction of LPS can be accelerated in tobacco users. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Involvement of nitric oxide in lipopolysaccharide induced anorexia.

PubMed

Riediger, Thomas; Cordani, Caroline; Potes, Catarina Soares; Lutz, Thomas A

2010-11-01

Treatment with the bacterial endotoxin lipopolysaccharide (LPS) is a commonly used model to induce disease-related anorexia. Following LPS treatment inducible nitric oxide synthase (iNOS) is expressed in the hypothalamic arcuate nucleus (ARC), where nitric oxide (NO) inhibits orexigenic neurons. Intracellular STAT signaling is triggered by inflammatory stimuli and has been linked to the transcriptional regulation of iNOS. We evaluated whether pharmacological blockade of iNOS by the specific inhibitor 1400W attenuates LPS-induced anorexia. Furthermore, we hypothesized that the tolerance to the anorectic effect occurring after repeated LPS treatment is paralleled by a blunted STAT3 phosphorylation in the ARC. Rats treated with a subcutaneous injection of 1400W (10 mg/kg) showed an attenuated anorectic LPS response relative to control rats receiving only LPS (100 µg/kg; i.p.). Similarly, iNOS blockade attenuated LPS-induced adipsia, hyperthermia, inactivity and the concomitant drop in energy expenditure. While single LPS treatment increased STAT3 phosphorylation in the ARC, rats treated repeatedly with LPS showed no anorectic response and also no STAT3 phosphorylation in the ARC after the second and third LPS injections, respectively. Hence, pSTAT3 signaling in the ARC might be part of the intracellular cascades translating pro-inflammatory stimuli into suppression of food intake. The current findings substantiate a role of iNOS dependent NO formation in disease-related anorexia. Copyright © 2010 Elsevier Inc. All rights reserved.

Coexposure of mice to trovafloxacin and lipopolysaccharide, a model of idiosyncratic hepatotoxicity, results in a unique gene expression profile and interferon gamma-dependent liver injury.

PubMed

Shaw, Patrick J; Ditewig, Amy C; Waring, Jeffrey F; Liguori, Michael J; Blomme, Eric A; Ganey, Patricia E; Roth, Robert A

2009-01-01

The antibiotic trovafloxacin (TVX) has caused severe idiosyncratic hepatotoxicity in people, whereas levofloxacin (LVX) has not. Mice cotreated with TVX and lipopolysaccharide (LPS), but not with LVX and LPS, develop severe hepatocellular necrosis. Mice were treated with TVX and/or LPS, and hepatic gene expression changes were measured before liver injury using gene array. Hepatic gene expression profiles from mice treated with TVX/LPS clustered differently from those treated with LPS or TVX alone. Several of the probe sets expressed differently in TVX/LPS-treated mice were involved in interferon (IFN) signaling and the janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. A time course of plasma concentrations of IFN-gamma and interleukin (IL)-18, which directly induces IFN-gamma production, revealed that both cytokines were selectively increased in TVX/LPS-treated mice. Both IL-18(-/-) and IFN-gamma(-/-) mice were significantly protected from TVX/LPS-induced liver injury. In addition, IFN-gamma(-/-) mice had decreased plasma concentrations of tumor necrosis factor-alpha, IL-18, and IL-1beta when compared to wild-type mice. In conclusion, the altered expression of genes involved in IFN signaling in TVX/LPS-treated mice led to the finding that IL-18 and IFN-gamma play a critical role in TVX/LPS-induced liver injury.

Sulforaphane suppresses LPS-induced inflammation in primary rat microglia.

PubMed

Brandenburg, Lars-Ove; Kipp, Markus; Lucius, Ralph; Pufe, Thomas; Wruck, Christoph J

2010-06-01

The aim of this study was to investigate the signal transduction pathways involved in sulforaphane (SF) mediated inhibition of the inflammatory response to lipopolysaccharide (LPS). Additionally, we investigated the effects of SF and LPS on the activity of Nrf2. Primary rat microglia and the murine microglia cell line BV2 were used. Cells were treated with LPS with or without SF. Cell viability was measured via WST-assay. Real-time RT-PCR was performed to analyze cytokine mRNA levels. The nitric oxide (NO) release was measured in LPS-stimulated microglia. The induction of various signal transduction pathways and Nrf2 was determined by Western blotting. NF-kappaB and AP-1 activation was measured by dual luciferase assay. We showed that SF attenuates the LPS-induced increase of IL-1beta, IL-6, and TNF-alpha expression in microglia. In addition, SF significantly decreases the NO in a concentration-dependent manner. SF inhibits LPS-stimulated ERK1/2 and JNK phosphorylation and thereby inhibits the LPS-induced activation of NF-kappaB- and activator protein-1 (AP-1). Moreover, SF and LPS together are able to induce Nrf2 activation. We showed that SF, and also LPS by itself, are able to activate the cell's defence against oxidative and electrophilic stress. We conclude that SF could be a candidate agent for anti-inflammatory treatment of the central nervous system.

The Transport and Inactivation Kinetics of Bacterial Lipopolysaccharide Influence its Immunological Potency in vivo1

PubMed Central

Lu, Mingfang; Munford, Robert S.

2011-01-01

The extraordinary potency and pathological relevance of Gram-negative bacterial lipopolysaccharides (LPSs) have made them very popular experimental agonists, yet little is known about what happens to these stimulatory molecules within animal tissues. We tracked fluorescent and radiolabeled-LPS from a subcutaneous inoculation site to its draining lymph nodes (DLN), blood and liver. Although we found FITC-labeled LPS in DLN within minutes of injection, drainage of radiolabeled LPS continued for more than six weeks. Within the DLN, most of the LPS was found in the subcapsular sinus or medulla, near or within lymphatic endothelial cells and CD169+ macrophages. Whereas most of the LPS seemed to pass through the DLN without entering B cell follicles, by 24 hrs after injection a small amount of LPS was found in the paracortex. In wildtype mice, ≥70% of the injected radiolabeled-LPS underwent inactivation by deacylation before it left the footpad; in animals that lacked acyloxyacyl hydrolase, the LPS-deacylating enzyme, prolonged drainage of fully acylated (active) LPS boosted polyclonal IgM and IgG3 antibody titers. LPS egress from a subcutaneous injection site thus occurred over many weeks and was mainly via lymphatic channels. Its immunological potency, as measured by its ability to stimulate polyclonal antibody production, was greatly influenced by the kinetics of both lymphatic drainage and enzymatic inactivation. PMID:21849675

Effects and mechanisms of cavidine protecting mice against LPS-induced endotoxic shock

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Weifeng, E-mail: liwf@mail.xjtu.edu.cn; Zhang,

LPS sensitized mice are usually considered as an experimental model of endotoxin shock. The present study aims to evaluate effects of cavidine on LPS-induced endotoxin shock. Mice were intraperitoneally administrated with cavidine (1, 3 and 10 mg/kg) or DEX (5 mg/kg) at 1 and 12 h before injecting LPS (30 mg/kg) intraperitoneally. Blood samples, liver, lung and kidney tissues were harvested after LPS injection. The study demonstrated that pretreatment with cavidine reduced the mortality of mice during 72 h after endotoxin injection. In addition, cavidine administration significantly attenuated histological pathophysiology features of LPS-induced injury in lung, liver and kidney. Furthermore,more » cavidine administration inhibited endotoxin-induced production of pro-inflammatory cytokines including TNF-α, IL-6 and HMGB1. Moreover, cavidine pretreatment attenuated the phosphorylation of mitogen-activated protein kinase primed by LPS. In summary, cavidine protects mice against LPS-induced endotoxic shock via inhibiting early pro-inflammatory cytokine TNF-α, IL-6 and late-phase cytokine HMGB1, and the modulation of HMGB1 may be related with MAPK signal pathway. - Highlights: • Cavidine significantly reduced mortality in mice during 72 h after LPS injection. • Cavidine attenuated histopathological changes in lung, liver and kidney. • Cavidine decreased the level of early inflammatory cytokine TNF-α, IL-6 in LPS- stimulated mice. • Cavidine inhibited late inflammatory cytokine HMGB1 through MAPK pathway.« less

The Deep-Sea Polyextremophile Halobacteroides lacunaris TB21 Rough-Type LPS: Structure and Inhibitory Activity towards Toxic LPS

PubMed Central

Di Lorenzo, Flaviana; Palmigiano, Angelo; Paciello, Ida; Pallach, Mateusz; Garozzo, Domenico; Bernardini, Maria-Lina; La Cono, Violetta; Yakimov, Michail M.; Molinaro, Antonio; Silipo, Alba

2017-01-01

The structural characterization of the lipopolysaccharide (LPS) from extremophiles has important implications in several biomedical and therapeutic applications. The polyextremophile Gram-negative bacterium Halobacteroides lacunaris TB21, isolated from one of the most extreme habitats on our planet, the deep-sea hypersaline anoxic basin Thetis, represents a fascinating microorganism to investigate in terms of its LPS component. Here we report the elucidation of the full structure of the R-type LPS isolated from H. lacunaris TB21 that was attained through a multi-technique approach comprising chemical analyses, NMR spectroscopy, and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Furthermore, cellular immunology studies were executed on the pure R-LPS revealing a very interesting effect on human innate immunity as an inhibitor of the toxic Escherichia coli LPS. PMID:28653982

Pushing the lipid envelope: using bio-inspired nanocomposites to understand and exploit lipid membrane limitations

NASA Astrophysics Data System (ADS)

Montano, Gabriel

Lipids serve as the organizing matrix material for biological membranes, the site of interaction of cells with the external environment. . As such, lipids play a critical role in structure/function relationships of an extraordinary number of critical biological processes. In this talk, we will look at bio-inspired membrane assemblies to better understand the roles of lipids in biological systems as well as attempt to generate materials that can mimic and potentially advance upon biological membrane processes. First, we will investigate the response of lipids to adverse conditions. In particular, I will present data that demonstrates the response of lipids to harsh conditions and how such responses can be exploited to generate nanocomposite rearrangements. I will also show the effect of adding the endotoxin lipopolysaccharide (LPS) to lipid bilayer assemblies and describe implications on our understanding of LPS organization in biological systems as well as describe induced lipid modifications that can be exploited to organize membrane composites with precise, two-dimensional geometric control. Lastly, I will describe the use of amphiphilic block copolymers to create membrane nanocomposites capable of mimicking biological systems. In particular, I will describe the use of our polymer-based membranes in creating artificial photosynthetic assemblies that rival biological systems in function in a more flexible, dynamic matrix.

Lipopolysaccharide mitagates methamphetamine-induced striatal dopamine depletion via modulating local TNF-alpha and dopamine transporter expression.

PubMed

Lai, Yu-Ting; Tsai, Yen-Ping N; Cherng, Chianfang G; Ke, Jing-Jer; Ho, Ming-Che; Tsai, Chia-Wen; Yu, Lung

2009-04-01

Systemic lipopolysaccharide (LPS) treatment may affect methamphetamine (MA)-induced nigrostriatal dopamine (DA) depletion. This study was undertaken to determine the critical time window for the protective effects of LPS treatment and the underlying mechanisms. An LPS injection (1 mg/kg) 72 h before or 2 h after MA treatment [three consecutive, subcutaneous injections of MA (10 mg/kg each) at 2-h intervals] diminished the MA-induced DA depletion in mouse striatum. Such an LPS-associated effect was independent of MA-produced hyperthermia. TNF-alpha, IL-1beta, IL-6 expressions were all elevated in striatal tissues following a systemic injection with LPS, indicating that peripheral LPS treatment affected striatal pro-inflammatory cytokine expression. Striatal TNF-alpha expression was dramatically increased at 72 and 96 h after the MA treatment, while such TNF-alpha elevation was abolished by the LPS pretreatment protocol. Moreover, MA-produced activation of nuclear NFkappaB, a transcription factor following TNF-alpha activation, in striatum was abolished by the LPS (1 mg/kg) pretreatment. Furthermore, thalidomide, a TNF-alpha antagonist, treatment abolished the LPS pretreatment-associated protective effects. Pretreatment with mouse recombinant TNF-alpha in striatum diminished the MA-produced DA depletion. Finally, single LPS treatment caused a rapid down-regulation of dopamine transporter (DAT) in striatum. Taken together, we conclude that peripheral LPS treatment protects nigrostriatal DA neurons against MA-induced toxicity, in part, by reversing elevated TNF-alpha expression and subsequent signaling cascade and causing a rapid DAT down-regulation in striatum.

Involvement of JNK and NF-κB pathways in lipopolysaccharide (LPS)-induced BAG3 expression in human monocytic cells.

PubMed

Wang, Hua-Qin; Meng, Xin; Liu, Bao-Qin; Li, Chao; Gao, Yan-Yan; Niu, Xiao-Fang; Li, Ning; Guan, Yifu; Du, Zhen-Xian

2012-01-01

Lipopolysaccharide (LPS) is an outer-membrane glycolipid component of Gram-negative bacteria known for its fervent ability to activate monocytic cells and for its potent proinflammatory capabilities. Bcl-2-associated athanogene 3 (BAG3) is a survival protein that has been shown to be stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, proteasome inhibition, and human immunodeficiency virus 1 (HIV-1) infection. In addition, BAG3 regulates replication of Varicella-Zoster Virus (VZV) and Herpes Simplex Virus (HSV) replication, suggesting that BAG3 could participate in the host response to infection. In the current study, we found that LPS increased the expression of BAG3 in a dose- and time-dependent manner. Actinomycin D completely blocked the LPS-induced BAG3 accumulation, as well as LPS activated the proximal promoter of BAG3 gene, supported that the induction by LPS occurred at the level of gene transcription. LPS-induced BAG3 expression was blocked by JNK or NF-κB inhibition, suggesting that JNK and NF-κB pathways participated in BAG3 induction by LPS. In addition, we also found that induction of BAG3 was implicated in monocytic cell adhesion to extracellular matrix induced by LPS. Overall, the data support that BAG3 is induced by LPS via JNK and NF-κB-dependent signals, and involved in monocytic cell-extracellular matrix interaction, suggesting that BAG3 may have a role in the host response to LPS stimulation. Copyright © 2011 Elsevier Inc. All rights reserved.

A butyrolactone derivative suppressed lipopolysaccharide-induced autophagic injury through inhibiting the autoregulatory loop of p8 and p53 in vascular endothelial cells.

PubMed

Meng, Ning; Zhao, Jing; Su, Le; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

2012-02-01

Lipopolysaccharide (LPS)-induced vascular endothelial cell (VEC) dysfunction is an important contributing factor in vascular diseases. Recently, we found that LPS impaired VEC by inducing autophagy. Our previous researches showed that a butyrolactone derivative, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) selectively protected VEC function. The objective of the present study is to investigate whether and how 3BDO inhibits LPS-induced VEC autophagic injury. Our results showed that LPS induced autophagy and led to increase of reactive oxygen species (ROS) and decrease of mitochondrial membrane potential (MMP) in Human umbilical vein vascular endothelial cells (HUVECs). Furthermore, LPS significantly increased p8 and p53 protein levels and the nuclear translocation of p53. All of these effects of LPS on HUVECs were strongly inhibited by 3BDO. Importantly, the ROS scavenger N-acetylcysteine (NAC) could inhibited LPS-induced autophagy and knockdown of p8 by RNA interference inhibited the autophagy, p53 protein level increase, the translocation of p53 into nuclei and the ROS level increase induced by LPS in HUVECs. The data suggested that 3BDO inhibited LPS-induced autophagy in HUVECs through inhibiting the ROS overproduction, the increase of p8 and p53 expression and the nuclear translocation of p53. Our findings provide a potential tool for understanding the mechanism underlying LPS-induced autophagy in HUVECs and open the door to a novel therapeutic drug for LPS-induced vascular diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

Extracellular vesicles are key intercellular mediators in the development of immune dysfunction to allergens in the airways.

PubMed

Shin, T-S; Kim, J H; Kim, Y-S; Jeon, S G; Zhu, Z; Gho, Yong Song; Kim, Yoon-Keun

2010-10-01

Previous evidence indicates that inhalation of lipopolysaccharide (LPS)-containing with allergens induced mixed Th1 and Th17 cell responses in the airways. Extracellular vesicles (EVs) are nanometer-sized spherical, lipid-bilayered structures and are recently in the public eye as an intercellular communicator in immune responses. To evaluate the role of EVs secreted by LPS inhalation in the development of airway immune dysfunction in response to allergens. Extracellular vesicles in bronchoalveolar lavage fluids of BALB/c mice were isolated and characterized 24 h after applications to the airway of 10 μg of LPS for 3 days. To evaluate the role of LPS-induced EVs on the development of airway immune dysfunction, in vivo and in vitro experiments were performed using the isolated LPS-induced EVs. The inhalation of LPS enhanced EVs release into the BAL fluid, when compared to the application of PBS. Airway sensitization with allergens and LPS-induced EVs resulted in a mixed Th1 and Th17 cell responses, although that with allergens and PBS-induced EVs induced immune tolerance. In addition, LPS-induced EVs enhanced the production of Th1- and Th17-polarizing cytokines (IL-12p70 and IL-6, respectively) by lung dendritic cells. Moreover, the immune responses induced by the LPS-induced EVs were blocked by denaturation of the EV-bearing proteins. These data suggest that EVs (especially, the protein components) secreted by LPS inhalation are a key intercellular communicator in the development of airway immune dysfunction to inhaled LPS-containing allergens.

Extracellular vesicles are key intercellular mediators in the development of immune dysfunction to allergens in the airways

PubMed Central

Shin, T-S; Kim, J H; Kim, Y-S; Jeon, S G; Zhu, Z; Gho, Y S; Kim, Y-K

2010-01-01

Background Previous evidence indicates that inhalation of lipopolysaccharide (LPS)-containing with allergens induced mixed Th1 and Th17 cell responses in the airways. Extracellular vesicles (EVs) are nanometer-sized spherical, lipid-bilayered structures and are recently in the public eye as an intercellular communicator in immune responses. Objective To evaluate the role of EVs secreted by LPS inhalation in the development of airway immune dysfunction in response to allergens. Methods Extracellular vesicles in bronchoalveolar lavage fluids of BALB/c mice were isolated and characterized 24 h after applications to the airway of 10 μg of LPS for 3 days. To evaluate the role of LPS-induced EVs on the development of airway immune dysfunction, in vivo and in vitro experiments were performed using the isolated LPS-induced EVs. Results The inhalation of LPS enhanced EVs release into the BAL fluid, when compared to the application of PBS. Airway sensitization with allergens and LPS-induced EVs resulted in a mixed Th1 and Th17 cell responses, although that with allergens and PBS-induced EVs induced immune tolerance. In addition, LPS-induced EVs enhanced the production of Th1- and Th17-polarizing cytokines (IL-12p70 and IL-6, respectively) by lung dendritic cells. Moreover, the immune responses induced by the LPS-induced EVs were blocked by denaturation of the EV-bearing proteins. Conclusion These data suggest that EVs (especially, the protein components) secreted by LPS inhalation are a key intercellular communicator in the development of airway immune dysfunction to inhaled LPS-containing allergens. PMID:20337607

Anti-inflammatory effects of methylthiouracil in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ku, Sae-Kwang; Baek, Moon-Chang, E-mail: mcbaek@knu.ac.kr; Bae, Jong-Sup, E-mail: baejs@knu.ac.kr

The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases. Here, methylthiouracil (MTU), an antithyroid drug, was examined for its effects on lipopolysaccharide (LPS)-mediated vascular inflammatory responses. The anti-inflammatory activities of MTU were determined by measuring permeability, human neutrophil adhesion and migration, and activation of pro-inflammatory proteins in LPS-activated human umbilical vein endothelial cells and mice. We found that post-treatment with MTU inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs), and adhesion/transendothelial migration of human neutrophils to human endothelial cells. MTU induced potent inhibition of LPS-inducedmore » endothelial cell protein C receptor (EPCR) shedding. It also suppressed LPS-induced hyperpermeability and neutrophil migration in vivo. Furthermore, MTU suppressed the production of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, and the activation of nuclear factor-κB (NF-κB) and extracellular regulated kinases (ERK) 1/2 by LPS. Moreover, post-treatment with MTU resulted in reduced LPS-induced lethal endotoxemia. These results suggest that MTU exerts anti-inflammatory effects by inhibiting hyperpermeability, expression of CAMs, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases. - Highlights: • MTU reduced LPS-mediated hyperpermeability in vitro and in vivo. • MTU inhibited LPS-mediated leukocyte adhesion and migration. • MTU inhibited LPS-mediated production of IL-6 and TNF-α. • MTU reduced LPS-mediated mortality and lung injury.« less

Modular Engineering of l-Tyrosine Production in Escherichia coli

PubMed Central

Juminaga, Darmawi; Baidoo, Edward E. K.; Redding-Johanson, Alyssa M.; Batth, Tanveer S.; Burd, Helcio; Mukhopadhyay, Aindrila; Petzold, Christopher J.

2012-01-01

Efficient biosynthesis of l-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for l-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to l-tyrosine on two plasmids. Rational engineering to improve l-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to l-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter l-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways. PMID:22020510

Improving the production of acetyl-CoA-derived chemicals in Escherichia coli BL21(DE3) through iclR and arcA deletion.

PubMed

Liu, Min; Ding, Yamei; Chen, Hailin; Zhao, Zhe; Liu, Huizhou; Xian, Mo; Zhao, Guang

2017-01-07

Acetyl-CoA-derived chemicals are suitable for multiple applications in many industries. The bio-production of these chemicals has become imperative owing to the economic and environmental problems. However, acetate overflow is the major drawback for acetyl-CoA-derived chemicals production. Approaches for overcoming acetate overflow may be beneficial for the production of acetyl-CoA-derived chemicals. In this study, a transcriptional regulator iclR was knocked out in E.coli BL21(DE3) to overcome acetate overflow and improve the chemicals production. Two important acetyl-CoA-derived chemicals, phloroglucinol (PG) and 3-hydroxypropionate (3HP) were used to evaluate it. It is revealed that knockout of iclR significantly increased expressions of aceBAK operon. The cell yields and glucose utilization efficiencies were higher than those of control strains. The acetate concentrations were decreased by more than 50% and the productions of PG and 3HP were increased more than twice in iclR mutants. The effects of iclR knockout on cell physiology, cell metabolism and production of acetyl-CoA-derived chemicals were similar to those of arcA knockout in our previous study. However, the arcA-iclR double mutants couldn't gain higher productions of PG and 3HP. The mechanisms are unclear and needed to be resolved in future. Knockout of iclR significantly increased gene expression of aceBAK operon and concomitantly activated glyoxylate pathway. This genetic modification may be a good way to overcome acetate overflow, and improve the production of a wide range of acetyl-CoA-derived chemicals.

Co-spread of metal and antibiotic resistance within ST3-IncHI2 plasmids from E. coli isolates of food-producing animals.

PubMed

Fang, Liangxing; Li, Xingping; Li, Liang; Li, Shumin; Liao, Xiaoping; Sun, Jian; Liu, Yahong

2016-05-04

Concerns have been raised in recent years regarding co-selection for antibiotic resistance among bacteria exposed to heavy metals, particularly copper and zinc, used as growth promoters for some livestock species. In this study, 25 IncHI2 plasmids harboring oqxAB (20/25)/blaCTX-M (18/25) were found with sizes ranging from ∼260 to ∼350 kb and 22 belonged to the ST3-IncHI2 group. In addition to blaCTX-M and oqxAB, pcoA-E (5/25) and silE-P (5/25), as well as aac(6')-Ib-cr (18/25), floR (16/25), rmtB (6/25), qnrS1(3/25) and fosA3 (2/25), were also identified on these IncHI2 plasmids. The plasmids carried pco and sil contributed to increasing in the MICs of CuSO4 and AgNO3. The genetic context surrounding the two operons was well conserved except some variations within the pco operon. The ~32 kb region containing the two operons identified in the IncHI2 plasmids was also found in chromosomes of different Enterobacteriaceae species. Further, phylogenetic analysis of this structure showed that Tn7-like transposon might play an important role in cross-genus transfer of the sil and pco operons among Enterobacteriaceae. In conclusion, co-existence of the pco and sil operons, and oqxAB/blaCTX-M as well as other antibiotic resistance genes on IncHI2 plasmids may promote the development of multidrug-resistant bacteria.

BadR and BadM Proteins Transcriptionally Regulate Two Operons Needed for Anaerobic Benzoate Degradation by Rhodopseudomonas palustris

PubMed Central

Hirakawa, Hidetada; Hirakawa, Yuko; Greenberg, E. Peter

2015-01-01

The bacterium Rhodopseudomonas palustris grows with the aromatic acid benzoate and the alicyclic acid cyclohexanecarboxylate (CHC) as sole carbon sources. The enzymatic steps in an oxygen-independent pathway for CHC degradation have been elucidated, but it was unknown how the CHC operon (badHI aliAB badK) encoding the enzymes for CHC degradation was regulated. aliA and aliB encode enzymes for the conversion of CHC to cyclohex-1-enecarboxyl–coenzyme A (CHene-CoA). At this point, the pathway for CHC degradation merges with the pathway for anaerobic benzoate degradation, as CHene-CoA is an intermediate in both degradation pathways. Three enzymes, encoded by badK, badH, and badI, prepare and cleave the alicyclic ring of CHene-CoA to yield pimelyl-CoA. Here, we show that the MarR transcription factor family member, BadR, represses transcription of the CHC operon by binding near the transcription start site of badH. 2-Ketocyclohexane-1-carboxyl–CoA, an intermediate of CHC and benzoate degradation, interacts with BadR to abrogate repression. We also present evidence that the transcription factor BadM binds to the promoter of the badDEFGAB (Bad) operon for the anaerobic conversion of benzoate to CHene-CoA to repress its expression. Contrary to previous reports, BadR does not appear to control expression of the Bad operon. These data enhance our view of the transcriptional regulation of anaerobic benzoate degradation by R. palustris. PMID:25888170

Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity

PubMed Central

Recinos, David A.; Sekedat, Matthew D.; Hernandez, Adriana; Cohen, Taylor Sitarik; Sakhtah, Hassan; Prince, Alice S.; Price-Whelan, Alexa; Dietrich, Lars E. P.

2012-01-01

Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2, although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2, but not of phz1, decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments. PMID:23129634

Structure and regulation of the Yersinia pestis yscBCDEF operon.

PubMed Central

Haddix, P L; Straley, S C

1992-01-01

We have investigated the physical and genetic structure and regulation of the Yersinia pestis yscBCDEF region, previously called lcrC. DNA sequence analysis showed that this region is homologous to the corresponding part of the ysc locus of Yersinia enterocolitica and suggested that the yscBCDEF cistrons belong to a single operon on the low-calcium response virulence plasmid pCD1. Promoter activity measurements of ysc subclones indicated that yscBCDEF constitutes a suboperon of the larger ysc region by revealing promoter activity in a clone containing the 3' end of yscD, intact yscE and yscF, and part of yscG. These experiments also revealed an additional weak promoter upstream of yscD. Northern (RNA) analysis with a yscD probe showed that operon transcription is thermally induced and downregulated in the presence of Ca2+. Primer extension of operon transcripts suggested that two promoters, a moderate-level constitutive one and a stronger, calcium-downregulated one, control full-length operon transcription at 37 degrees C. Primer extension provided additional support for the proposed designation of a yscBCDEF suboperon by identifying a 5' end within yscF, for which relative abundances in the presence and absence of Ca2+ revealed regulation that is distinct from that for transcripts initiating farther upstream. YscB and YscC were expressed in Escherichia coli by using a high-level transcription system. Attempts to express YscD were only partially successful, but they revealed interesting regulation at the translational level. Images PMID:1624469

Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.

PubMed

Entcheva, P; Liebl, W; Johann, A; Hartsch, T; Streit, W R

2001-01-01

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

Transcriptome analysis of the responses of Staphylococcus aureus to antimicrobial peptides and characterization of the roles of vraDE and vraSR in antimicrobial resistance

PubMed Central

Pietiäinen, Milla; François, Patrice; Hyyryläinen, Hanne-Leena; Tangomo, Manuela; Sass, Vera; Sahl, Hans-Georg; Schrenzel, Jacques; Kontinen, Vesa P

2009-01-01

Background Understanding how pathogens respond to antimicrobial peptides, and how this compares to currently available antibiotics, is crucial for optimizing antimicrobial therapy. Staphylococcus aureus has several known resistance mechanisms against human cationic antimicrobial peptides (CAMPs). Gene expression changes in S. aureus strain Newman exposed to linear CAMPs were analyzed by DNA microarray. Three antimicrobial peptides were used in the analysis, two are derived from frog, temporin L and dermaseptin K4-S4(1-16), and the ovispirin-1 is obtained from sheep. Results The peptides induced the VraSR cell-wall regulon and several other genes that are also up-regulated in cells treated with vancomycin and other cell wall-active antibiotics. In addition to this similarity, three genes/operons were particularly strongly induced by the peptides: vraDE, SA0205 and SAS016, encoding an ABC transporter, a putative membrane-bound lysostaphin-like peptidase and a small functionally unknown protein, respectively. Ovispirin-1 and dermaseptin K4-S4(1-16), which disrupt lipid bilayers by the carpet mechanism, appeared to be strong inducers of the vraDE operon. We show that high level induction by ovispirin-1 is dependent on the amide modification of the peptide C-terminus. This suggests that the amide group has a crucial role in the activation of the Aps (GraRS) sensory system, the regulator of vraDE. In contrast, temporin L, which disrupts lipid bilayers by forming pores, revealed a weaker inducer of vraDE despite the C-terminal amide modification. Sensitivity testing with CAMPs and other antimicrobials suggested that VraDE is a transporter dedicated to resist bacitracin. We also showed that SA0205 belongs to the VraSR regulon. Furthermore, VraSR was shown to be important for resistance against a wide range of cell wall-active antibiotics and other antimicrobial agents including the amide-modified ovispirin-1, bacitracin, teicoplanin, cefotaxime and 10 other β-lactam antibiotics, chlorpromazine, thioridazine and EGTA. Conclusion Defense against different CAMPs involves not only general signaling pathways but also CAMP-specific ones. These results suggest that CAMPs or a mixture of CAMPs could constitute a potential additive to standard antibiotic treatment. PMID:19751498

Structural and physiological studies of the Escherichia coli histidine operon inserted into plasmid vectors.

PubMed Central

Bruni, C B; Musti, A M; Frunzio, R; Blasi, F

1980-01-01

A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells have shown that the fragment contains the regulatory region of the operon, the hisG, hisD genes, and part of the hisC gene. Another plasmid (pCB5) contained the hisG gene and part of the hisD gene. Expression of the hisG gene in the latter plasmid was under control of the tetracycline promoter of the pBR313 plasmid. The in vivo expression of the two groups of plasmids described above, as well as their effect on the expression of the histidine genes not carried by the plasmids but present on the host chromosome, has been studied. The presence of multiple copies of pCB2 or pCB3, but not of pCB5, prevented derepression of the chromosomal histidine operon. Possible interpretations of this phenomenon are discussed. Images PMID:6246067

GCView: the genomic context viewer for protein homology searches

PubMed Central

Grin, Iwan; Linke, Dirk

2011-01-01

Genomic neighborhood can provide important insights into evolution and function of a protein or gene. When looking at operons, changes in operon structure and composition can only be revealed by looking at the operon as a whole. To facilitate the analysis of the genomic context of a query in multiple organisms we have developed Genomic Context Viewer (GCView). GCView accepts results from one or multiple protein homology searches such as BLASTp as input. For each hit, the neighboring protein-coding genes are extracted, the regions of homology are labeled for each input and the results are presented as a clear, interactive graphical output. It is also possible to add more searches to iteratively refine the output. GCView groups outputs by the hits for different proteins. This allows for easy comparison of different operon compositions and structures. The tool is embedded in the framework of the Bioinformatics Toolkit of the Max-Planck Institute for Developmental Biology (MPI Toolkit). Job results from the homology search tools inside the MPI Toolkit can be forwarded to GCView and results can be subsequently analyzed by sequence analysis tools. Results are stored online, allowing for later reinspection. GCView is freely available at http://toolkit.tuebingen.mpg.de/gcview. PMID:21609955

The rec A operon: a novel stress response gene cluster in Bacteroides fragilis

PubMed Central

Nicholson, Samantha A; Smalley, Darren; Smith, C. Jeffrey; Abratt, Valerie R

2014-01-01

Bacteroides fragilis, an opportunistic pathogen of humans, is a leading cause of bacteraemias and anaerobic abscesses which are often treated with metronidazole, a drug which damages DNA. This study investigated the responses of the B. fragilis recA three gene operon to the stress experienced during metronidazole treatment and exposure to reactive oxygen species simulating those generated by the host immune system during infection. A transcriptionally regulated response was observed using quantitative RT-PCR after metronidazole and hydrogen peroxide treatment, with all three genes being upregulated under stress conditions. In vivo and in vitro analysis of the functional role of the second gene of the operon was done using heterologous complementation and protein expression (in Escherichia coli), with subsequent biochemical assay. This gene encoded a functional bacterioferritin co-migratory protein (BCP) which was thiol-specific and had antioxidant properties, including protection of the glutamine synthetase III enzyme. This in vitro data supports the hypothesis that the genes of the operon may be involved in protection of the bacteria from the oxidative burst during tissue invasion and may play a significant role in bacterial survival and metronidazole resistance during treatment of B. fragilis infections. PMID:24703997

Amplification of the groESL operon in Pseudomonas putida increases siderophore gene promoter activity.

PubMed

Venturi, V; Wolfs, K; Leong, J; Weisbeek, P J

1994-10-17

Pseudobactin 358 is the yellow-green fluorescent siderophore [microbial iron(III) transport agent] produced by Pseudomonas putida WCS358 under iron-limiting conditions. The genes encoding pseudobactin 358 biosynthesis are iron-regulated at the level of transcription. In this study, the molecular characterization is reported of a cosmid clone of WCS358 DNA that can stimulate, in an iron-dependent manner, the activity of a WCS358 siderophore gene promoter in the heterologous Pseudomonas strain A225. The functional region in the clone was identified by subcloning, transposon mutagenesis and DNA sequencing as the groESL operon of strain WCS358. This increase in promoter activity was not observed when the groESL genes of strain WCS358 were integrated via a transposon vector into the genome of Pseudomonas A225, indicating that multiple copies of the operon are necessary for the increase in siderophore gene promoter activity. Amplification of the Escherichia coli and WCS358 groESL genes also increased iron-regulated promoter activity in the parent strain WCS358. The groESL operon codes for the chaperone proteins GroES and GroEL, which are responsible for mediating the folding and assembly of many proteins.

Functional genomics of corrinoid starvation in the organohalide-respiring bacterium Dehalobacter restrictus strain PER-K23

PubMed Central

Rupakula, Aamani; Lu, Yue; Kruse, Thomas; Boeren, Sjef; Holliger, Christof; Smidt, Hauke; Maillard, Julien

2015-01-01

De novo corrinoid biosynthesis represents one of the most complicated metabolic pathways in nature. Organohalide-respiring bacteria (OHRB) have developed different strategies to deal with their need of corrinoid, as it is an essential cofactor of reductive dehalogenases, the key enzymes in OHR metabolism. In contrast to Dehalococcoides mccartyi, the genome of Dehalobacter restrictus strain PER-K23 contains a complete set of corrinoid biosynthetic genes, of which cbiH appears to be truncated and therefore non-functional, possibly explaining the corrinoid auxotrophy of this obligate OHRB. Comparative genomics within Dehalobacter spp. revealed that one (operon-2) of the five distinct corrinoid biosynthesis associated operons present in the genome of D. restrictus appeared to be present only in that particular strain, which encodes multiple members of corrinoid transporters and salvaging enzymes. Operon-2 was highly up-regulated upon corrinoid starvation both at the transcriptional (346-fold) and proteomic level (46-fold on average), in line with the presence of an upstream cobalamin riboswitch. Together, these data highlight the importance of this operon in corrinoid homeostasis in D. restrictus and the augmented salvaging strategy this bacterium adopted to cope with the need for this essential cofactor. PMID:25610435

Laser pulse sampler

DOEpatents

Vann, C.

1998-03-24

The Laser Pulse Sampler (LPS) measures temporal pulse shape without the problems of a streak camera. Unlike the streak camera, the laser pulse directly illuminates a camera in the LPS, i.e., no additional equipment or energy conversions are required. The LPS has several advantages over streak cameras. The dynamic range of the LPS is limited only by the range of its camera, which for a cooled camera can be as high as 16 bits, i.e., 65,536. The LPS costs less because there are fewer components, and those components can be mass produced. The LPS is easier to calibrate and maintain because there is only one energy conversion, i.e., photons to electrons, in the camera. 5 figs.

Heterologous Expression of the Desulfovibrio gigas [NiFe] Hydrogenase in Desulfovibrio fructosovorans MR400

PubMed Central

Rousset, Marc; Magro, Valérie; Forget, Nicole; Guigliarelli, Bruno; Belaich, Jean-Pierre; Hatchikian, E. Claude

1998-01-01

The ability of Desulfovibrio fructosovorans MR400 ΔhynABC to express the heterologous cloned [NiFe] hydrogenase of Desulfovibrio gigas was investigated. The [NiFe] hydrogenase operon from D. gigas, hynABCD, was cloned, sequenced, and introduced into D. fructosovorans MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native D. gigas protein. A chimeric operon containing hynAB from D. gigas and hynC from D. fructosovorans placed under the control of the D. fructosovorans hynAp promoter was constructed and expressed in D. fructosovorans MR400. Under these conditions, the same level of activity was obtained as with the D. gigas hydrogenase operon. PMID:9733707

Molecular analysis of mercury-resistant Bacillus isolates from sediment of Minamata Bay, Japan.

PubMed Central

Nakamura, K; Silver, S

1994-01-01

Bacillus isolates from Minamata Bay sediment were selected for the ability to volatilize mercury from a range of organomercurials (including methylmercury). Chromosomal DNA from 74 of 78 such strains hybridized with the mer mercury resistance operon DNA from marine Bacillus sp. strain RC607 (Y. Wang, M. Moore, H. S. Levinson, S. Silver, C. Walsh, and I. Mahler, J. Bacteriol. 171:83-92, 1989). The most frequent classes with regard to restriction nuclease site maps of the mer operon for the new isolates were identical to or closely related to the mer determinant of strain RC607. PCR amplification analysis with primers designed from the strain RC607 mer operon gave products of precisely the predicted size with the 74 Minamata Bay isolates. Images PMID:7811095

NEMO-Binding Domain Peptide Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting the NF-κB Signaling Pathway

PubMed Central

Huang, Jianhua; Li, Li; Yuan, Weifeng; Zheng, Linxin

2016-01-01

The aim of the present study is to investigate the protective effects and relevant mechanisms exerted by NEMO-binding domain peptide (NBD) against lipopolysaccharide- (LPS-) induced acute lung injury (ALI) in mice. The ALI model was induced by intratracheally administered atomized LPS (5 mg/kg) to BABL/c mice. Half an hour before LPS administration, we treated the mice with increasing concentrations of intratracheally administered NBD or saline aerosol. Two hours after LPS administration, each group of mice was sacrificed. We observed that NBD pretreatment significantly attenuated LPS-induced lung histopathological injury in a dose-dependent manner. Western blotting established that NBD pretreatment obviously attenuated LPS-induced IκB-α and NF-κBp65 activation and NOX1, NOX2, and NOX4 overexpression. Furthermore, NBD pretreatment increased SOD and T-AOC activity and decreased MDA levels in lung tissue. In addition, NBD also inhibited TNF-α and IL-1β secretion in BALF after LPS challenge. In conclusion, NBD protects against LPS-induced ALI in mice. PMID:27956761

Production of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta by human polymorphonuclear neutrophils stimulated with Porphyromonas endodontalis lipopolysaccharide.

PubMed

Ko, Hyun Jung; Lim, Sung Sam

2002-11-01

This study was undertaken to investigate the capacity of polymorphonuclear neutrophils (PMNs) to secrete Macrophage Inflammatory Protein (MIP)-1alpha and MIP-1beta after stimulation with Porphyromonas endodontalis lipopolysaccharide (LPS). Escherichia coli LPS was used as a positive control. Venous blood was collected and PMNs were isolated from healthy volunteers. Cells were cultured with various concentrations of LPS for different periods of time. Cell supernatants were assayed by enzyme-linked immunosorbent assay. The levels of chemokine secretion in PMNs stimulated with each LPS were found to be significantly higher than in the unstimulated control cells (p < 0.05), and this expression occurred in a time- and dose-dependent manner. E. coli LPS induced higher levels of cytokines than P. endodontalis LPS. These findings demonstrated that P. endodontalis LPS is capable of stimulating PMNs to produce chemotactic cytokines and suggested that PMNs stimulated with P. endodontalis LPS may play a crucial role in the inflammatory and immunopathological reactions of pulpal and periapical diseases.

The PI3K/Akt pathway is required for LPS activation of microglial cells.

PubMed

Saponaro, Concetta; Cianciulli, Antonia; Calvello, Rosa; Dragone, Teresa; Iacobazzi, Francesco; Panaro, Maria Antonietta

2012-10-01

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.

Associations of Escherichia coli K-12 OmpF trimers with rough and smooth lipopolysaccharides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diedrich, D.L.; Stein, M.A.; Schnaitman, C.A.

1990-09-01

The associations of both rough and smooth lipopolysaccharides (LPS) with the OmpF porin of Escherichia coli K-12 were examined in galE strains deleted for ompC. Transformation with pSS37 and growth with galactose conferred the ability to assemble a Shigella dysenteriae O antigen onto the core oligosaccharide of E. coli K-12 LPS. The association of LPS with OmpF trimers was assessed by staining, autoradiography of LPS specifically labeled with (1-14C)galactose, and Western immunoblotting with a monoclonal antibody specific for OmpF trimers. These techniques revealed that the migration distances and multiple banding patterns of OmpF porin trimers in sodium dodecyl sulfate-polyacrylamide gelsmore » were dictated by the chemotype of associated LPS. Expression of smooth LPS caused almost all of the trimeric OmpF to run in gels with a slower mobility than trimers from rough strains. The LPS associated with trimers from a smooth strain differed from the bulk-phase LPS by consisting almost exclusively of molecules with O antigen.« less

6-Shogaol attenuates LPS-induced inflammation in BV2 microglia cells by activating PPAR-γ.

PubMed

Han, Qinghe; Yuan, Qinghai; Meng, Xiaolin; Huo, Junyuan; Bao, Yuxin; Xie, Guanghong

2017-06-27

6-Shogaol, a pungent agent isolated from Zingiber officinale Roscoe, has been known to have anti-tumor and anti-inflammatory effects. However, the anti-inflammatory effects and biological mechanism of 6-Shogaol in LPS-activated BV2 microglia remains largely unknown. In this study, we evaluated the anti-inflammatory effects of 6-Shogaol in LPS-activated BV2 microglia. 6-Shogaol was administrated 1 h before LPS treatment. The production of inflammatory mediators were detected by ELISA. The expression of NF-κB and PPAR-γ were detected by western blot analysis. Our results revealed that 6-Shogaol inhibited LPS-induced TNF-α, IL-1β, IL-6, and PGE2 production in a concentration dependent manner. Furthermore, 6-Shogaol inhibited LPS-induced NF-κB activation by inhibiting phosphorylation and nuclear translocation of NF-κB p65. In addition, 6-Shogaol could increase the expression of PPAR-γ. Moreover, inhibition of PPAR-γ by GW9662 could prevent the inhibition of 6-Shogaol on LPS-induced inflammatory mediator production. In conclusion, 6-Shogaol inhibits LPS-induced inflammation by activating PPAR-γ.

SMAD4 is Involved in the Development of Endotoxin Tolerance in Microglia.

PubMed

Liu, Xiaorong; Qin, Yongwei; Dai, Aihua; Zhang, Yu; Xue, Huaqing; Ni, Haidan; Han, Lijian; Zhu, Liang; Yuan, Debin; Tao, Tao; Cao, Maohong

2016-07-01

Initial exposure of macrophages to LPS induces hyporesponsiveness to a second challenge with LPS, a phenomenon termed LPS tolerance. Smad4 plays important roles in the induction of LPS tolerance. However, the function of Smad4 in microglia remains unknown. Here we show that expression of Smad4 was highly up-regulated in LPS-tolerized mouse cerebral cortex. Smad4 was mostly colocalized with microglia, rarely with neurons. Using a microglia cell line, BV2, we find that LPS activates endogenous Smad4, inducing its migration into the nucleus and increasing its expression. Smad4 significantly suppressed TLR-triggered production of proinflammatory cytokines (IL-6), increased anti-inflammatory cytokine in LPS-tolerized microglia. Moreover, IL-6 concentrations in culture supernatants after second LPS challenge are higher in SMAD4 small interfering RNA (siRNA) BV2 cells than control siRNA BV2 cells, indicating failure to induce tolerance in absence of Smad4 signaling. In our study, we conclude that both in vivo and in vitro, Smad4 signaling is required for maximal induction of endotoxin tolerance.

Lipopolysaccharide reduces food passage rate from the crop by a prostaglandin-independent mechanism in chickens

PubMed Central

Tachibana, T.; Ogino, M.; Makino, R.; Khan, M. S. I.; Cline, M. A.

2017-01-01

ABSTRACT 1. We examined the effect of lipopolysaccharide (LPS), a component of Gram-negative bacteria, on food passage in the digestive tract of chickens (Gallus gallus) in order to clarify whether bacterial infection affects food passage in birds. 2. Food passage in the crop was significantly reduced by intraperitoneal (IP) injection of LPS while it did not affect the number of defecations, suggesting that LPS may affect food passage only in the upper digestive tract. 3. Similar to LPS, prostaglandin E2 (PGE2), one of the mediators of LPS, also reduced crop-emptying rate in chickens while it had no effect on the number of defecations. 4. Pretreatment with indomethacin, which is an inhibitor of cyclooxygenase (COX), a prostaglandin synthase, had no effect on LPS-induced inhibition of crop emptying. 5. IP injection of LPS did not affect the mRNA expression of COX2 in the upper digestive tract of chickens. 6. It is therefore likely that LPS and PGE2 reduced food passage rate in the crop by a prostaglandin-independent pathway in chickens. PMID:27871194

Lipopolysaccharide reduces food passage rate from the crop by a prostaglandin-independent mechanism in chickens.

PubMed

Tachibana, T; Ogino, M; Makino, R; Khan, M S I; Cline, M A

2017-02-01

1. We examined the effect of lipopolysaccharide (LPS), a component of Gram-negative bacteria, on food passage in the digestive tract of chickens (Gallus gallus) in order to clarify whether bacterial infection affects food passage in birds. 2. Food passage in the crop was significantly reduced by intraperitoneal (IP) injection of LPS while it did not affect the number of defecations, suggesting that LPS may affect food passage only in the upper digestive tract. 3. Similar to LPS, prostaglandin E2 (PGE2), one of the mediators of LPS, also reduced crop-emptying rate in chickens while it had no effect on the number of defecations. 4. Pretreatment with indomethacin, which is an inhibitor of cyclooxygenase (COX), a prostaglandin synthase, had no effect on LPS-induced inhibition of crop emptying. 5. IP injection of LPS did not affect the mRNA expression of COX2 in the upper digestive tract of chickens. 6. It is therefore likely that LPS and PGE2 reduced food passage rate in the crop by a prostaglandin-independent pathway in chickens.

Lactobacillus pentosus strain LPS16 produces lactic acid, inhibiting multidrug-resistant Helicobacter pylori.

PubMed

Zheng, Po-Xing; Fang, Hsin-Yi; Yang, Hsiao-Bai; Tien, Nai-Yueh; Wang, Ming-Cheng; Wu, Jiunn-Jong

2016-04-01

Helicobacter pylori is a human gastric pathogen. Antibiotic resistance of H. pylori has become a problem increasing the failure of H. pylori eradication. Therefore alternative approaches are required. The aim of this study was to evaluate the anti-H. pylori activity of Lactobacillus pentosus strain LPS16 and the mechanism of its killing effect. The anti-H. pylori activity of LPS16 was determined by the disc diffusion test and time killing assay. High-performance liquid chromatography analysis was used to analyze the secreted compounds of LPS16. Sixty H. pylori strains isolated from different gastric diseases, having different antibiotic susceptibility were collected to analyze the spectrum of anti-H. pylori activity of LPS16. Adhesion ability of LPS16 to gastric epithelial cell lines was assayed by flow cytometry. The anti-H. pylori activity of LPS16 depended on the secreted component, and lactic acid mediated bactericidal activity against H. pylori. The bactericidal activity did not vary significantly among the strains isolated from different diseases having different antibiotic susceptibility. Moreover, LPS16 can adhere on gastric epithelial cell lines AKG and MKN45. L. pentosus strain LPS16 had the broad-spectrum anti-H. pylori activity, suggesting that it can be used to prevent H. pylori infection. Copyright © 2014. Published by Elsevier B.V.

Lipopolysaccharide (LPS) induced sickness in adolescent female rats alters the acute-phase response and lithium chloride (LiCl)- induced impairment of conditioned place avoidance/aversion learning, following a homotypic LPS challenge in adulthood.

PubMed

Cloutier, Caylen J; Kavaliers, Martin; Ossenkopp, Klaus-Peter

2018-10-01

The multi-variable locomotor activity effects of LiCl treatment in female rats were examined in a conditioned place avoidance/aversion (CPA) paradigm. In addition, the sickness effects of an LPS injection (200 μg/kg), given during adolescents, on CPA learning in adulthood were examined, as were the effects of a homotypic LPS injection (200 μg/kg) just prior to CPA acquisition trials. Female rats were injected with LPS or saline during adolescents (6 weeks of age) and later pretreated with LPS again or saline in an automated two-chamber CPA paradigm with LiCl (95 mg/kg) treatments as the aversive toxin. Results showed that, while adolescent LPS treatment had no long-term effect on the establishment of CPA, it did interfere with the ability of a second LPS challenge in adulthood to impair CPA learning, an effect obtained in subjects pretreated with LPS in the CPA procedure in adulthood only. The results of this study demonstrate the importance of considering the adolescent stage of development when evaluating the effects of environmental challenges on adult behavior. Copyright © 2018 Elsevier B.V. All rights reserved.

Nitric Oxide-Mediated Maintenance of Redox Homeostasis Contributes to NPR1-Dependent Plant Innate Immunity Triggered by Lipopolysaccharides1[C][W

PubMed Central

Sun, Aizhen; Nie, Shengjun; Xing, Da

2012-01-01

The perception of lipopolysaccharides (LPS) by plant cells can lead to nitric oxide (NO) production and defense gene induction. However, the signaling cascades underlying these cellular responses have not yet been resolved. This work investigated the biosynthetic origin of NO and the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) to gain insight into the mechanism involved in LPS-induced resistance of Arabidopsis (Arabidopsis thaliana). Analysis of inhibitors and mutants showed that LPS-induced NO synthesis was mainly mediated by an arginine-utilizing source of NO generation. Furthermore, LPS-induced NO caused transcript accumulation of alternative oxidase genes and increased antioxidant enzyme activity, which enhanced antioxidant capacity and modulated redox state. We also analyzed the subcellular localization of NPR1 to identify the mechanism for protein-modulated plant innate immunity triggered by LPS. LPS-activated defense responses, including callose deposition and defense-related gene expression, were found to be regulated through an NPR1-dependent pathway. In summary, a significant NO synthesis induced by LPS contributes to the LPS-induced defense responses by up-regulation of defense genes and modulation of cellular redox state. Moreover, NPR1 plays an important role in LPS-triggered plant innate immunity. PMID:22926319

An evaluation of the inflammatory response of lipopolysaccharide-treated primary dental pulp cells with regard to calcium silicate-based cements

PubMed Central

Lai, Wei-Yun; Kao, Chia-Tze; Hung, Chi-Jr; Huang, Tsui-Hsien; Shie, Ming-You

2014-01-01

This study compared the biological changes of lipopolysaccharide (LPS)-treated dental pulp (DP) cells directly cultured on mineral trioxide aggregate (MTA) and calcium silicate (CS) cements. DP cells were treated with LPS for 24 h. Then, the LPS-treated DP cells were cultured on MTA or CS cements. Cell viability, cell death mechanism and interleukin (IL)-1β expressions were analysed. A one-way analysis of variance was used to evaluate the significance of the differences between the means. A significantly higher IL-1β expression (2.9-fold) was found for LPS-treated cells (P<0.05) compared with DP cells without LPS treatment at 24 h. Absorbance values of LPS-treated cells cultured on CS cement were higher than a tissue culture plate. A significant difference (P<0.05) in cell viability was observed between cells on CS and MTA cements 24 h after seeding. At 48 h, a high concentration of Si (5 mM) was released from MTA, which induced LPS-treated DP cell apoptosis. The present study demonstrates that CS cement is biocompatible with cultured LPS-treated DP cells. MTA stimulates inflammation in LPS-treated DP cells, which leads to greater IL-1β expression and apoptosis. PMID:24556955

High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system.

PubMed

Jiang, Hui; Jiang, Donglei; Shao, Jingdong; Sun, Xiulan; Wang, Jiasheng

2016-11-14

Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.

Oral arginine reduces gut mucosal injury caused by lipopolysaccharide endotoxemia in rat.

PubMed

Sukhotnik, Igor; Mogilner, Jorge; Krausz, Michael M; Lurie, Michael; Hirsh, Mark; Coran, Arnold G; Shiloni, Eitan

2004-12-01

The objective of this study was to evaluate the effects of lipopolysaccharide (LPS) endotoxemia and enteral arginine (ARG) supplementation on intestinal structural changes, enterocyte proliferation, and apoptosis in rat. Male Sprague-Dawley rats, weighing 250-280 g, were divided into three experimental groups: control rats, LPS rats treated with lipopolysaccharide given ip at a dose of 10 mg/kg every 24 h (two injections), and LPS-ARG rats treated with enteral arginine given in drinking water (2%) 72 h before and following injection of LPS. Intestinal structural changes, enterocyte proliferation, and enterocyte apoptosis were determined on day 3 following the first LPS injection. LPS rats demonstrated a significant decrease in bowel weight in duodenum, mucosal weight in duodenum, jejunum, and ileum, mucosal DNA and protein in jejunum and ileum, and villus height in jejunum and ileum compared to control animals. LPS rats also had a significantly lower cell proliferation index in jejunum and ileum and a higher apoptotic index in jejunum and ileum compared to control rats. LPS-ARG animals demonstrated greater duodenal bowel weight, duodenal and ileal mucosal weight, ileal mucosal DNA and protein, ileal villus height, and jejunal and ileal cell proliferation index compared to LPS animals. LPS endotoxemia impairs the integrity of the gastrointestinal mucosa in rat. Decreased cell proliferation and increased apoptosis may be considered the main mechanisms responsible for the decreased cell mass. Enteral arginine administration decreases the mucosal injury caused by lipopolysaccharide.

Adjuvant Effects Elicited by Novel Oligosaccharide Variants of Detoxified Meningococcal Lipopolysaccharides on Neisseria meningitidis Recombinant PorA Protein: A Comparison in Mice

PubMed Central

Mehta, Ojas H.; Norheim, Gunnstein; Hoe, J . Claire; Rollier, Christine S.; Nagaputra, Jerry C.; Makepeace, Katherine; Saleem, Muhammad; Chan, Hannah; Ferguson, David J. P.; Jones, Claire; Sadarangani, Manish; Hood, Derek W.; Feavers, Ian; Derrick, Jeremy P.; Pollard, Andrew J.; Moxon, E . Richard

2014-01-01

Neisseria meningitidis lipopolysaccharide (LPS) has adjuvant properties that can be exploited to assist vaccine immunogenicity. The modified penta-acylated LPS retains the adjuvant properties of hexa-acylated LPS but has a reduced toxicity profile. In this study we investigated whether two modified glycoform structures (LgtE and IcsB) of detoxified penta-acylated LPS exhibited differential adjuvant properties when formulated as native outer membrane vesicles (nOMVs) as compared to the previously described LgtB variant. Detoxified penta-acylated LPS was obtained by disruption of the lpxL1 gene (LpxL1 LPS), and three different glycoforms were obtained by disruption of the lgtB, lgtE or icsB genes respectively. Mice (mus musculus) were immunized with a recombinant PorA P1.7-2,4 (rPorA) protein co-administered with different nOMVs (containing a different PorA serosubtype P1.7,16), each of which expressed one of the three penta-acylated LPS glycoforms. All nOMVs induced IgG responses against the rPorA, but the nOMVs containing the penta-acylated LgtB-LpxL1 LPS glycoform induced significantly greater bactericidal activity compared to the other nOMVs or when the adjuvant was Alhydrogel. Compared to LgtE or IcsB LPS glycoforms, these data support the use of nOMVs containing detoxified, modified LgtB-LpxL1 LPS as a potential adjuvant for future meningococcal protein vaccines. PMID:25545241

COX-2 plays a role in angiogenic DBA(+) uNK cell subsets activation and pregnancy protection in LPS-exposed mice.

PubMed

Zavan, Bruno; De Almeida, Eliana Martins; Salles, Évila da Silva Lopes; do Amarante-Paffaro, Andréa Mollica; Paffaro, Valdemar Antonio

2016-08-01

Although uterine Natural Killer (uNK) cells have cytoplasmic granules rich in perforin and granzymes, these cells do not degranulate in normal pregnancy. DBA lectin(+) uNK cells produce angiogenic factors which stimulate remodeling of uterine arterioles to increase blood flow within the growing feto-placental unit. We sought to investigate the importance of COX-2 on mouse pregnancy inoculated with Gram-negative bacteria Lipopolysaccharide (LPS) by treating with a selective COX-2 inhibitor (nimesulide). We have combined histochemical, immunohistochemical, stereological, morphometric, behavioral, and litter analyses to investigate mouse pregnancy inoculated with LPS with or without pre-treatment with nimesulide 30 min before LPS injections, focusing on DBA(+) uNK cell response and viability of the pregnancy. LPS caused sickness behavior, an immature DBA(+) uNK influx, decreased mature DBA(+) uNK cell numbers, and triggered a new DBA(low) uNK appearance. These effects of LPS, except the sickness behavior, were prevented by nimesulide. COX-2 inhibition also prevented the down-regulation of uNK perforin and spiral arteriole α-actin expression stimulated by LPS. While the litter size from Nimesulide + LPS-treated mothers was significantly smaller compared to those from LPS-treated group, nimesulide alone showed no effect on the offspring. Collectively, our data indicate that COX-2 changes angiogenic DBA(+) uNK cells in order to protect mouse pregnancy after LPS injection. Copyright © 2016 Elsevier Ltd. All rights reserved.

COX-2 contributes to LPS-induced Stat3 activation and IL-6 production in microglial cells

PubMed Central

Zhu, Jie; Li, Shuzhen; Zhang, Yue; Ding, Guixia; Zhu, Chunhua; Huang, Songming; Zhang, Aihua; Jia, Zhanjun; Li, Mei

2018-01-01

Many stimuli including lipopolysaccharide (LPS) could activate microglial cells to subsequently cause inflammatory nerve injury. However, the mechanism of LPS-induced neuroinflammation in microglial cells is still elusive. Thus, the present study was undertaken to examine the role of COX-2 in mediating the activation of Stat3 and the production of IL-6 in BV2 cells challenged with LPS. After 24 h treatment, LPS dose-dependently enhanced COX-2 expression at both mRNA and protein levels. Meanwhile, IL-6 with other inflammatory cytokines including IL-1β, TNF-α, and MCP-1 were similarly enhanced by LPS. Then a specific COX-2 inhibitor (NS-398) was administered to BV2 before LPS treatment. Significantly, COX-2 inhibition suppressed the upregulation of IL-6 at both mRNA and protein levels in line with the trend blockade on IL-1β, TNF-α, and MCP-1. Stat3 drives proinflammatory signaling pathways and contributes to IL-6 production via a transcriptional mechanism in many diseases. Here we found that inhibition of COX-2 entirely blocked LPS-induced Stat3 phosphorylation, which might contribute to the blockade of IL-6 production to some extent. Meanwhile, COX-2 siRNA approach largely reproduced the phenotypes shown by specific COX-2 inhibitor in LPS-treated BV2 cells. Together, these findings suggested that COX-2 might contribute to LPS-induced IL-6 production possibly through activating Stat3 signaling pathway in microglial cells. PMID:29636886

Soluble β-(1,3)-glucans enhance LPS-induced response in the monocyte activation test, but inhibit LPS-mediated febrile response in rabbits: Implications for pyrogenicity tests.

PubMed

Pardo-Ruiz, Zenia; Menéndez-Sardiñas, Dalia E; Pacios-Michelena, Anabel; Gabilondo-Ramírez, Tatiana; Montero-Alejo, Vivian; Perdomo-Morales, Rolando

2016-01-01

In the present study, we aimed to determine the influence of β-(1,3)-d-glucans on the LPS-induced pro-inflammatory cytokine response in the Monocyte Activation Test (MAT) for pyrogens, and on the LPS-induced febrile response in the Rabbit Pyrogen Test (RPT), thus evaluating the resulting effect in the outcome of each test. It was found that β-(1,3)-d-glucans elicited the production of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, also known as endogenous pyrogens, but not enough to classify them as pyrogenic according to MAT. The same β-(1,3)-d-glucans samples were non-pyrogenic by RPT. However, β-(1,3)-d-glucans significantly enhanced the LPS-induced pro-inflammatory cytokines response in MAT, insomuch that samples containing non-pyrogenic concentrations of LPS become pyrogenic. On the other hand, β-(1,3)-d-glucans had no effect on sub-pyrogenic LPS doses in the RPT, but surprisingly, inhibited the LPS-induced febrile response of pyrogenic LPS concentrations. Thus, while β-(1,3)-d-glucans could mask the LPS pyrogenic activity in the RPT, they exerted an overstimulation of pro-inflammatory cytokines in the MAT. Hence, MAT provides higher safety since it evidences an unwanted biological response, which is not completely controlled and is overlooked by the RPT. Copyright © 2015 Elsevier B.V. All rights reserved.

Dose Response of Endotoxin on Hepatocyte and Muscle Mitochondrial Respiration In Vitro

PubMed Central

Brandt, Sebastian; Porta, Francesca; Jakob, Stephan M.; Takala, Jukka; Djafarzadeh, Siamak

2015-01-01

Introduction. Results on mitochondrial dysfunction in sepsis are controversial. We aimed to assess effects of LPS at wide dose and time ranges on hepatocytes and isolated skeletal muscle mitochondria. Methods. Human hepatocellular carcinoma cells (HepG2) were exposed to placebo or LPS (0.1, 1, and 10 μg/mL) for 4, 8, 16, and 24 hours and primary human hepatocytes to 1 μg/mL LPS or placebo (4, 8, and 16 hours). Mitochondria from porcine skeletal muscle samples were exposed to increasing doses of LPS (0.1–100 μg/mg) for 2 and 4 hours. Respiration rates of intact and permeabilized cells and isolated mitochondria were measured by high-resolution respirometry. Results. In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration. Stimulated complex II respiration was reduced time-dependently using 1 μg/mL LPS. In primary human hepatocytes, stimulated mitochondrial complex II respiration was reduced time-dependently using 1 μg/mL LPS. In isolated porcine skeletal muscle mitochondria, stimulated respiration decreased at high doses (50 and 100 μg/mL LPS). Conclusion. LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential. LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner. PMID:25649304

[Gallic acid inhibits inflammatory response of RAW264.7 macrophages by blocking the activation of TLR4/NF-κB induced by LPS].

PubMed

Huang, Lihua; Hou, Lin; Xue, Hainan; Wang, Chunjie

2016-12-01

Objective To observe the influence of gallic acid on Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) pathway in the RAW264.7 macrophages stimulated by lipopolysaccharide (LPS). Methods RAW264.7 macrophages were divided into the following groups: control group, LPS group, LPS combined with gallic acid group, LPS combined with pyrrolidine dithiocarbamate (PDTC) group and LPS combined with dexamethasone (DM) group. RAW264.7 cells were cultured for 24 hours after corresponding treatments. The levels of tumor necrosis factor α (TNF-α), interleukin-1 (IL-1) and IL-6 were detected by ELISA. The levels of TLR4 and NF-κB mRNAs were tested by real-time PCR. The levels of p-IκBα, p65, p-p65 and TLR4 proteins were examined by Western blotting. Results The expression levels of TNF-α, IL-1 and IL-6 were up-regulated in the RAW264.7 macrophages after stimulated by LPS. Gallic acid could reduce the elevated expression levels of TNF-α, IL-1 and IL-6 induced by LPS. The expression of TLR4 significantly increased after stimulated by LPS and NF-κB was activated. Gallic acid could reverse the above changes and prevent the activation of NF-κB. Conclusion Gallic acid could inhibit LPS-induced inflammatory response in RAW264.7 macrophages via TLR4/NF-κB pathway.

Adsorption of endotoxins on Ca2+ -iminodiacetic acid by metal ion affinity chromatography.

PubMed

Lopes, André Moreni; Romeu, Jorge Sánchez; Meireles, Rolando Páez; Perera, Gabriel Marquez; Morales, Rolando Perdomo; Pessoa, Adalberto; Cárdenas, Lourdes Zumalacárregui

2012-11-01

Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by-products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ -iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+ was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than 100 EU/mL and 100 000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.

Deacylation of Purified Lipopolysaccharides by Cellular and Extracellular Components of a Sterile Rabbit Peritoneal Inflammatory Exudate

PubMed Central

Weinrauch, Yvette; Katz, Seth S.; Munford, Robert S.; Elsbach, Peter; Weiss, Jerrold

1999-01-01

The extent to which the mammalian host is capable of enzymatic degradation and detoxification of bacterial lipopolysaccharides (LPS) is still unknown. Partial deacylation of LPS by the enzyme acyloxyacyl hydrolase (AOAH) provides such a mechanism, but its participation in the disposal of LPS under physiological conditions has not been established. In this study, deacylation of isolated radiolabeled LPS by both cellular and extracellular components of a sterile inflammatory peritoneal exudate elicited in rabbits was examined ex vivo. AOAH-like activity, tested under artificial conditions (pH 5.4, 0.1% Triton X-100), was evident in all components of the exudate (mononuclear cells [MNC] > polymorphonuclear leukocytes [PMN] > inflammatory [ascitic] fluid [AF]). Under more physiological conditions, in a defined medium containing purified LPS-binding protein, the LPS-deacylating activity of MNC greatly exceeded that of PMN. In AF, MNC (but not PMN) also produced rapid and extensive CD14-dependent LPS deacylation. Under these conditions, almost all MNC-associated LPS underwent deacylation within 1 h, a rate greatly exceeding that previously found in any cell type. The remaining extracellular LPS was more slowly subject to CD14-independent deacylation in AF. Quantitative analysis showed a comparable release of laurate and myristate but no release of 3-hydroxymyristate, consistent with an AOAH-like activity. These findings suggest a major role for CD14+ MNC and a secondary role for AF in the deacylation of cell-free LPS at extravascular inflammatory sites. PMID:10377115

Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica.

PubMed

Hajjar, Adeline M; Ernst, Robert K; Fortuno, Edgardo S; Brasfield, Alicia S; Yam, Cathy S; Newlon, Lindsay A; Kollmann, Tobias R; Miller, Samuel I; Wilson, Christopher B

2012-01-01

Although lipopolysaccharide (LPS) stimulation through the Toll-like receptor (TLR)-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of Yersinia pestis LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, Y. pestis LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, Y. pestis LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to Y. pestis for "humanized" TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including Salmonella enterica or Escherichia coli, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, Y. pestis exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with "humanized" TLR4/MD-2 transgenic mice.

The anti-inflammatory effects of baicalin through suppression of NLRP3 inflammasome pathway in LPS-challenged piglet mononuclear phagocytes.

PubMed

Ye, Chun; Li, Sali; Yao, Wenxu; Xu, Lei; Qiu, Yinsheng; Liu, Yu; Wu, Zhongyuan; Hou, Yongqing

2016-04-01

In this study, the anti-inflammatory effects and mechanisms of baicalin on LPS-induced NLRP3 inflammatory pathway were investigated in piglet mononuclear phagocytes (control, LPS stimulation, LPS stimulation + 12.5 µg/ml baicalin, LPS stimulation + 25 µg/ml baicalin, LPS stimulation + 50 µg/ml baicalin and LPS stimulation + 100 µg/ml baicalin). The levels of reactive oxygen species (ROS), the secretion levels of IL-1β, IL-18 and TNF-α, mRNA expression levels of IL-1β, IL-18, TNF-α and NLRP3, as well as the protein levels of cleaved caspase-1 p20 were significantly increased after LPS-challengein vitro However, LPS stimulation did not influence apoptosis-associated speck-like protein and caspase-1 mRNA levels, which are also components of the NLRP3 inflammasome. Baicalin at 50 µg/ml and 100 µg/ml could inhibit the production of ROS, TNF-α, IL-1β and IL-18, and down-regulate mRNA expression of IL-1β, IL-18, TNF-α and NLRP3, as well as expression of cleaved caspase-1 p20. These results showed that the anti-inflammatory effects of baicalin occurred via the regulation of the release of ROS and mRNA expression of NLRP3. The anti-inflammatory activity of baicalin could be related to the suppression of NLRP3 inflammasome pathway under LPS stimulation. © The Author(s) 2016.

Dietary arginine supplementation alleviates intestinal mucosal disruption induced by Escherichia coli lipopolysaccharide in weaned pigs.

PubMed

Liu, Yulan; Huang, Jingjing; Hou, Yongqing; Zhu, Huiling; Zhao, Shengjun; Ding, Binying; Yin, Yulong; Yi, Ganfeng; Shi, Junxia; Fan, Wei

2008-09-01

This study evaluated whether arginine (Arg) supplementation could attenuate gut injury induced by Escherichia coli lipopolysaccharide (LPS) challenge through an anti-inflammatory role in weaned pigs. Pigs were allotted to four treatments including: (1) non-challenged control; (2) LPS-challenged control; (3) LPS+0.5 % Arg; (4) LPS+1.0 % Arg. On day 16, pigs were injected with LPS or sterile saline. At 6 h post-injection, pigs were killed for evaluation of small intestinal morphology and intestinal gene expression. Within 48 h of challenge, 0.5 % Arg alleviated the weight loss induced by LPS challenge (P = 0.025). In all three intestinal segments, 0.5 or 1.0 % Arg mitigated intestinal morphology impairment (e.g. lower villus height and higher crypt depth) induced by LPS challenge (P < 0.05), and alleviated the decrease of crypt cell proliferation and the increase of villus cell apoptosis after LPS challenge (P < 0.01). The 0.5 % Arg prevented the elevation of jejunal IL-6 mRNA abundance (P = 0.082), and jejunal (P = 0.030) and ileal (P = 0.039) TNF-alpha mRNA abundance induced by LPS challenge. The 1.0 % Arg alleviated the elevation of jejunal IL-6 mRNA abundance (P = 0.053) and jejunal TNF-alpha mRNA abundance (P = 0.003) induced by LPS challenge. The 0.5 % Arg increased PPARgamma mRNA abundance in all three intestinal segments (P < 0.10), and 1.0 % Arg increased duodenal PPARgamma mRNA abundance (P = 0.094). These results indicate that Arg supplementation has beneficial effects in alleviating gut mucosal injury induced by LPS challenge. Additionally, it is possible that the protective effects of Arg on the intestine are associated with decreasing the expression of intestinal pro-inflammatory cytokines through activating PPARgamma expression.

Effect of Lipopolysaccharide on Inflammation and Insulin Action in Human Muscle

PubMed Central

Liang, Hanyu; Hussey, Sophie E.; Sanchez-Avila, Alicia; Tantiwong, Puntip; Musi, Nicolas

2013-01-01

Accumulating evidence from animal studies suggest that chronic elevation of circulating intestinal-generated lipopolysaccharide (LPS) (i.e., metabolic endotoxemia) could play a role in the pathogenesis of insulin resistance. However, the effect of LPS in human muscle is unclear. Moreover, it is unknown whether blockade/down regulation of toll-like receptor (TLR)4 can prevent the effect of LPS on insulin action and glucose metabolism in human muscle cells. In the present study we compared plasma LPS concentration in insulin resistant [obese non-diabetic and obese type 2 diabetic (T2DM)] subjects versus lean individuals. In addition, we employed a primary human skeletal muscle cell culture system to investigate the effect of LPS on glucose metabolism and whether these effects are mediated via TLR4. Obese non-diabetic and T2DM subjects had significantly elevated plasma LPS and LPS binding protein (LBP) concentrations. Plasma LPS (r = −0.46, P = 0.005) and LBP (r = −0.49, P = 0.005) concentrations negatively correlated with muscle insulin sensitivity (M). In human myotubes, LPS increased JNK phosphorylation and MCP-1 and IL-6 gene expression. This inflammatory response led to reduced insulin-stimulated IRS-1, Akt and AS160 phosphorylation and impaired glucose transport. Both pharmacologic blockade of TLR4 with TAK-242, and TLR4 gene silencing, suppressed the inflammatory response and insulin resistance caused by LPS in human muscle cells. Taken together, these findings suggest that elevations in plasma LPS concentration found in obese and T2DM subjects could play a role in the pathogenesis of insulin resistance and that antagonists of TLR4 may improve insulin action in these individuals. PMID:23704966

Effect of lipopolysaccharide on inflammation and insulin action in human muscle.

PubMed

Liang, Hanyu; Hussey, Sophie E; Sanchez-Avila, Alicia; Tantiwong, Puntip; Musi, Nicolas

2013-01-01

Accumulating evidence from animal studies suggest that chronic elevation of circulating intestinal-generated lipopolysaccharide (LPS) (i.e., metabolic endotoxemia) could play a role in the pathogenesis of insulin resistance. However, the effect of LPS in human muscle is unclear. Moreover, it is unknown whether blockade/down regulation of toll-like receptor (TLR)4 can prevent the effect of LPS on insulin action and glucose metabolism in human muscle cells. In the present study we compared plasma LPS concentration in insulin resistant [obese non-diabetic and obese type 2 diabetic (T2DM)] subjects versus lean individuals. In addition, we employed a primary human skeletal muscle cell culture system to investigate the effect of LPS on glucose metabolism and whether these effects are mediated via TLR4. Obese non-diabetic and T2DM subjects had significantly elevated plasma LPS and LPS binding protein (LBP) concentrations. Plasma LPS (r = -0.46, P = 0.005) and LBP (r = -0.49, P = 0.005) concentrations negatively correlated with muscle insulin sensitivity (M). In human myotubes, LPS increased JNK phosphorylation and MCP-1 and IL-6 gene expression. This inflammatory response led to reduced insulin-stimulated IRS-1, Akt and AS160 phosphorylation and impaired glucose transport. Both pharmacologic blockade of TLR4 with TAK-242, and TLR4 gene silencing, suppressed the inflammatory response and insulin resistance caused by LPS in human muscle cells. Taken together, these findings suggest that elevations in plasma LPS concentration found in obese and T2DM subjects could play a role in the pathogenesis of insulin resistance and that antagonists of TLR4 may improve insulin action in these individuals.

Edaravone abrogates LPS-induced behavioral anomalies, neuroinflammation and PARP-1.

PubMed

Sriram, Chandra Shaker; Jangra, Ashok; Gurjar, Satendra Singh; Mohan, Pritam; Bezbaruah, Babul Kumar

2016-02-01

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA nick-sensor enzyme that functions at the center of cellular stress response and affects the immune system at several key points, and thus modulates inflammatory diseases. Our previous study demonstrated that lipopolysaccharide (LPS)-induced depressive-like behavior in mice can be ameliorated by 3-aminobenzamide, which is a PARP-1 inhibitor. In the present study we've examined the effect of a free radical scavenger, edaravone pretreatment against LPS-induced anxiety and depressive-like behavior as well as various hippocampal biochemical parameters including PARP-1. Male Swiss albino mice were treated with edaravone (3 & 10mg/kgi.p.) once daily for 14days. On the 14th day 30min after edaravone treatment mice were challenged with LPS (1mg/kgi.p.). After 3h and 24h of LPS administration we've tested mice for anxiety and depressive-like behaviors respectively. Western blotting analysis of PARP-1 in hippocampus was carried out after 12h of LPS administration. Moreover, after 24h of LPS administration serum corticosterone, hippocampal BDNF, oxido-nitrosative stress and pro-inflammatory cytokines were estimated by ELISA. Results showed that pretreatment of edaravone (10mg/kg) ameliorates LPS-induced anxiety and depressive-like behavior. Western blotting analysis showed that LPS-induced anomalous expression of PARP-1 significantly reverses by the pretreatment of edaravone (10mg/kg). Biochemical analyses revealed that LPS significantly diminishes BDNF, increases pro-inflammatory cytokines and oxido-nitrosative stress in the hippocampus. However, pretreatment with edaravone (10mg/kg) prominently reversed all these biochemical alterations. Our study emphasized that edaravone pretreatment prevents LPS-induced anxiety and depressive-like behavior, mainly by impeding the inflammation, oxido-nitrosative stress and PARP-1 overexpression. Copyright © 2015. Published by Elsevier Inc.

Protective effects of aerobic exercise on acute lung injury induced by LPS in mice

PubMed Central

2012-01-01

Introduction The regular practice of physical exercise has been associated with beneficial effects on various pulmonary conditions. We investigated the mechanisms involved in the protective effect of exercise in a model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Methods Mice were divided into four groups: Control (CTR), Exercise (Exe), LPS, and Exercise + LPS (Exe + LPS). Exercised mice were trained using low intensity daily exercise for five weeks. LPS and Exe + LPS mice received 200 µg of LPS intratracheally 48 hours after the last physical test. We measured exhaled nitric oxide (eNO); respiratory mechanics; neutrophil density in lung tissue; protein leakage; bronchoalveolar lavage fluid (BALF) cell counts; cytokine levels in BALF, plasma and lung tissue; antioxidant activity in lung tissue; and tissue expression of glucocorticoid receptors (Gre). Results LPS instillation resulted in increased eNO, neutrophils in BALF and tissue, pulmonary resistance and elastance, protein leakage, TNF-alpha in lung tissue, plasma levels of IL-6 and IL-10, and IL-1beta, IL-6 and KC levels in BALF compared to CTR (P ≤0.02). Aerobic exercise resulted in decreases in eNO levels, neutrophil density and TNF-alpha expression in lung tissue, pulmonary resistance and elastance, and increased the levels of IL-6, IL-10, superoxide dismutase (SOD-2) and Gre in lung tissue and IL-1beta in BALF compared to the LPS group (P ≤0.04). Conclusions Aerobic exercise plays important roles in protecting the lungs from the inflammatory effects of LPS-induced ALI. The effects of exercise are mainly mediated by the expression of anti-inflammatory cytokines and antioxidants, suggesting that exercise can modulate the inflammatory-anti-inflammatory and the oxidative-antioxidative balance in the early phase of ALI. PMID:23078757

Lemongrass and citral effect on cytokines production by murine macrophages.

PubMed

Bachiega, Tatiana Fernanda; Sforcin, José Maurício

2011-09-01

Cymbopogon citratus (DC) Stapf (Poaceae-Gramineae), an herb commonly known as lemongrass (LG), is an important source of ethnomedicines as well as citral, the major constituent of Cymbopogon citratus, used in perfumery, cosmetic and pharmaceutical industries for controlling pathogens. Thus, the goal of this work was to analyze the effect of LG and citral on cytokines production (IL-1β, IL-6 and IL-10) in vitro, as well as before or after LPS incubation. Peritoneal macrophages from BALB/c mice were treated with LG or citral in different concentrations for 24h. The concentrations that inhibited cytokines production were tested before or after macrophages challenge with LPS, in order to evaluate a possible anti-inflammatory action. Supernatants of cell cultures were used for cytokines determination by ELISA. As to IL-1β, only citral inhibited its release, exerting an efficient action before LPS challenge. LG and citral inhibited IL-6 release. Cymbopogon citratus showed inhibitory effects only after LPS challenge, whereas citral prevented efficiently LPS effects before and after LPS addition. Citral inhibited IL-10 production and although LG did not inhibit its production, the concentration of 100 μg/well was tested in the LPS-challenge protocol, because it inhibited IL-6 production. LG inhibited LPS action after macrophages incubation with LPS, while citral counteracted LPS action when added before or after LPS incubation. LG exerted an anti-inflammatory action and citral may be involved in its inhibitory effects on cytokines production. We suggest that a possible mechanism involved in such results could be the inhibition of the transcription factor NF-κB. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis.

PubMed

Dong, Wen-Wen; Zhang, Yun-Qian; Zhu, Xiao-Yan; Mao, Yan-Fei; Sun, Xue-Jun; Liu, Yu-Jian; Jiang, Lai

2017-05-19

BACKGROUND Fibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis. MATERIAL AND METHODS Male ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-β1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined. RESULTS Mice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-β1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress. CONCLUSIONS Hydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

Lipopolysaccharide affects exploratory behaviors toward novel objects by impairing cognition and/or motivation in mice: Possible role of activation of the central amygdala.

PubMed

Haba, Ryota; Shintani, Norihito; Onaka, Yusuke; Wang, Hyper; Takenaga, Risa; Hayata, Atsuko; Baba, Akemichi; Hashimoto, Hitoshi

2012-03-17

Lipopolysaccharide (LPS) produces a series of systemic and psychiatric changes called sickness behavior. In the present study, we characterized the LPS-induced decrease in novel object exploratory behaviors in BALB/c mice. As already reported, LPS (0.3-5 μg/mouse) induced dose- and time-dependent decreases in locomotor activity, food intake, social interaction, and exploration for novel objects, and an increase in immobility in the forced-swim test. Although the decrease in locomotor activity was ameliorated by 10h postinjection, novel object exploratory behaviors remained decreased at 24h and were observed even with the lowest dose of LPS. In an object exploration test, LPS shortened object exploration time but did not affect moving time or the frequency of object exploration. Although pre-exposure to the same object markedly decreased the duration of exploration and LPS did not change this reduction, LPS significantly impaired the exploration of a novel object that replaced the familiar one. LPS did not affect anxiety-like behaviors in open-field and elevated plus-maze tests. An LPS-induced increase in the number of c-Fos-immunoreactive cells was observed in several brain regions within 6h of LPS administration, but the number of cells quickly returned to control levels, except in the central amygdala where the increase continued for 24h. These results suggest that LPS most prominently affects object exploratory behaviors by impairing cognition and/or motivation including continuous attention and curiosity toward objects, and that this may be associated with activation of brain nuclei such as the central amygdala. Copyright © 2012 Elsevier B.V. All rights reserved.

In vitro and in vivo protective effect of arginine against lipopolysaccharide induced inflammatory response in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian).

PubMed

Jiang, Jun; Shi, Dan; Zhou, Xiao-Qiu; Hu, Yi; Feng, Lin; Liu, Yang; Jiang, Wei-Dan; Zhao, Ye

2015-02-01

The present study was designed to assess the possible protective effects of arginine (Arg) against lipopolysaccharide (LPS) induced inflammatory response in juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and in enterocytes in vitro. Firstly, inflammatory response was established by exposing enterocytes to different concentrations of LPS for 24 h. Secondly, the protective effects of Arg against subsequent LPS exposure were studied in enterocytes. Finally, we investigated whether dietary Arg supplementation could attenuate immune challenge induced by LPS in vivo. The result indicated that 10 mg/L LPS could induced inflammatory response in enterocytes. Cells exposed to LPS (10-30 mg/L) alone for 24 h resulted in a significant increase in lactate dehydrogenase release (LDH) (P < 0.05). The cell viability, protein content, alkaline phosphatase activity were decreased by LPS (P < 0.05). Moreover, LPS exposure significantly increased TNF-α, IL-1β, and IL-6 mRNA expression in vitro (P < 0.05). However, pre-treatment with Arg remarkably prevented the increase of TNF-α, IL-1β, and IL-6 by inhibiting the excessive activation of TLR4-Myd88 signaling pathway through down-regulating TLR4, Myd88, NFκB p65, and MAPK p38 mRNA expression (P < 0.05). Interestingly, the experiment in vivo showed that Arg pre-supplementation could attenuate immune challenge induced by LPS via TLR4-Myd88 signaling pathway, and thus protect fish against LPS-induced inflammatory response. In conclusion, all of these results indicated pre-supplementation with Arg decreased LPS induced immune damage and regulated TLR4-Myd88 signaling pathway in juvenile Jian carp in vivo and in enterocytes in vitro. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway.

PubMed

Wang, Jun; Si, Yanfang; Wu, Chen; Sun, Lu; Ma, Yudong; Ge, Aili; Li, Baomin

2012-10-17

Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS) to imitate inflammatory conditions. In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP) as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4) antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB), which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1), compared with transforming growth factor-β1 (TGF-β1). Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial infection contributes to the pathological process of atherosclerosis.

Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway

PubMed Central

2012-01-01

Background Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS) to imitate inflammatory conditions. Results In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP) as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4) antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB), which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1), compared with transforming growth factor-β1 (TGF-β1). Conclusions Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial infection contributes to the pathological process of atherosclerosis. PMID:23072373

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling.

PubMed

Kukolj, Tamara; Trivanović, Drenka; Djordjević, Ivana Okić; Mojsilović, Slavko; Krstić, Jelena; Obradović, Hristina; Janković, Srdja; Santibanez, Juan Francisco; Jauković, Aleksandra; Bugarski, Diana

2018-01-01

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast-like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF-β, fibronectin (FN), α-SMA, and NG2. LPS also increased protein and gene expression levels of anti-inflammatory COX-2 and pro-inflammatory IL-6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS-treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen-stimulated proliferation of CD4 + and the ratio of CD4 + CD25 high /CD4 + CD25 low lymphocytes. LPS-treated PDLSCs did not change the frequency of CD34 + and CD45 + cells, but decreased the frequency of CD33 + and CD14 + myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU-GM number. The results indicated that LPS-activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features. © 2017 Wiley Periodicals, Inc.

Supramolecular structure of enterobacterial wild-type lipopolysaccharides (LPS), fractions thereof, and their neutralization by Pep19-2.5.

PubMed

Brandenburg, Klaus; Heinbockel, Lena; Correa, Wilmar; Fukuoka, Satoshi; Gutsmann, Thomas; Zähringer, Ulrich; Koch, Michel H J

2016-04-01

Lipopolysaccharides (LPS) belong to the strongest immune-modulating compounds known in nature, and are often described as pathogen-associated molecular patterns (PAMPs). In particular, at higher concentrations they are responsible for sepsis and the septic shock syndrome associated with high lethality. Since most data are indicative that LPS aggregates are the bioactive units, their supramolecular structures are considered to be of outmost relevance for deciphering the molecular mechanisms of its bioactivity. So far, however, most of the data available addressing this issue, were published only for the lipid part (lipid A) and the core-oligosaccharide containing rough LPS, representing the bioactive unit. By contrast, it is well known that most of the LPS specimen identified in natural habitats contain the smooth-form (S-form) LPS, which carry additionally a high-molecular polysaccharide (O-chain). To fill this lacuna and going into a more natural system, here various wild-type (smooth form) LPS including also some LPS fractions were investigated by small-angle X-ray scattering with synchrotron radiation to analyze their aggregate structure. Furthermore, the influence of a recently designed synthetic anti-LPS peptide (SALP) Pep19-2.5 on the aggregate structure, on the binding thermodynamics, and on the cytokine-inducing activity of LPS were characterized, showing defined aggregate changes, high affinity binding and inhibition of cytokine secretion. The data obtained are suitable to refine our view on the preferences of LPS for non-lamellar structures, representing the highest bioactive forms which can be significantly influenced by the binding with neutralizing peptides such as Pep19-2.5. Copyright © 2016 Elsevier Inc. All rights reserved.

[Effect of Ca(OH)2 on the cytotoxicity of lipopolysaccharide extracted from Porphyromonas endodontalis in vitro].

PubMed

Guo, Jia-jie; Qiu, Li-hong; Yu, Ya-qiong; Xu, Li-ya; Fan, Yun-qian; Zhong, Ming

2014-02-01

To detect the degradation of Ca(OH)2 on lipopolysaccharide (LPS) extracted from Porphyromonas endodontalis (P.e) in vitro and estimate the influence of P.e LPS pretreated with Ca(OH)2 on the proliferation of MC3T3-E1 cells. The effect of Ca(OH)2 on MC3T3-E1 cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay. Then P.e LPS was treated with Ca(OH)2 for 30 mins or 60 mins at 37 degrees centigrade in vitro and the activity of P.e LPS was evaluated by Chromogenic End-point Tachypleus Amebocyte Lysate (CE TAL) test. Finally, MC3T3-E1 cells were exposed to P.e LPS pretreated with 15% Ca(OH)2 for 1, 3 and 5 d, and the cell proliferation was measured using the MTT assay comparing with the P.e LPS control group. SPSS 13.0 software package was used for statistical analysis. Compared with the negative control, exposing cells to 5%, 10% and 15% Ca(OH)2 had greatly promoted MC3T3-E1 cell proliferation. P.e LPS treated with 10% and 15% Ca(OH)2 both presented the best results by CE TAL and significant difference compared with P.e LPS control group. When 10 μg/mL P.e LPS was pretreated with 15% Ca(OH)2, no inhibition of MC3T3-E1 cell proliferation was noted. Ca(OH)2 detoxifies P.e LPS in vitro, mitigates the impact of P.e LPS on MC3T3-E1 cell proliferation. Supported by Science and Technology Projects of Liaoning Province (2011225020).

Transiently enhanced LPS-induced fever following hyperthermic stress in rabbits

NASA Astrophysics Data System (ADS)

Shibata, Masaaki; Uno, Tadashi; Riedel, Walter; Nishimaki, Michiyo; Watanabe, Kaori

2005-11-01

Hyperthermia has been shown to induce an enhanced febrile response to the bacterial-derived endotoxin lipopolysaccharide (LPS). The aim of the present study was to test the hypothesis that the enhanced LPS-induced fever seen in heat stressed (HS) animals is caused by leakage of intestinal bacterial LPS into the circulation. Male rabbits were rendered transiently hyperthermic (a maximum rectal temperature of 43°C) and divided into three groups. They were then allowed to recover in a room at 24°C for 1, 2 or 3 days post-HS. One day after injection with LPS, the post-HS rabbits exhibited significantly higher fevers than the controls, though this was not seen in rabbits at either 2 or 3 days post-HS. The plasma levels of endogenous LPS were significantly increased during the HS as compared to those seen in normothermic rabbits prior to HS. LPS fevers were not induced in these animals. One day post-HS, rabbits that had been pretreated with oral antibiotics exhibited significantly attenuated LPS levels. When challenged with human recombinant interleukin-1β instead of LPS, the 1-day post-HS rabbits did not respond with enhanced fevers. The plasma levels of TNFα increased similarly during LPS-induced fevers in both the control and 1-day post-HS rabbits, while the plasma levels of corticosterone and the osmolality of the 1-day post-HS rabbits showed no significant differences to those seen prior to the HS. These results suggest that the enhanced fever in the 1-day post-HS rabbits is LPS specific, and may be caused by increased leakage of intestinal endotoxin into blood circulation.

Wool and grain dusts stimulate TNF secretion by alveolar macrophages in vitro.

PubMed Central

Brown, D M; Donaldson, K

1996-01-01

OBJECTIVE: The aim of the study was to investigate the ability of two organic dusts, wool and grain, and their soluble leachates to stimulate secretion of tumour necrosis factor (TNF) by rat alveolar macrophages with special reference to the role of lipopolysaccharide (LPS). METHODS: Rat alveolar macrophages were isolated by bronchoalveolar lavage (BAL) and treated in vitro with whole dust, dust leachates, and a standard LPS preparation. TNF production was measured in supernatants with the L929 cell line bioassay. RESULTS: Both wool and grain dust samples were capable of stimulating TNF release from rat alveolar macrophages in a dose-dependent manner. The standard LPS preparation caused a dose-dependent secretion of TNF. Leachates prepared from the dusts contained LPS and also caused TNF release but leachable LPS could not account for the TNF release and it was clear that non-LPS leachable activity was present in the grain dust and that wool dust particles themselves were capable of causing release of TNF. The role of LPS in wool dust leachates was further investigated by treating peritoneal macrophages from two strains of mice, LPS responders (C3H) and LPS non-responders (C3H/HEJ), with LPS. The non-responder mouse macrophages produced very low concentrations of TNF in response to the wool dust leachates compared with the responders. CONCLUSIONS: LPS and other unidentified leachable substances present on the surface of grain dust, and to a lesser extent on wool dust, are a trigger for TNF release by lung macrophages. Wool dust particles themselves stimulate TNF. TNF release from macrophages could contribute to enhancement of inflammatory responses and symptoms of bronchitis and breathlessness in workers exposed to organic dusts such as wool and grain. PMID:8758033

Wool and grain dusts stimulate TNF secretion by alveolar macrophages in vitro.

PubMed

Brown, D M; Donaldson, K

1996-06-01

The aim of the study was to investigate the ability of two organic dusts, wool and grain, and their soluble leachates to stimulate secretion of tumour necrosis factor (TNF) by rat alveolar macrophages with special reference to the role of lipopolysaccharide (LPS). Rat alveolar macrophages were isolated by bronchoalveolar lavage (BAL) and treated in vitro with whole dust, dust leachates, and a standard LPS preparation. TNF production was measured in supernatants with the L929 cell line bioassay. Both wool and grain dust samples were capable of stimulating TNF release from rat alveolar macrophages in a dose-dependent manner. The standard LPS preparation caused a dose-dependent secretion of TNF. Leachates prepared from the dusts contained LPS and also caused TNF release but leachable LPS could not account for the TNF release and it was clear that non-LPS leachable activity was present in the grain dust and that wool dust particles themselves were capable of causing release of TNF. The role of LPS in wool dust leachates was further investigated by treating peritoneal macrophages from two strains of mice, LPS responders (C3H) and LPS non-responders (C3H/HEJ), with LPS. The non-responder mouse macrophages produced very low concentrations of TNF in response to the wool dust leachates compared with the responders. LPS and other unidentified leachable substances present on the surface of grain dust, and to a lesser extent on wool dust, are a trigger for TNF release by lung macrophages. Wool dust particles themselves stimulate TNF. TNF release from macrophages could contribute to enhancement of inflammatory responses and symptoms of bronchitis and breathlessness in workers exposed to organic dusts such as wool and grain.

Dietary l-threonine supplementation attenuates lipopolysaccharide-induced inflammatory responses and intestinal barrier damage of broiler chickens at an early age.

PubMed

Chen, Yueping; Zhang, Hao; Cheng, Yefei; Li, Yue; Wen, Chao; Zhou, Yanmin

2018-06-01

This study was conducted to investigate the protective effects of l-threonine (l-Thr) supplementation on growth performance, inflammatory responses and intestinal barrier function of young broilers challenged with lipopolysaccharide (LPS). A total of 144 1-d-old male chicks were allocated to one of three treatments: non-challenged broilers fed a basal diet (control group), LPS-challenged broilers fed a basal diet without l-Thr supplementation and LPS-challenged broilers fed a basal diet supplemented with 3·0 g/kg l-Thr. LPS challenge was performed intraperitoneally at 17, 19 and 21 d of age, whereas the control group received physiological saline injection. Compared with the control group, LPS challenge impaired growth performance of broilers, and l-Thr administration reversed LPS-induced increase in feed/gain ratio. LPS challenge elevated blood cell counts related to inflammation, and pro-inflammatory cytokine concentrations in serum (IL-1β and TNF-α), spleen (IL-1β and TNF-α) and intestinal mucosa (jejunal interferon-γ (IFN-γ) and ileal IL-1β). The concentrations of intestinal cytokines in LPS-challenged broilers were reduced by l-Thr supplementation. LPS administration increased circulating d-lactic acid concentration, whereas it reduced villus height, the ratio between villus height and crypt depth and goblet density in both jejunum and ileum. LPS-induced decreases in jejunal villus height, intestinal villus height:crypt depth ratio and ileal goblet cell density were reversed with l-Thr supplementation. Similarly, LPS-induced alterations in the intestinal mRNA abundances of genes related to intestinal inflammation and barrier function (jejunal toll-like receptor 4, IFN- γ and claudin-3, and ileal IL-1 β and zonula occludens-1) were normalised with l-Thr administration. It can be concluded that l-Thr supplementation could attenuate LPS-induced inflammatory responses and intestinal barrier damage of young broilers.

Protective effect of thalidomide on endotoxin-induced liver injury.

PubMed

Enomoto, Nobuyuki; Takei, Yoshiyuki; Hirose, Miyoko; Kitamura, Tsuneo; Ikejima, Kenichi; Sato, Nobuhiro

2003-08-01

Activation of Kupffer cells by lipopolysaccharide (LPS) plays a pivotal role in the onset of pathophysiological events that occur during endotoxemia, and intracellular calcium concentration ([Ca2+]i) is involved in LPS-stimulated cytokine production. Tumor necrosis factor (TNF)-alpha is produced exclusively by the monocyte-macrophage lineage, which is mostly made up of Kupffer cells, and thalidomide has been shown to reduce TNF-alpha production from macrophages. However, there is increasing evidence that TNF-alpha may play a role in the initiation or progression of multiple organ failure syndrome. Therefore, the purpose of this work was to determine whether thalidomide could prevent LPS-induced liver injury. Rats were given a single oral dose of thalidomide (5 mg/kg). To assess the sensitization of Kupffer cells, LPS (5 or 10 mg/kg) was administered intravenously, and mortality, liver histology, and transaminases were evaluated 24 hr later. Kupffer cells were isolated 2 hr after thalidomide treatment. After the addition of LPS, [Ca2+]i was measured by using a microspectrofluorometer with the fluorescent indicator fura-2, and TNF-alpha was measured by enzyme-linked immunosorbent assay. LPS caused focal necrosis with neutrophil infiltration in the liver. Moreover, LPS dramatically increased transaminases. These pathologic parameters and increases of serum transaminases were diminished markedly by thalidomide. In isolated Kupffer cells, LPS-induced increases in [Ca2+]i and TNF-alpha production were suppressed by treatment with thalidomide. To further explore the mechanism by which thalidomide directly abrogated Kupffer cell sensitivity to LPS, we determined the effect of thalidomide (5 microM) in vitro on LPS-induced [Ca2+]i response and TNF-alpha production. With the addition of thalidomide (5 microM) in vitro to the culture media for 2 hr before LPS, these parameters were suppressed. Thalidomide prevents LPS-induced liver injury via mechanisms dependent on the suppression of TNF-alpha production from Kupffer cells.

Febuxostat protects rats against lipopolysaccharide-induced lung inflammation in a dose-dependent manner.

PubMed

Fahmi, Alaa N A; Shehatou, George S G; Shebl, Abdelhadi M; Salem, Hatem A

2016-03-01

The aim of the present work was to investigate possible protective effects of febuxostat, a highly potent xanthine oxidase inhibitor, against acute lung injury (ALI) induced by lipopolysaccharide (LPS) in rats. Male Sprague Dawley rats were randomly divided into six groups, as follows: (i) vehicle control group; (ii) and (iii) febuxostat 10 and febuxostat 15 groups, drug-treated controls; (iv) LPS group, receiving an intraperitoneal injection of LPS (7.5 mg/kg); (v) and (vi) febuxostat 10-LPS and febuxostat 15-LPS groups, receiving oral treatment of febuxostat (10 and 15 mg/kg/day, respectively) for 7 days before LPS. After 18 h administration of LPS, blood was collected for C-reactive protein (CRP) measurement. Bronchoalveolar lavage fluid (BALF) was examined for leukocyte infiltration, lactate dehydrogenase (LDH) activity, protein content, and total nitrate/nitrite. Lung weight gain was determined, and lung tissue homogenate was prepared and evaluated for oxidative stress. Tumor necrosis factor-α (TNF-α) was assessed in BALF and lung homogenate. Moreover, histological changes of lung tissues were evaluated. LPS elicited lung injury characterized by increased lung water content (by 1.2 fold), leukocyte infiltration (by 13 fold), inflammation and oxidative stress (indicated by increased malondialdehyde (MDA), by 3.4 fold), and reduced superoxide dismutase (SOD) activity (by 34 %). Febuxostat dose-dependently decreased LPS-induced lung edema and elevations in BALF protein content, infiltration of leukocytes, and LDH activity. Moreover, the elevated levels of TNF-α in BALF and lung tissue of LPS-treated rats were attenuated by febuxostat pretreatment. Febuxostat also displayed a potent antioxidant activity by decreasing lung tissue levels of MDA and enhancing SOD activity. Histological analysis of lung tissue further demonstrated that febuxostat dose-dependently reversed LPS-induced histopathological changes. These findings demonstrate a significant dose-dependent protection by febuxostat against LPS-induced lung inflammation in rats.

In vivo and in vitro effects of lysine clonixinate on nitric oxide synthase in LPS-treated and untreated rat lung preparations.

PubMed

Franchi, A M; Di Girolamo, G; Farina, M; de los Santos, A R; Martí, M L; Gimeno, M A

2001-04-01

Recent studies have shown that some nonsteroidal antiinflammatory drugs (NSAIDS) inhibited the inducible NO synthase (iNOS) without direct effect on the catalytic activity of this enzyme. This study was conducted to investigate the in vitro and in vivo effects of lysine clonixinate (LC) and indomethacin (INDO) on NOS activity in rat lung preparation. LC is a drug with antiinflammatory, antipyretic, and analgesic action. In the in vitro experiments, rats were injected with saline or lipopolysaccharide (LPS) and killed 6 h after treatment. Lung preparations were incubated with LC at 2.3 x 10(-5) M or 3.8 x 10(-5) M. The minimum concentration did not modify NOS activity in control or LPS-treated rats but the maximum dose inhibited increased NO production induced by LPS. Furthermore, INDO at 10(-6) M had no effect on enzymatic activity in control or LPS-treated rats. In the in vivo experiments, 40 mg/kg of LC were injected ip. Such a dose did not affect basal production of NO. When LC and LPS were injected simultaneously 6 h before sacrifice, a significant decrease in LPS-induced NOS activity was observed. INDO 10 mg/kg injected in control animals had no effect on NOS activity and did not block LPS induced stimulation of NO production when injected simultaneously. Finally, when LC (40 mg/kg) was injected 3 h after LPS, the enzymatic activity remained unchanged. Expression of iNOS was detected by Western blotting in rats treated with LPS plus 4, 10, 20, and 40 mg/kg of LC. The lowest dose was the only one showing no effect on LPS-induced increase of iNOS. In short, LC is a NSAID with inhibitory action on the expression of LPS-induced NOS, effect that was not seen with INDO in our experimental conditions. Copyright 2001 Academic Press.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through a TLR4-TRIF-PI3K signaling pathway.

PubMed

Watts, Bruns A; George, Thampi; Sherwood, Edward R; Good, David W

2017-07-01

Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that induces tolerance to LPS and augments host resistance to bacterial infections. Previously, we demonstrated that LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a basolateral Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-ERK pathway. Here we examined whether pretreatment with MPLA would attenuate LPS inhibition. MTALs from rats were perfused in vitro with MPLA (1 µg/ml) in bath and lumen or bath alone for 2 h, and then LPS was added to (and MPLA removed from) the bath solution. Pretreatment with MPLA eliminated LPS-induced inhibition of [Formula: see text] absorption. In MTALs pretreated with MPLA plus a phosphatidylinositol 3-kinase (PI3K) or Akt inhibitor, LPS decreased [Formula: see text] absorption. MPLA increased Akt phosphorylation in dissected MTALs. The Akt activation was eliminated by a PI3K inhibitor and in MTALs from TLR4 -/- or Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF) -/- mice. The effect of MPLA to prevent LPS inhibition of [Formula: see text] absorption also was TRIF dependent. Pretreatment with MPLA prevented LPS-induced ERK activation; this effect was dependent on PI3K. MPLA alone had no effect on [Formula: see text] absorption, and MPLA pretreatment did not prevent ERK-mediated inhibition of [Formula: see text] absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit [Formula: see text] absorption in the MTAL. This protective effect is mediated directly through MPLA stimulation of a TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced ERK activation. These studies identify detoxified TLR4-based immunomodulators as novel potential therapeutic agents to prevent or treat renal tubule dysfunction in response to bacterial infections. Copyright © 2017 the American Physiological Society.

Comprehensive adipocytic and neurogenic tissue microarray analysis of NY-ESO-1 expression - a promising immunotherapy target in malignant peripheral nerve sheath tumor and liposarcoma

PubMed Central

Shurell, Elizabeth; Vergara-Lluri, Maria E.; Li, Yunfeng; Crompton, Joseph G.; Singh, Arun; Bernthal, Nicholas; Wu, Hong; Eilber, Fritz C.; Dry, Sarah M.

2016-01-01

Background Immunotherapy targeting cancer-testis antigen NY-ESO-1 shows promise for tumors with poor response to chemoradiation. Malignant peripheral nerve sheath tumors (MPNSTs) and liposarcomas (LPS) are chemoresistant and have few effective treatment options. Materials Methods Using a comprehensive tissue microarray (TMA) of both benign and malignant tumors in primary, recurrent, and metastatic samples, we examined NY-ESO-1 expression in peripheral nerve sheath tumor (PNST) and adipocytic tumors. The PNST TMA included 42 MPNSTs (spontaneous n = 26, NF1-associated n = 16), 35 neurofibromas (spontaneous n = 22, NF-1 associated n = 13), 11 schwannomas, and 18 normal nerves. The LPS TMA included 48 well-differentiated/dedifferentiated (WD/DD) LPS, 13 myxoid/round cell LPS, 3 pleomorphic LPS, 8 lipomas, 1 myelolipoma, and 3 normal adipocytic tissue samples. Stained in triplicate, NY-ESO-1 intensity and density were scored. Results NY-ESO-1 expression was exclusive to malignant tumors. 100% of myxoid/round cell LPS demonstrated NY-ESO-1 expression, while only 6% of WD/DD LPS showed protein expression, one of which was WD LPS. Of MPNST, 4/26 (15%) spontaneous and 2/16 (12%) NF1-associated MPNSTs demonstrated NY-ESO-1 expression. Strong NY-ESO-1 expression was observed in myxoid/round cell and dedifferentiated LPS, and MPNST in primary, neoadjuvant, and metastatic settings. Conclusions We found higher prevalence of NY-ESO-1 expression in MPNSTs than previously reported, highlighting a subset of MPNST patients who may benefit from immunotherapy. This study expands our understanding of NY-ESO-1 in WD/DD LPS and is the first demonstration of staining in a WD LPS and metastatic/recurrent myxoid/round cell LPS. These results suggest immunotherapy targeting NY-ESO-1 may benefit patients with aggressive tumors resistant to conventional therapy. PMID:27655679

Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88

PubMed Central

Guo, Shuhong; Nighot, Meghali; Al-Sadi, Rana; Alhmoud, Tarik; Nighot, Prashant; Ma, Thomas Y.

2015-01-01

Gut-derived bacterial lipopolysaccharides (LPS) play an essential role in inducing intestinal and systemic inflammatory responses and have been implicated as a pathogenic factor of necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD). The defective intestinal tight junction (TJ) barrier has been shown to be an important factor contributing to the development of intestinal inflammation. LPS, at physiological concentrations, cause an increase in intestinal tight junction permeability (TJP) via a TLR-4 dependent process; however the intracellular mechanisms that mediate LPS regulation of intestinal TJP remain unclear. The aim of this study was to investigate the adaptor proteins and the signaling interactions that mediate LPS modulation of intestinal TJ barrier using an in-vitro and in-vivo model system. LPS caused a TLR-4 dependent activation of membrane-associated adaptor protein FAK in Caco-2 monolayers. LPS caused an activation of both MyD88-dependent and –independent pathways. SiRNA silencing of MyD88 prevented LPS-induced increase in TJP. LPS caused a MyD88-dependent activation of IRAK4. TLR-4, FAK and MyD88 were co-localized. SiRNA silencing of TLR-4 inhibited TLR-4 associated FAK activation; and FAK knockdown prevented MyD88 activation. In-vivo studies also confirmed that LPS-induced increase in mouse intestinal permeability was associated with FAK and MyD88 activation; knockdown of intestinal epithelial FAK prevented LPS-induced increase in intestinal permeability. Additionally, high dose LPS-induced intestinal inflammation was also dependent on TLR-4/FAK/MyD88 signal-transduction axis. Our data show for the first time that LPS-induced increase in intestinal TJP and intestinal inflammation was regulated by TLR-4 dependent activation of FAK-MyD88-IRAK4 signaling pathway. PMID:26466961

Role of innate immunity and altered intestinal motility in LPS- and MnCl2-induced intestinal intussusception in mice

PubMed Central

Killoran, Kristin E.; Miller, Amber D.; Uray, Karen S.; Weisbrodt, Norman W.; Pautler, Robia G.; Goyert, Sanna M.; van Rooijen, Nico

2014-01-01

Intestinal intussusception (ISS) commonly causes intestinal obstruction in children. One mechanism that has been proposed to cause ISS is inflammation-induced alteration of intestinal motility. We investigated whether innate inflammatory factors or altered motility is required for induction of ISS by LPS. We compared rates of ISS among BALB/c and C57BL/6 mice, mice lacking lymphocytes or depleted of phagocytes, or mice with defects in the Toll-like receptor 4 (TLR4) signaling pathway following administration of LPS or the Ca2+ analog MnCl2. At 6 or 2 h after administration of LPS or MnCl2, respectively, mice underwent image analysis to assess intestinal contraction rate or laparotomy to identify ISS. LPS-induced ISS (LPS-ISS) was observed in BALB/c mice, but not in C57BL/6 mice or any BALB/c mice with disruptions of TLR4 signaling. LPS-induced serum TNF-α, IL-6, and nitric oxide (NO) and intestinal NO levels were similar in BALB/c and C57BL/6 mice. The rate of LPS-ISS was significantly reduced in phagocyte-depleted, but not lymphocyte-deficient, mice. Intestinal contraction rates were reduced in LPS-ISS-susceptible BALB/c mice, but not in LPS-ISS-resistant C57BL/6 or TLR4 mutant mice, suggesting a role for reduced intestinal contraction rate in LPS-ISS susceptibility. This was tested with MnCl2, a Ca2+ antagonist that reduced intestinal contraction rates and induced ISS, irrespective of mouse strain. Therefore, LPS-ISS is initiated by innate immune signaling that requires TLR4 and phagocytes but may be independent of TNF-α, IL-6, and NO levels. Furthermore, alteration of intestinal motility, specifically, reduced intestinal contraction rate, is a key factor in the development of ISS. PMID:24407593

LYATK1 potently inhibits LPS-mediated pro-inflammatory response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xi, Feng; Liu, Yuan; Wang, Xiujuan

Lipopolysaccharide (LPS)-primed monocytes/macrophages produce pro-inflammatory cytokines, which could lead to endotoxin shock. TGF-β-activated kinase1 (TAK1) activation is involved in the process. In the current study, we studied the potential effect of a selective TAK1 inhibitor, LYTAK1, on LPS-stimulated response both in vitro and in vivo. We demonstrated that LYTAK1 inhibited LPS-induced mRNA expression and production of several pro-inflammatory cytokines [interleukin 1β (IL-1β), tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6)] in RAW 264.7 macrophages. LYTAK1's activity was almost nullified with TAK1 shRNA-knockdown. Meanwhile, in both primary mouse bone marrow derived macrophages (BMDMs) and human peripheral blood mononuclear cells (PBMCs), LPS-induced pro-inflammatory cytokine productionmore » was again attenuated with LYTAK1 co-treatment. Molecularly, LYTAK1 dramatically inhibited LPS-induced TAK1-nuclear factor kappa B (NFκB) and mitogen-activated protein kinase (Erk, Jnk and p38) activation in RAW 264.7 cells, mouse BMDMs and human PBMCs. In vivo, oral administration of LYTAK1 inhibited LPS-induced activation of TAK1-NFκB-p38 in ex-vivo cultured PBMCs, and cytokine production and endotoxin shock in mice. Together, these results demonstrate that LYTAK1 inhibits LPS-induced production of several pro-inflammatory cytokines and endotoxin shock probably through blocking TAK1-regulated signalings. - Highlights: • LYTAK1 inhibits LPS-induced pro-inflammatory cytokine production in RAW 264.7 cells. • The effect by LYTAK1 is more potent than other known TAK1 inhibitors. • LYTAK1 inhibits LPS-induced cytokine production in primary macrophages/monocytes. • LYTAK1 inhibits LPS-induced TAK1-NFκB and MAPK activation in macrophages/monocytes. • LYTAK1 gavage inhibits LPS-induced endotoxin shock and cytokine production in mice.« less

Ketamine inhibits tumor necrosis factor-{alpha} and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, G.-J.; Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 {mu}M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 {mu}M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-{alpha}more » and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-{alpha} and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 {mu}M) significantly inhibited LPS-induced TNF-{alpha} and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-{alpha} and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-{alpha} and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated sequential activations of c-Jun N-terminal kinase and activator protein-1.« less

Ketamine inhibits tumor necrosis factor-alpha and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation.

PubMed

Wu, Gone-Jhe; Chen, Ta-Liang; Ueng, Yune-Fang; Chen, Ruei-Ming

2008-04-01

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 microM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 microM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-alpha and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-alpha and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 microM) significantly inhibited LPS-induced TNF-alpha and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-alpha and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-alpha and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated sequential activations of c-Jun N-terminal kinase and activator protein-1.

Relating the physical properties of Pseudomonas aeruginosa lipopolysaccharides to virulence by atomic force microscopy.

PubMed

Ivanov, Ivan E; Kintz, Erica N; Porter, Laura A; Goldberg, Joanna B; Burnham, Nancy A; Camesano, Terri A

2011-03-01

Lipopolysaccharides (LPS) are an important class of macromolecules that are components of the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa. P. aeruginosa contains two different sugar chains, the homopolymer common antigen (A band) and the heteropolymer O antigen (B band), which impart serospecificity. The characteristics of LPS are generally assessed after isolation rather than in the context of whole bacteria. Here we used atomic force microscopy (AFM) to probe the physical properties of the LPS of P. aeruginosa strain PA103 (serogroup O11) in situ. This strain contains a mixture of long and very long polymers of O antigen, regulated by two different genes. For this analysis, we studied the wild-type strain and four mutants, ΔWzz1 (producing only very long LPS), ΔWzz2 (producing only long LPS), DΔM (with both the wzz1 and wzz2 genes deleted), and Wzy::GM (producing an LPS core oligosaccharide plus one unit of O antigen). Forces of adhesion between the LPS on these strains and the silicon nitride AFM tip were measured, and the Alexander and de Gennes model of steric repulsion between a flat surface and a polymer brush was used to calculate the LPS layer thickness (which we refer to as length), compressibility, and spacing between the individual molecules. LPS chains were longest for the wild-type strain and ΔWzz1, at 170.6 and 212.4 nm, respectively, and these values were not statistically significantly different from one another. Wzy::GM and DΔM have reduced LPS lengths, at 34.6 and 37.7 nm, respectively. Adhesion forces were not correlated with LPS length, but a relationship between adhesion force and bacterial pathogenicity was found in a mouse acute pneumonia model of infection. The adhesion forces with the AFM probe were lower for strains with LPS mutations, suggesting that the wild-type strain is optimized for maximal adhesion. Our research contributes to further understanding of the role of LPS in the adhesion and virulence of P. aeruginosa.

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling.

PubMed

Buntin, Nirunya; Hongpattarakere, Tipparat; Ritari, Jarmo; Douillard, François P; Paulin, Lars; Boeren, Sjef; Shetty, Sudarshan A; de Vos, Willem M

2017-01-15

The draft genomes of Lactobacillus plantarum strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of L. plantarum, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. β-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of fosRABCDXE, which encodes β-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover, pts1BCA genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the fosRABCDXE operon involved in the inulin utilization of L. plantarum The expression levels of the fos operon and pst genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored β-fructosidase were induced to a high abundance when inulin was present as a carbon source. Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development. Copyright © 2016 American Society for Microbiology.

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling

PubMed Central

Buntin, Nirunya; Hongpattarakere, Tipparat; Ritari, Jarmo; Douillard, François P.; Paulin, Lars; Boeren, Sjef; Shetty, Sudarshan A.

2016-01-01

ABSTRACT The draft genomes of Lactobacillus plantarum strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of L. plantarum, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. β-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of fosRABCDXE, which encodes β-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover, pts1BCA genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the fosRABCDXE operon involved in the inulin utilization of L. plantarum. The expression levels of the fos operon and pst genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored β-fructosidase were induced to a high abundance when inulin was present as a carbon source. IMPORTANCE Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development. PMID:27815279

PccD Regulates Branched-Chain Amino Acid Degradation and Exerts a Negative Effect on Erythromycin Production in Saccharopolyspora erythraea.

PubMed

Xu, Zhen; Liu, Yong; Ye, Bang-Ce

2018-04-15

Branched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis in Saccharopolyspora erythraea In this study, we found that the bkd operon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of the bkd operon was upregulated 5-fold in a pccD gene deletion strain (Δ pccD strain) and decreased 3-fold in a pccD overexpression strain (WT/pIB- pccD ), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion of pccD significantly improved the Δ pccD strain's growth rate, whereas pccD overexpression repressed WT/pIB- pccD growth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion of gdhA1 and the BcdhE1 gene (genes in the bkd operon) resulted in lower growth rates of Δ gdhA1 and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that the bkd operon is involved in BCAA degradation. Both bkd overexpression (WT/pIB- bkd ) and pccD inactivation (Δ pccD strain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB- pccD was decreased by 48%. Lastly, we explored the applications of engineering pccD and bkd in an industrial high-erythromycin-producing strain. pccD deletion in industrial strain S. erythraea E3 (E3 pccD ) improved erythromycin production by 20%, and the overexpression of bkd in E3Δ pccD (E3Δ pccD /pIB- bkd ) increased erythromycin production by 39% compared with S. erythraea E3 in an industrial fermentation medium. Addition of 30 mM valine to industrial fermentation medium further improved the erythromycin production by 23%, a 72% increase from the initial strain S. erythraea E3. IMPORTANCE We describe a bkd operon involved in BCAA degradation in S. erythraea The genes of the operon are repressed by a TetR regulator, PccD. The results demonstrated that PccD controlled the supply of precursors for biosynthesis of erythromycin via regulating the BCAA degradation and propionyl-CoA assimilation and exerted a negative effect on erythromycin production. The findings reveal a regulatory mechanism in feeder pathways and provide new strategies for designing metabolic engineering to increase erythromycin yield. Copyright © 2018 American Society for Microbiology.

Administration of LPS three times during gestation alters the postnatal acute phase and metabolic responses to an LPS challenge in weaned beef heifers

USDA-ARS?s Scientific Manuscript database

This study evaluated whether three administrations of lipopolysaccharide (LPS) during gestation would alter the acute phase (APR) and metabolic responses to a postnatal LPS challenge in weaned heifers. Pregnant crossbred cows (n=50) were randomized into prenatal immune stimulation (PIS; n=24; admini...

Fenbendazole treatment may influence lipopolysaccharide effects in rat brain.

PubMed

Hunter, Randy L; Choi, Dong-Young; Kincer, Jeanie F; Cass, Wayne A; Bing, Guoying; Gash, Don M

2007-10-01

In evaluating discrepant results between experiments in our laboratory, we collected data that challenge the notion that anthelminthic drugs like FBZ do not alter inflammatory responses. We found that FBZ significantly modulates inflammation in F344 rats intrastriatally injected with LPS. FBZ treatment of LPS-injected rats significantly increased weight loss, microglial activation, and dopamine loss; in addition, FBZ attenuated the LPS-induced loss of astrocytes. Therefore, FBZ treatment altered the effects of LPS injection. Caution should be used in interpreting data collected from rats treated with LPS and FBZ.

Genome analysis of the freshwater planktonic Vulcanococcus limneticus sp. nov. reveals horizontal transfer of nitrogenase operon and alternative pathways of nitrogen utilization.

PubMed

Di Cesare, Andrea; Cabello-Yeves, Pedro J; Chrismas, Nathan A M; Sánchez-Baracaldo, Patricia; Salcher, Michaela M; Callieri, Cristiana

2018-04-16

Many cyanobacteria are capable of fixing atmospheric nitrogen, playing a crucial role in biogeochemical cycling. Little is known about freshwater unicellular cyanobacteria Synechococcus spp. at the genomic level, despite being recognised of considerable ecological importance in aquatic ecosystems. So far, it has not been shown whether these unicellular picocyanobacteria have the potential for nitrogen fixation. Here, we present the draft-genome of the new pink-pigmented Synechococcus-like strain Vulcanococcus limneticus. sp. nov., isolated from the volcanic Lake Albano (Central Italy). The novel species Vulcanococcus limneticus sp. nov. falls inside the sub-cluster 5.2, close to the estuarine/marine strains in a maximum-likelihood phylogenetic tree generated with 259 marker genes with representatives from marine, brackish, euryhaline and freshwater habitats. V.limneticus sp. nov. possesses a complete nitrogenase and nif operon. In an experimental setup under nitrogen limiting and non-limiting conditions, growth was observed in both cases. However, the nitrogenase genes (nifHDK) were not transcribed, i.e., V.limneticus sp. nov. did not fix nitrogen, but instead degraded the phycobilisomes to produce sufficient amounts of ammonia. Moreover, the strain encoded many other pathways to incorporate ammonia, nitrate and sulphate, which are energetically less expensive for the cell than fixing nitrogen. The association of the nif operon to a genomic island, the relatively high amount of mobile genetic elements (52 transposases) and the lower observed GC content of V.limneticus sp. nov. nif operon (60.54%) compared to the average of the strain (68.35%) support the theory that this planktonic strain may have obtained, at some point of its evolution, the nif operon by horizontal gene transfer (HGT) from a filamentous or heterocystous cyanobacterium. In this study, we describe the novel species Vulcanococcus limneticus sp. nov., which possesses a complete nif operon for nitrogen fixation. The finding that in our experimental conditions V.limneticus sp. nov. did not express the nifHDK genes led us to reconsider the actual ecological meaning of these accessory genes located in genomic island that have possibly been acquired via HGT.

Regulation of the yjjQ-bglJ Operon, Encoding LuxR-Type Transcription Factors, and the Divergent yjjP Gene by H-NS and LeuO▿ †

PubMed Central

Stratmann, Thomas; Madhusudan, S.; Schnetz, Karin

2008-01-01

The yjjQ and bglJ genes encode LuxR-type transcription factors conserved in several enterobacterial species. YjjQ is a potential virulence factor in avian pathogenic Escherichia coli. BglJ counteracts the silencing of the bgl (β-glucoside) operon by H-NS in E. coli K-12. Here we show that yjjQ and bglJ form an operon carried by E. coli K-12, whose expression is repressed by the histone-like nucleoid structuring (H-NS) protein. The LysR-type transcription factor LeuO counteracts this repression. Furthermore, the yjjP gene, encoding a membrane protein of unknown function and located upstream in divergent orientation to the yjjQ-bglJ operon, is likewise repressed by H-NS. Mapping of the promoters as well as the H-NS and LeuO binding sites within the 555-bp intergenic region revealed that H-NS binds to the center of the AT-rich regulatory region and distal to the divergent promoters. LeuO sites map to the center and to positions distal to the yjjQ promoters, while one LeuO binding site overlaps with the divergent yjjP promoter. This latter LeuO site is required for full derepression of the yjjQ promoters. The arrangement of regulatory sites suggests that LeuO restructures the nucleoprotein complex formed by H-NS. Furthermore, the data support the conclusion that LeuO, whose expression is likewise repressed by H-NS and which is a virulence factor in Salmonella enterica, is a master regulator that among other loci, also controls the yjjQ-bglJ operon and thus indirectly the presumptive targets of YjjQ and BglJ. PMID:18055596

Co-spread of metal and antibiotic resistance within ST3-IncHI2 plasmids from E. coli isolates of food-producing animals

PubMed Central

Fang, Liangxing; Li, Xingping; Li, Liang; Li, Shumin; Liao, Xiaoping; Sun, Jian; Liu, Yahong

2016-01-01

Concerns have been raised in recent years regarding co-selection for antibiotic resistance among bacteria exposed to heavy metals, particularly copper and zinc, used as growth promoters for some livestock species. In this study, 25 IncHI2 plasmids harboring oqxAB (20/25)/blaCTX-M (18/25) were found with sizes ranging from ∼260 to ∼350 kb and 22 belonged to the ST3-IncHI2 group. In addition to blaCTX-M and oqxAB, pcoA-E (5/25) and silE-P (5/25), as well as aac(6′)-Ib-cr (18/25), floR (16/25), rmtB (6/25), qnrS1(3/25) and fosA3 (2/25), were also identified on these IncHI2 plasmids. The plasmids carried pco and sil contributed to increasing in the MICs of CuSO4 and AgNO3. The genetic context surrounding the two operons was well conserved except some variations within the pco operon. The ~32 kb region containing the two operons identified in the IncHI2 plasmids was also found in chromosomes of different Enterobacteriaceae species. Further, phylogenetic analysis of this structure showed that Tn7-like transposon might play an important role in cross-genus transfer of the sil and pco operons among Enterobacteriaceae. In conclusion, co-existence of the pco and sil operons, and oqxAB/blaCTX-M as well as other antibiotic resistance genes on IncHI2 plasmids may promote the development of multidrug-resistant bacteria. PMID:27143648

Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing

PubMed Central

Eastman, Alexander W.; Yuan, Ze-Chun

2015-01-01

Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects. PMID:25653642

Role of BkdR, a Transcriptional Activator of the SigL-Dependent Isoleucine and Valine Degradation Pathway in Bacillus subtilis

PubMed Central

Debarbouille, Michel; Gardan, Rozenn; Arnaud, Maryvonne; Rapoport, George

1999-01-01

A new gene, bkdR (formerly called yqiR), encoding a regulator with a central (catalytic) domain was found in Bacillus subtilis. This gene controls the utilization of isoleucine and valine as sole nitrogen sources. Seven genes, previously called yqiS, yqiT, yqiU, yqiV, bfmBAA, bfmBAB, and bfmBB and now referred to as ptb, bcd, buk, lpd, bkdA1, bkdA2, and bkdB, are located downstream from the bkdR gene in B. subtilis. The products of these genes are similar to phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1α (dehydrogenase), E1β (decarboxylase), and E2 (dihydrolipoamide acyltransferase). Isoleucine and valine utilization was abolished in bcd and bkdR null mutants of B. subtilis. The seven genes appear to be organized as an operon, bkd, transcribed from a −12, −24 promoter. The expression of the bkd operon was induced by the presence of isoleucine or valine in the growth medium and depended upon the presence of the sigma factor SigL, a member of the sigma 54 family. Transcription of this operon was abolished in strains containing a null mutation in the regulatory gene bkdR. Deletion analysis showed that upstream activating sequences are involved in the expression of the bkd operon and are probably the target of bkdR. Transcription of the bkd operon is also negatively controlled by CodY, a global regulator of gene expression in response to nutritional conditions. PMID:10094682

An homolog of the Frz Phosphoenolpyruvate:carbohydrate phosphoTransferase System of extraintestinal pathogenic Escherichia coli is encoded on a genomic island in specific lineages of Streptococcus agalactiae.

PubMed

Patron, Kévin; Gilot, Philippe; Camiade, Emilie; Mereghetti, Laurent

2015-06-01

We identified a Streptococcus agalactiae metabolic region (fru2) coding for a Phosphoenolpyruvate:carbohydrate phosphoTransferase System (PTS) homologous to the Frz system of extraintestinal pathogenic Escherichia coli strains. The Frz system is involved in environmental sensing and regulation of the expression of adaptation and virulence genes in E. coli. The S. agalactiae fru2 region codes three subunits of a PTS transporter of the fructose-mannitol family, a transcriptional activator of PTSs of the MtlR family, an allulose-6 phosphate-3-epimerase, a transaldolase and a transketolase. We demonstrated that all these genes form an operon. The fru2 operon is present in a 17494-bp genomic island. We analyzed by multilocus sequence typing a population of 492 strains representative of the S. agalactiae population and we showed that the presence of the fru2 operon is linked to the phylogeny of S. agalactiae. The fru2 operon is always present within strains of clonal complexes CC 1, CC 7, CC 10, CC 283 and singletons ST 130 and ST 288, but never found in other CCs and STs. Our results indicate that the fru2 operon was acquired during the evolution of the S. agalactiae species from a common ancestor before the divergence of CC 1, CC 7, CC 10, CC 283, ST 130 and ST 288. As S. agalactiae strains of CC 1 and CC 10 are frequently isolated from adults with invasive disease, we hypothesize that the S. agalactiae Fru2 system senses the environment to allow the bacterium to adapt to new conditions encountered during the infection of adults. Copyright © 2015 Elsevier B.V. All rights reserved.

Whole-Transcriptome Shotgun Sequencing (RNA-seq) Screen Reveals Upregulation of Cellobiose and Motility Operons of Lactobacillus ruminis L5 during Growth on Tetrasaccharides Derived from Barley β-Glucan

PubMed Central

Lawley, Blair; Sims, Ian M.

2013-01-01

Lactobacillus ruminis is an inhabitant of human bowels and bovine rumens. None of 10 isolates (three from bovine rumen, seven from human feces) of L. ruminis that were tested could utilize barley β-glucan for growth. Seven of the strains of L. ruminis were, however, able to utilize tetrasaccharides (3-O-β-cellotriosyl-d-glucose [LDP4] or 4-O-β-laminaribiosyl-d-cellobiose [CDP4]) present in β-glucan hydrolysates for growth. The tetrasaccharides were generated by the use of lichenase or cellulase, respectively. To learn more about the utilization of tetrasaccharides by L. ruminis, whole-transcriptome shotgun sequencing (RNA-seq) was tested as a transcriptional screen to detect altered gene expression when an autochthonous human strain (L5) was grown in medium containing CDP4. RNA-seq results were confirmed and extended by reverse transcription-quantitative PCR assays of selected genes in two upregulated operons when cells were grown as batch cultures in medium containing either CDP4 or LDP4. The cellobiose utilization operon had increased transcription, particularly in early growth phase, whereas the chemotaxis/motility operon was upregulated in late growth phase. Phenotypic changes were seen in relation to upregulation of chemotaxis/flagellar operons: flagella were rarely seen by electron microscopy on glucose-grown cells but cells cultured in tetrasaccharide medium were commonly flagellated. Chemotactic movement toward tetrasaccharides was demonstrated in capillary cultures. L. ruminis utilized 3-O-β-cellotriosyl-d-glucose released by β-glucan hydrolysis due to bowel commensal Coprococcus sp., indicating that cross feeding of tetrasaccharide between bacteria could occur. Therefore, the RNA-seq screen and subsequent experiments had utility in revealing foraging attributes of gut commensal Lactobacillus ruminis. PMID:23851085

Construction of a β-galactosidase-gene-based fusion is convenient for screening candidate genes involved in regulation of pyrrolnitrin biosynthesis in Pseudomonas chlororaphis G05.

PubMed

Luo, Wangtai; Miao, Jing; Feng, Zhibin; Lu, Ruiyang; Sun, Xiaoqiang; Zhang, Baoshen; Ding, Weiqiu; Lu, Yang; Wang, Yanhua; Chi, Xiaoyan; Ge, Yihe

2018-05-28

In our recent work, we found that pyrrolnitrin, and not phenazines, pyrrolnitrin contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain. To obtain new candidate genes involved in regulation of the prn operon expression, we successfully constructed a fusion mutant G05ΔphzΔprn::lacZ, in which most of the coding regions of the prn operon and the phzABCDEFG (the phz operon, phenazine biosynthetic locus) were deleted, and the promoter region plus the first thirty condons of the prnA was in-frame fused with the truncated lacZ gene on its chromosome. The expression of the fused lacZ reporter gene driven by the promoter of the prn operon made it easy for us to detect the level of the prn expression in terms of the color variation of colonies on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). With this fusion mutant as a recipient strain, mini-Tn5-based random insertional mutagenesis was then conducted. By picking up colonies with color change, it is possible for us to screen and identify new candidate genes involved in regulation of the prn expression. Identification of additional regulatory genes in further work could reasonably be expected to increase pyrrolnitrin production in G05 and to improve its biological control function.

More than just a metabolic regulator - elucidation and validation of new targets of PdhR in Escherichia coli

PubMed Central

2011-01-01

Background The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulatory network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach. Results 22 new genes contained in two operons controlled by PdhR (previously only 20 regulatory targets in eight operons were known) were identified by analysing a large-scale dataset of E. coli from the Many Microbes Microarray Database and novel expression data from a pdhR knockout strain, as well as a PdhR overproducing strain. We identified a regulation of the glycolate utilization operon glcDEFGBA using chromatin immunoprecipitation and gel shift assays. We show that this regulation could be part of a cross-induction between genes necessary for acetate and pyruvate utilisation controlled through PdhR. Moreover, a link of PdhR regulation to the replication machinery of the cell via control of the transcription of the dcw-cluster was verified in experiments. This augments our knowledge of the functions of the PdhR-regulon and demonstrates its central importance for further cellular processes in E. coli. Conclusions We extended the PdhR regulon by 22 new genes contained in two operons and validated the regulation of the glcDEFGBA operon for glycolate utilisation and the dcw-cluster for cell division proteins experimentally. Our results provide, for the first time, a plausible regulatory link between the nutritional status of the cell and cell replication mediated by PdhR. PMID:22168595

Annexin A5 Binds to Lipopolysaccharide and Reduces Its Endotoxin Activity

PubMed Central

Rand, Jacob H.; Wu, Xiao-Xuan; Lin, Elaine Y.; Griffel, Alexander; Gialanella, Philip; McKitrick, John C.

2012-01-01

ABSTRACT Annexin A5 (AnxA5) has a high affinity for phosphatidylserine. The protein is widely used to detect apoptotic cells because phosphatidylserine, a phospholipid that is normally present in the inner leaflets of cytoplasmic membranes, becomes translocated to the outer leaflets during programmed cell death. Here we report the novel observation that AnxA5 binds to Gram-negative bacteria via the lipid A domain of lipopolysaccharide (LPS). Binding of AnxA5 to bacteria was measured quantitatively, confirmed by fluorescence microscopy, and found to be inhibited by antibodies against lipid A. AnxA5 also bound to purified dot-blotted LPS and lipid A. Through ellipsometry, we found that the binding of AnxA5 to purified LPS was calcium dependent and rapid and showed a high affinity—characteristics similar to those of AnxA5 binding to phosphatidylserine. Initial functional studies indicated that AnxA5 can affect LPS activities. AnxA5 inhibited LPS-mediated gelation in the Limulus amebocyte lysate assay. Incubation of LPS with the protein reduced the quantity of tumor necrosis factor alpha (TNF-α) released by cultured monocytes compared to that released upon incubation with LPS alone. Initial in vivo experiments indicated that injection of mice with LPS preincubated with AnxA5 produced serum TNF-α levels lower than those seen after injection of LPS alone. These data demonstrate that AnxA5 binds to LPS and open paths to investigation of the potential biological and therapeutic implications of this interaction. PMID:22415004

MHC class II molecules control murine B cell responsiveness to lipopolysaccharide stimulation.

PubMed

Rodo, Joana; Gonçalves, Lígia A; Demengeot, Jocelyne; Coutinho, António; Penha-Gonçalves, Carlos

2006-10-01

LPS is a strong stimulator of the innate immune system and inducer of B lymphocyte activation. Two TLRs, TLR4 and RP105 (CD180), have been identified as mediators of LPS signaling in murine B cells, but little is known about genetic factors that are able to control LPS-induced cell activation. We performed a mouse genome-wide screen that aside from identifying a controlling locus mapping in the TLR4 region (logarithm of odds score, 2.77), also revealed that a locus closely linked to the MHC region (logarithm of odds score, 3.4) governed B cell responsiveness to LPS stimulation. Using purified B cells obtained from MHC congenic strains, we demonstrated that the MHC(b) haplotype is accountable for higher cell activation, cell proliferation, and IgM secretion, after LPS stimulation, when compared with the MHC(d) haplotype. Furthermore, B cells from MHC class II(-/-) mice displayed enhanced activation and proliferation in response to LPS. In addition, we showed that the MHC haplotype partially controls expression of RP105 (a LPS receptor molecule), following a pattern that resembles the LPS responsiveness phenotype. Together, our results strongly suggest that murine MHC class II molecules play a role in constraining the B cell response to LPS and that genetic variation at the MHC locus is an important component in controlling B cell responsiveness to LPS stimulation. This work raises the possibility that constraining of B cell responsiveness by MHC class II molecules may represent a functional interaction between adaptive and innate immune systems.

LPS-induced expression of CD14 in the TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages.

PubMed

Yang, Qin; Pröll, Maren J; Salilew-Wondim, Dessie; Zhang, Rui; Tesfaye, Dawit; Fan, Huitao; Cinar, Mehmet U; Große-Brinkhaus, Christine; Tholen, Ernst; Islam, Mohammad A; Hölker, Michael; Schellander, Karl; Uddin, Muhammad J; Neuhoff, Christiane

2016-11-01

Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro. CD14 gene expression was induced by 5 µg/ml LPS at the time point of 12 h and suppressed by 5 µM SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-κB, TRAF3, IRF7, TNF-α, IL-1β, IL-6, and IFN-β. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-α, IL-1β and IFN-β were suppressed by 5 µM SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 µM SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs.

The LptD chaperone LptE is not directly involved in lipopolysaccharide transport in Neisseria meningitidis.

PubMed

Bos, Martine P; Tommassen, Jan

2011-08-19

The biosynthesis of lipopolysaccharide (LPS) in gram-negative bacteria is well understood, in contrast to the transport to its destination, the outer leaflet of the outer membrane. In Escherichia coli, synthesis and transport of LPS are essential processes. Neisseria meningitidis, conversely, can survive without LPS and tolerates inactivation of genes involved in LPS synthesis and transport. Here, we analyzed whether the LptA, LptB, LptC, LptE, LptF, and LptG proteins, recently implicated in LPS transport in E. coli, function similarly in N. meningitidis. None of the analyzed proteins was essential in N. meningitidis, consistent with their expected roles in LPS transport and additionally demonstrating that they are not required for an essential process such as phospholipid transport. As expected, the absence of most of the Lpt proteins resulted in a severe defect in LPS transport. However, the absence of LptE did not disturb transport of LPS to the cell surface. LptE was found to be associated with LptD, and its absence affected total levels of LptD, suggesting a chaperone-like role for LptE in LptD biogenesis. The absence of a direct role of LptE in LPS transport was substantiated by bioinformatic analyses showing a low conservation of LptE in LPS-producing bacteria. Apparently, the role of LptE in N. meningitidis deviates from that in E. coli, suggesting that the Lpt system does not function in a completely conserved manner in all gram-negative bacteria.

The LptD Chaperone LptE Is Not Directly Involved in Lipopolysaccharide Transport in Neisseria meningitidis*

PubMed Central

Bos, Martine P.; Tommassen, Jan

2011-01-01

The biosynthesis of lipopolysaccharide (LPS) in Gram-negative bacteria is well understood, in contrast to the transport to its destination, the outer leaflet of the outer membrane. In Escherichia coli, synthesis and transport of LPS are essential processes. Neisseria meningitidis, conversely, can survive without LPS and tolerates inactivation of genes involved in LPS synthesis and transport. Here, we analyzed whether the LptA, LptB, LptC, LptE, LptF, and LptG proteins, recently implicated in LPS transport in E. coli, function similarly in N. meningitidis. None of the analyzed proteins was essential in N. meningitidis, consistent with their expected roles in LPS transport and additionally demonstrating that they are not required for an essential process such as phospholipid transport. As expected, the absence of most of the Lpt proteins resulted in a severe defect in LPS transport. However, the absence of LptE did not disturb transport of LPS to the cell surface. LptE was found to be associated with LptD, and its absence affected total levels of LptD, suggesting a chaperone-like role for LptE in LptD biogenesis. The absence of a direct role of LptE in LPS transport was substantiated by bioinformatic analyses showing a low conservation of LptE in LPS-producing bacteria. Apparently, the role of LptE in N. meningitidis deviates from that in E. coli, suggesting that the Lpt system does not function in a completely conserved manner in all Gram-negative bacteria. PMID:21705335

Structural diversity of Burkholderia pseudomallei lipopolysaccharides affects innate immune signaling

PubMed Central

Norris, Michael H.; Schweizer, Herbert P.

2017-01-01

Burkholderia pseudomallei (Bp) causes the disease melioidosis. The main cause of mortality in this disease is septic shock triggered by the host responding to lipopolysaccharide (LPS) components of the Gram-negative outer membrane. Bp LPS is thought to be a weak inducer of the host immune system. LPS from several strains of Bp were purified and their ability to induce the inflammatory mediators TNF-α and iNOS in murine macrophages at low concentrations was investigated. Innate and adaptive immunity qPCR arrays were used to profile expression patterns of 84 gene targets in response to the different LPS types. Additional qPCR validation confirmed large differences in macrophage response. LPS from a high-virulence serotype B strain 576a and a virulent rough central nervous system tropic strain MSHR435 greatly induced the innate immune response indicating that the immunopathogenesis of these strains is different than in infections with strains similar to the prototype strain 1026b. The accumulation of autophagic vesicles was also increased in macrophages challenged with highly immunogenic Bp LPS. Gene induction and concomitant cytokine secretion profiles of human PBMCs in response to the various LPS were also investigated. MALDI-TOF/TOF was used to probe the lipid A portions of the LPS, indicating substantial structural differences that likely play a role in host response to LPS. These findings add to the evolving knowledge of host-response to bacterial LPS, which can be used to better understand septic shock in melioidosis patients and in the rational design of vaccines. PMID:28453531

Cardiac-Specific Overexpression of Catalase Attenuates Lipopolysaccharide-Induced Myocardial Contractile Dysfunction: Role of Autophagy

PubMed Central

Turdi, Subat; Han, Xuefeng; Huff, Anna F.; Roe, Nathan D.; Hu, Nan; Gao, Feng; Ren, Jun

2012-01-01

Lipopolysaccharide (LPS) from Gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complication in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged with LPS (6 mg/kg) and cardiac function was evaluated. Levels of oxidative stress, autophagy, apoptosis and protein damage were examined using fluorescence microscopy, Western blot, TUNEL assay, caspase-3 activity and carbonyl formation. Kaplan-Meier curve was constructed for survival following LPS treatment. Our results revealed a lower mortality in catalase mice compared with FVB mice following LPS challenge. LPS injection led to depressed cardiac contractile capacity as evidenced by echocardiography and cardiomyocyte contractile function, the effect of which was ablated by catalase overexpression. LPS treatment induced elevated TNF-α level, autophagy, apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), production of ROS and O2−, and protein carbonyl formation, the effects of which were significantly attenuated by catalase overexpression. Electron microscopy revealed focal myocardial damage characterized by mitochondrial injury following LPS treatment, which was less severe in catalase mice. Interestingly, LPS-induced cardiomyocyte contractile dysfunction was prevented by antioxidant NAC and the autophagy inhibitor 3-methyladenine. Taken together, our data revealed that catalase protects against LPS-induced cardiac dysfunction and mortality, which may be associated with inhibition of oxidative stress and autophagy. PMID:22902401

Lipopolysaccharide endotoxemia induces amyloid-β and p-tau formation in the rat brain.

PubMed

Wang, Li-Ming; Wu, Qi; Kirk, Ryan A; Horn, Kevin P; Ebada Salem, Ahmed H; Hoffman, John M; Yap, Jeffrey T; Sonnen, Joshua A; Towner, Rheal A; Bozza, Fernando A; Rodrigues, Rosana S; Morton, Kathryn A

2018-01-01

Amyloid beta (Aβ) plaques are not specific to Alzheimer's disease and occur with aging and neurodegenerative disorders. Soluble brain Aβ may be neuroprotective and increases in response to neuroinflammation. Sepsis is associated with neurocognitive compromise. The objective was to determine, in a rat endotoxemia model of sepsis, whether neuroinflammation and soluble Aβ production are associated with Aβ plaque and hyperphosphorylated tau deposition in the brain. Male Sprague Dawley rats received a single intraperitoneal injection of 10 mg/kg of lipopolysaccharide endotoxin (LPS). Brain and blood levels of IL-1β, IL-6, and TNFα and cortical microglial density were measured in LPS-injected and control animals. Soluble brain Aβ and p-tau were compared and Aβ plaques were quantified and characterized. Brain uptake of [ 18 F]flutemetamol was measured by phosphor imaging. LPS endotoxemia resulted in elevations of cytokines in blood and brain. Microglial density was increased in LPS-treated rats relative to controls. LPS resulted in increased soluble Aβ and in p-tau levels in whole brain. Progressive increases in morphologically-diffuse Aβ plaques occurred throughout the interval of observation (to 7-9 days post LPS). LPS endotoxemia resulted in increased [ 18 F]flutemetamol in the cortex and increased cortex: white matter ratios of activity. In conclusion, LPS endotoxemia causes neuroinflammation, increased soluble Aβ and Aβ diffuse plaques in the brain. Aβ PET tracers may inform this neuropathology. Increased p-tau in the brain of LPS treated animals suggests that downstream consequences of Aβ plaque formation may occur. Further mechanistic and neurocognitive studies to understand the causes and consequences of LPS-induced neuropathology are warranted.

Heat stress prevents lipopolysaccharide-induced apoptosis in pulmonary microvascular endothelial cells by blocking calpain/p38 MAPK signalling.

PubMed

Liu, Zhi-Feng; Zheng, Dong; Fan, Guo-Chang; Peng, Tianqing; Su, Lei

2016-08-01

Pulmonary microvascular endothelial cells (PMECs) injury including apoptosis plays an important role in the pathogenesis of acute lung injury during sepsis. Our recent study has demonstrated that calpain activation contributes to apoptosis in PMECs under septic conditions. This study investigated how calpain activation mediated apoptosis and whether heat stress regulated calpain activation in lipopolysaccharides (LPS)-stimulated PMECs. In cultured mouse primary PMECs, incubation with LPS (1 μg/ml, 24 h) increased active caspase-3 fragments and DNA fragmentation, indicative of apoptosis. These effects of LPS were abrogated by pre-treatment with heat stress (43 °C for 2 h). LPS also induced calpain activation and increased phosphorylation of p38 MAPK. Inhibition of calpain and p38 MAPK prevented apoptosis induced by LPS. Furthermore, inhibition of calpain blocked p38 MAPK phosphorylation in LPS-stimulated PMECs. Notably, heat stress decreased the protein levels of calpain-1/2 and calpain activities, and blocked p38 MAPK phosphorylation in response to LPS. Additionally, forced up-regulation of calpain-1 or calpain-2 sufficiently induced p38 MAPK phosphorylation and apoptosis in PMECs, both of which were inhibited by heat stress. In conclusion, heat stress prevents LPS-induced apoptosis in PMECs. This effect of heat stress is associated with down-regulation of calpain expression and activation, and subsequent blockage of p38 MAPK activation in response to LPS. Thus, blocking calpain/p38 MAPK pathway may be a novel mechanism underlying heat stress-mediated inhibition of apoptosis in LPS-stimulated endothelial cells.

Heat stress prevents lipopolysaccharide-induced apoptosis in pulmonary microvascular endothelial cells by blocking calpain/p38 MAPK signalling

PubMed Central

Liu, Zhi-feng; Zheng, Dong; Fan, Guo-chang; Peng, Tianqing; Su, Lei

2016-01-01

Pulmonary microvascular endothelial cells (PMECs) injury including apoptosis plays an important role in the pathogenesis of acute lung injury during sepsis. Our recent study has demonstrated that calpain activation contributes to apoptosis in PMECs under septic conditions. This study investigated how calpain activation mediated apoptosis and whether heat stress regulated calpain activation in lipopolysaccharides (LPS)-stimulated PMECs. In cultured mouse primary PMECs, incubation with LPS (1 µg/ml, 24 h) increased active caspase-3 fragments and DNA fragmentation, indicative of apoptosis. These effects of LPS were abrogated by pre-treatment with heat stress (43 °C for 2 h). LPS also induced calpain activation and increased phosphorylation of p38 MAPK. Inhibition of calpain and p38 MAPK prevented apoptosis induced by LPS. Furthermore, inhibition of calpain blocked p38 MAPK phosphorylation in LPS-stimulated PMECs. Notably, heat stress decreased the protein levels of calpain-1/2 and calpain activities, and blocked p38 MAPK phosphorylation in response to LPS. Additionally, forced up-regulation of calpain-1 or calpain-2 sufficiently induced p38 MAPK phosphorylation and apoptosis in PMECs, both of which were inhibited by heat stress. In conclusion, heat stress prevents LPS-induced apoptosis in PMECs. This effect of heat stress is associated with down-regulation of calpain expression and activation, and subsequent blockage of p38 MAPK activation in response to LPS. Thus, blocking calpain/p38 MAPK pathway may be a novel mechanism underlying heat stress-mediated inhibition of apoptosis in LPS-stimulated endothelial cells. PMID:27325431

Requirement for STAT1 in LPS-induced gene expression in macrophages.

PubMed

Ohmori, Y; Hamilton, T A

2001-04-01

This study examines the role of the signal transducer and activator of transcription 1 (STAT1) in induction of lipopolysaccharide (LPS)-stimulated gene expression both in vitro and in vivo. LPS-induced expression of an interferon (IFN)-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) mRNAs was severely impaired in macrophages prepared from Stat1-/- mice, whereas levels of tumor necrosis factor alpha and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were unaffected. A similar deficiency in LPS-induced gene expression was observed in livers and spleens from Stat1-/- mice. The reduced LPS-stimulated gene expression seen in Stat1-/- macrophages was not the result of reduced activation of nuclear factor kappaB. LPS stimulated the delayed activation of both IFN-stimulated response element and IFN-gamma-activated sequence binding activity in macrophages from wild-type mice. Activation of these STAT1-containing transcription factors was mediated by the intermediate induction of type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA expression was markedly reduced in macrophages from IFN-alpha/betaR-/- mice and blocked by cotreatment with antibodies against type I IFN. These results indicate that indirect activation of STAT1 by LPS-induced type I IFN participates in promoting optimal expression of LPS-inducible genes, and they suggest that STAT1 may play a critical role in innate immunity against gram-negative bacterial infection.

Okanin, effective constituent of the flower tea Coreopsis tinctoria, attenuates LPS-induced microglial activation through inhibition of the TLR4/NF-κB signaling pathways

NASA Astrophysics Data System (ADS)

Hou, Yue; Li, Guoxun; Wang, Jian; Pan, Yingni; Jiao, Kun; Du, Juan; Chen, Ru; Wang, Bing; Li, Ning

2017-04-01

The EtOAc extract of Coreopsis tinctoria Nutt. significantly inhibited LPS-induced nitric oxide (NO) production, as judged by the Griess reaction, and attenuated the LPS-induced elevation in iNOS, COX-2, IL-1β, IL-6 and TNF-α mRNA levels, as determined by quantitative real-time PCR, when incubated with BV-2 microglial cells. Immunohistochemical results showed that the EtOAc extract significantly decreased the number of Iba-1-positive cells in the hippocampal region of LPS-treated mouse brains. The major effective constituent of the EtOAc extract, okanin, was further investigated. Okanin significantly suppressed LPS-induced iNOS expression and also inhibited IL-6 and TNF-α production and mRNA expression in LPS-stimulated BV-2 cells. Western blot analysis indicated that okanin suppressed LPS-induced activation of the NF-κB signaling pathway by inhibiting the phosphorylation of IκBα and decreasing the level of nuclear NF-κB p65 after LPS treatment. Immunofluorescence staining results showed that okanin inhibited the translocation of the NF-κB p65 subunit from the cytosol to the nucleus. Moreover, okanin significantly inhibited LPS-induced TLR4 expression in BV-2 cells. In summary, okanin attenuates LPS-induced activation of microglia. This effect may be associated with its capacity to inhibit the TLR4/NF-κB signaling pathways. These results suggest that okanin may have potential as a nutritional preventive strategy for neurodegenerative disorders.

Low level laser therapy reduces acute lung inflammation in a model of pulmonary and extrapulmonary LPS-induced ARDS.

PubMed

Oliveira, Manoel Carneiro; Greiffo, Flávia Regina; Rigonato-Oliveira, Nicole Cristine; Custódio, Ricardo Wesley Alberca; Silva, Vanessa Roza; Damaceno-Rodrigues, Nilsa Regina; Almeida, Francine Maria; Albertini, Regiane; Lopes-Martins, Rodrigo Álvaro B; de Oliveira, Luis Vicente Franco; de Carvalho, Paulo de Tarso Camillo; Ligeiro de Oliveira, Ana Paula; Leal, Ernesto César P; Vieira, Rodolfo P

2014-05-05

The present study aimed to investigate the effects low level laser therapy (LLLT) in a LPS-induced pulmonary and extrapulmonary acute respiratory distress syndrome (ARDS) in BALB/c mice. Laser (830nm laser, 9J/cm(2), 35mW, 80s per point, 3 points per application) was applied in direct contact with skin, 1h after LPS administration. Mice were distributed in control (n=6; PBS), ARDS IT (n=7; LPS orotracheally 10μg/mouse), ARDS IP (n=7; LPS intra-peritoneally 100μg/mouse), ARDS IT+Laser (n=9; LPS intra-tracheally 10μg/mouse), ARDS IP+Laser (n=9; LPS intra-peritoneally 100μg/mouse). Twenty-four hours after last LPS administration, mice were studied for pulmonary inflammation by total and differential cell count in bronchoalveolar lavage (BAL), cytokines (IL-1beta, IL-6, KC and TNF-alpha) levels in BAL fluid and also by quantitative analysis of neutrophils number in the lung parenchyma. LLLT significantly reduced pulmonary and extrapulmonary inflammation in LPS-induced ARDS, as demonstrated by reduced number of total cells (p<0.001) and neutrophils (p<0.001) in BAL, reduced levels of IL-1beta, IL-6, KC and TNF-alpha in BAL fluid and in serum (p<0.001), as well as the number of neutrophils in lung parenchyma (p<0.001). LLLT is effective to reduce pulmonary inflammation in both pulmonary and extrapulmonary model of LPS-induced ARDS. Copyright © 2014 Elsevier B.V. All rights reserved.

Minocycline protects against lipopolysaccharide-induced cognitive impairment in mice.

PubMed

Hou, Yue; Xie, Guanbo; Liu, Xia; Li, Guoxun; Jia, Congcong; Xu, Jinghua; Wang, Bing

2016-03-01

The role of glial cells, especially microglia and astrocytes, in neuroinflammation and cognition has been studied intensively. Lipopolysaccharide (LPS), a commonly used inducer of neuroinflammation, can cause cognitive impairment. Minocycline is known to possess potent neuroprotective activity, but its effect on LPS-induced cognitive impairment is unknown. This study aims to investigate the effects of minocycline on LPS-induced cognitive impairment and glial cell activation in mice. Behavioral tests were conducted for cognitive function, immunohistochemistry for microglial and astrocyte response, and quantitative PCR for mRNA expression of proinflammatory cytokines. Minocycline significantly reversed the decreased spontaneous alternation induced by intrahippocampal administration of LPS in the Y-maze task. In the Morris water maze place navigation test, minocycline decreased the escape latency and distance traveled compared to LPS-treated mice. In the probe test, minocycline-treated mice spent more time in the target quadrant and crossed the platform area more frequently than animals in the LPS-treated group. Minocycline produced a significant decrease in the number of Iba-1- and GFAP-positive hippocampal cells compared to the LPS-treated group. Minocycline-treated mice had significantly reduced hippocampal TNF-α and IL-1β mRNA levels compared with LPS-treated animals. Minocycline caused a significant increase in hippocampal BDNF expression compared to the LPS-treated group. Minocycline can attenuate LPS-induced cognitive impairments in mice. This effect may be associated with its action to suppress the activation of microglia and astrocytes and to normalize BDNF expression. Since neuroinflammatory processes and cognitive impairments are implicated in neurodegenerative disorders, minocycline may be a promising candidate for treating such diseases.

Crystallization behavior of the Li2S-P2S5 glass electrolyte in the LiNi1/3Mn1/3Co1/3O2 positive electrode layer.

PubMed

Tsukasaki, Hirofumi; Mori, Yota; Otoyama, Misae; Yubuchi, So; Asano, Takamasa; Tanaka, Yoshinori; Ohno, Takahisa; Mori, Shigeo; Hayashi, Akitoshi; Tatsumisago, Masahiro

2018-04-18

Sulfide-based all-solid-state lithium batteries are a next-generation power source composed of the inorganic solid electrolytes which are incombustible and have high ionic conductivity. Positive electrode composites comprising LiNi 1/3 Mn 1/3 Co 1/3 O 2 (NMC) and 75Li 2 S·25P 2 S 5 (LPS) glass electrolytes exhibit excellent charge-discharge cycle performance and are promising candidates for realizing all-solid-state batteries. The thermal stabilities of NMC-LPS composites have been investigated by transmission electron microscopy (TEM), which indicated that an exothermal reaction could be attributed to the crystallization of the LPS glass. To further understand the origin of the exothermic reaction, in this study, the precipitated crystalline phase of LPS glass in the NMC-LPS composite was examined. In situ TEM observations revealed that the β-Li 3 PS 4 precipitated at approximately 200 °C, and then Li 4 P 2 S 6 and Li 2 S precipitated at approximately 400 °C. Because the Li 4 P 2 S 6 and Li 2 S crystalline phases do not precipitate in the single LPS glass, the interfacial contact between LPS and NMC has a significant influence on both the LPS crystallization behavior and the exothermal reaction in the NMC-LPS composites.

Dexmedetomidine inhibits inflammatory reaction in the hippocampus of septic rats by suppressing NF-κB pathway.

PubMed

Zhang, Xiaobao; Yan, Fang; Feng, Jiying; Qian, Haitao; Cheng, Zhi; Yang, Qianqian; Wu, Yong; Zhao, Zhibin; Li, Aimin; Xiao, Hang

2018-01-01

Dexmedetomidine (DEX) is known to provide neuroprotective effect in the central nervous system. However, the detailed mechanism remains far more elusive. This study was designed to investigate the relevant mechanisms of DEX's neuroprotective effect. Sprague-Dawley (SD) rats were injected with dexmedetomidine and/or Lipopolysaccharide (LPS) intraperitoneally, and inflammatory cytokines in serum and in the hippocampus were measured by enzyme linked immunosorbent assay (ELISA). NF-κB in the brain tissue extracts was analyzed with western-blot. Then, we investigated whether NF-κB inhibitor prevents the elevation of inflammatory cytokines in rats injected with LPS. Our results indicated that compared with the control group, the rats exposed to LPS showed significant cognitive dysfunction. When compared to controls, the levels of TNF-α and IL-6 in the serum and hippocampus homogenate were increased in rats treated with LPS. DEX pretreatment inhibited the rats' TNF-α, IL-6 and NF-κB levels induced by LPS. In response to LPS, PDTC pretreatment restrains the production of proinflammatory cytokines (TNF-α and IL-6). Rats treated with PDTC and DEX alongside LPS exhibited less TNF-α and IL-6 than the LPS treated group. In combination, PDTC and DEX showed addictive effects. Our data suggest that DEX exerts a neuroprotective effect through NF-κB in part after LPS-induced cognitive dysfunction.

Effects of Citral on Lipopolysaccharide-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

PubMed

Song, Yan; Zhao, Hongfeng; Liu, Jinyang; Fang, Chao; Miao, Renying

2016-04-01

Citral is an active compound of lemongrass oil which has been reported to have anti-inflammatory effects. In this study, we investigated the effects of citral on lipopolysaccharide (LPS)-induced inflammatory response in a rat model of peritonitis and human umbilical vein endothelial cells (HUVECs). LPS was intraperitoneally injected into rats to establish a peritonitis model. The HUVECs were treated with citral for 12 h before exposure to LPS. The levels of TNF-α and IL-8 were measured using ELISA. Western blotting was used to detect the expression of VCAM-1, ICAM-1, NF-κB, and PPAR-γ. The results showed that citral had a protective effect against LPS-induced peritonitis. Citral decreased the levels of WBCs and inflammatory cytokines TNF-α and IL-6. Citral also inhibited LPS-induced myeloperoxidase (MPO) activity in the peritoneal tissue. Treatment of HUVECs with citral significantly inhibited TNF-α and IL-8 expression induced by LPS. LPS-induced VCAM-1 and ICAM-1 expression were also suppressed by citral. Meanwhile, we found that citral inhibited LPS-induced NF-κB activation in HUVECs. Furthermore, we found that citral activated PPAR-γ and the anti-inflammatory effects of citral can be reversed by PPAR-γ antagonist GW9662. In conclusion, citral inhibits LPS-induced inflammatory response via activating PPAR-γ which attenuates NF-κB activation and inflammatory mediator production.

Krill Oil-In-Water Emulsion Protects against Lipopolysaccharide-Induced Proinflammatory Activation of Macrophages In Vitro.

PubMed

Bonaterra, Gabriel A; Driscoll, David; Schwarzbach, Hans; Kinscherf, Ralf

2017-03-15

Parenteral nutrition is often a mandatory therapeutic strategy for cases of septicemia. Likewise, therapeutic application of anti-oxidants, anti-inflammatory therapy, and endotoxin lowering, by removal or inactivation, might be beneficial to ameliorate the systemic inflammatory response during the acute phases of critical illness. Concerning anti-inflammatory properties in this setting, omega-3 fatty acids of marine origin have been frequently described. This study investigated the anti-inflammatory and LPS-inactivating properties of krill oil (KO)-in-water emulsion in human macrophages in vitro. Differentiated THP-1 macrophages were activated using specific ultrapure-LPS that binds only on the toll-like receptor 4 (TLR4) in order to determine the inhibitory properties of the KO emulsion on the LPS-binding capacity, and the subsequent release of TNF-α. KO emulsion inhibited the macrophage binding of LPS to the TLR4 by 50% (at 12.5 µg/mL) and 75% (at 25 µg/mL), whereas, at 50 µg/mL, completely abolished the LPS binding. Moreover, KO (12.5 µg/mL, 25 µg/mL, or 50 µg/mL) also inhibited (30%, 40%, or 75%, respectively) the TNF-α release after activation with 0.01 µg/mL LPS in comparison with LPS treatment alone. KO emulsion influences the LPS-induced pro-inflammatory activation of macrophages, possibly due to inactivation of the LPS binding capacity.

Cystine improves survival rates in a LPS-induced sepsis mouse model.

PubMed

Tanaka, Kenji A K; Kurihara, Shigekazu; Shibakusa, Tetsuro; Chiba, Yasumasa; Mikami, Takashi

2015-12-01

The control of inflammation is important for suppressing severe sepsis. Oral administration of cystine and theanine have been shown to suppress inflammatory responses due to invasion. Furthermore, the uptake of cystine into monocytes is promoted by exposure to lipopolysaccharide (LPS). In the present study, the effects of cystine were examined in the context of inflammatory responses. Cystine was orally administered to mice, and the levels of interleukin (IL)-6 in the blood and spleen and the survival rates were calculated after the administration of LPS. The effects of cystine as well as neutralising anti-IL-10 antibodies on the LPS-induced production of IL-6 and IL-10 were examined in a monocyte cell line. The oral administration of cystine reduced IL-6 levels in the blood and spleen after LPS stimulation and improved survival rates. The addition of cystine to monocytes suppressed LPS-induced IL-6 production but enhanced IL-10 production. A neutralising anti-IL-10 antibody eliminated the inhibitory effects of cystine on the LPS-induced production of IL-6. The oral administration of cystine suppressed IL-6 production following LPS stimulation and improved survival rates in mice with LPS-induced sepsis. The enhanced production of IL-10 by monocytes may be involved in this anti-inflammatory response. Copyright © 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Induction of the chemotactic S100 protein, CP-10, in monocyte/macrophages by lipopolysaccharide.

PubMed

Hu, S P; Harrison, C; Xu, K; Cornish, C J; Geczy, C L

1996-05-01

The murine S100 protein CP-10 is a potent chemotactic factor for murine and human myeloid cells in vivo and in vitro. This is the first report describing regulations of the CP-10 gene by a proinflammatory stimulus, lipopolysaccharide (LPS), in cells of the monocyte/macrophage lineage. Murine monocyte/macrophage-like WEHI 265 and RAW 264.7 cells preexposed to 5 to 50 ng/mL LPS expressed significant levels of CP-10 mRNA 4 hours, and maximal at 20 hours, after a secondary LPS challenge. This was accompanied by increasing levels of cell-associated and released CP-10 protein. In contrast, a single dose of LPS upregulated CP-10 mRNA in elicited peritoneal macrophages, whereas mRNA and protein levels decreased following LPS challenge. The state of macrophage differentiation may control responsiveness as LPS had no effect on CP-10 basal levels in bone marrow derived macrophages. LPS-induced CP-10 expression was controlled at the transcriptional level and nuclear run-on and protein synthesis inhibition assays indicated that LPS priming and challenge of RAW cells occurred via distinct pathways. MRP14, another S100 protein generally coordinately expressed with human MRP8, was not induced by LPS under the same conditions. We propose that CP-10 may play a key role in recruitment of leukocytes into tissues in response to gram-negative bacterial infection.

α-Dihydroxychalcone-glycoside (α-DHC) isolated from the heartwood of Pterocarpus marsupium inhibits LPS induced MAPK activation and up regulates HO-1 expression in murine RAW 264.7 macrophage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, Prarthana; Saraswat, Ghungroo; Kabir, Syed N., E-mail: snkabir@iicb.res.in

2014-05-15

Three phenolic glycosides isolated from the heartwood of Pterocarpus marsupium showed significant free radical and superoxide ion scavenging activity and antioxidant potential that were comparable to, or several folds higher than those of standard antioxidants, trolox and ascorbic acid. The effective concentrations of these compounds were far below their cytotoxic levels. Compound 3, which was characterized to be α-dihydroxychalcone-glycoside (α-DHC), was the most potent one. Subsequent studies demonstrated that α-DHC effectively reduced nitric oxide and cytokine production by the LPS stimulated RAW 264.7 mouse macrophage cell line. The compound effectively attenuated the expression of inflammation-mediating enzymes COX-2 and iNOS atmore » the mRNA as well as protein levels in a concentration dependent manner. It prevented phosphorylation of all the three MAPKs (JNK, ERK, p38) and eventually blocked the activation of downstream elements contributing to inflammation. Phosphorylation of IκB-α and subsequent translocation of NF-κB into the nucleus were restricted, while the expression of stress responsive gene HO-1 was up-regulated. α-DHC targeted Keap-1 by modifying its cysteine thiols, dissociating it from Nrf-2 and facilitating nuclear entry of the latter; and this in turn induced HO-1 expression. Thus α-DHC exerts its anti-inflammatory activity in a dual manner: by down regulating MAPKs and restricting nuclear stabilization of NF-κB at one end, and by disrupting Nrf-2–Keap-1 complex on the other. In conclusion, the anti-inflammatory potential together with its high therapeutic index envisages α-DHC as a prospective candidate molecule for the development of therapeutic strategy against inflammatory disorders. - Highlights: • α-DHC isolated from Pterocarpus marsupium has significant antioxidant potential. • α-DHC inhibits NO, IL-6, IL-1β, TNF-α production in LPS-stimulated RAW 264.7 cells. • α-DHC down-regulates of COX-2, iNOS expression in LPS-stimulated RAW 264.7 cells. • α-DHC down-regulates MAPK phosphorylation and nuclear translocation of NF-κB. • α-DHC induces ARE-regulated gene expression via Keap-1 modification.« less

[Endoplasmic reticulum stress mediates lipopolysaccharide-induced apoptosis in rat hepatocyte].

PubMed

Ji, Ying-Lei; Yan, Jun; Wang, Yan-Sha; Liu, Yi-Chang; Gu, Zhen-Yong

2014-02-01

To investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced hepatocyte apoptosis. Cells of the rat hepatocyte line BRL were cultured. The hepatocytes were treated with LPS, ERS inducer thapsigargin (TG), and ERS inhibitor 4-phenylbutyric acid (4-PBA), respectively or in their different combination. The cell viability was measured by MTT assay. The cyto-nuclear morphological changes of apoptosis cells were detected by the fluorescent dye Hoechst 33258. The apoptosis rate was assessed by flow cytometry with Annexin V-FITC/PI double-staining. Expressions of GRP78 as ERS marker protein, CHOP, caspase-12 and cleaved-caspase-3 as ERS related protein were detected by Western blotting. LPS could cause a decrease in cell viability and an increase in apoptosis rate in a dose- and time-dependent manner. The expression of GRP78, CHOP, caspase-12 and cleaved-caspase-3 proteins were significantly increased with LPS treatment. TG led to a marked decrease in cell viability and an increase in apoptosis rate, which aggravated the hepatocyte injury induced by LPS; whereas 4-PBA alleviated LPS-induced apoptosis. ERS mediates LPS-induced hepatocyte injuries, indicating that ERS may play a vital role in the pathogenesis of LPS-induced hepatocyte injuries.

Identification of Novel Glycosyltransferases Required for Assembly of the Pasteurella multocida A:1 Lipopolysaccharide and Their Involvement in Virulence▿ †

PubMed Central

Boyce, John D.; Harper, Marina; St. Michael, Frank; John, Marietta; Aubry, Annie; Parnas, Henrietta; Logan, Susan M.; Wilkie, Ian W.; Ford, Mark; Cox, Andrew D.; Adler, Ben

2009-01-01

We previously determined the structure of the Pasteurella multocida Heddleston type 1 lipopolysaccharide (LPS) molecule and characterized some of the transferases essential for LPS biosynthesis. We also showed that P. multocida strains expressing truncated LPS display reduced virulence. Here, we have identified all of the remaining glycosyltransferases required for synthesis of the oligosaccharide extension of the P. multocida Heddleston type 1 LPS, including a novel α-1,6 glucosyltransferase, a β-1,4 glucosyltransferase, a putative bifunctional galactosyltransferase, and two heptosyltransferases. In addition, we identified a novel oligosaccharide extension expressed only in a heptosyltransferase (hptE) mutant background. All of the analyzed mutants expressing LPS with a truncated main oligosaccharide extension displayed reduced virulence, but those expressing LPS with an intact heptose side chain were able to persist for long periods in muscle tissue. The hptC mutant, which expressed LPS with the shortest oligosaccharide extension and no heptose side chain, was unable to persist on the muscle or cause any disease. Furthermore, all of the mutants displayed increased sensitivity to the chicken antimicrobial peptide fowlicidin 1, with mutants expressing highly truncated LPS being the most sensitive. PMID:19168738

6-Shogaol attenuates LPS-induced inflammation in BV2 microglia cells by activating PPAR-γ

PubMed Central

Han, Qinghe; Yuan, Qinghai; Meng, Xiaolin; Huo, Junyuan; Bao, Yuxin; Xie, Guanghong

2017-01-01

6-Shogaol, a pungent agent isolated from Zingiber officinale Roscoe, has been known to have anti-tumor and anti-inflammatory effects. However, the anti-inflammatory effects and biological mechanism of 6-Shogaol in LPS-activated BV2 microglia remains largely unknown. In this study, we evaluated the anti-inflammatory effects of 6-Shogaol in LPS-activated BV2 microglia. 6-Shogaol was administrated 1 h before LPS treatment. The production of inflammatory mediators were detected by ELISA. The expression of NF-κB and PPAR-γ were detected by western blot analysis. Our results revealed that 6-Shogaol inhibited LPS-induced TNF-α, IL-1β, IL-6, and PGE2 production in a concentration dependent manner. Furthermore, 6-Shogaol inhibited LPS-induced NF-κB activation by inhibiting phosphorylation and nuclear translocation of NF-κB p65. In addition, 6-Shogaol could increase the expression of PPAR-γ. Moreover, inhibition of PPAR-γ by GW9662 could prevent the inhibition of 6-Shogaol on LPS-induced inflammatory mediator production. In conclusion, 6-Shogaol inhibits LPS-induced inflammation by activating PPAR-γ. PMID:28410218

[The role of N-acetylcysteine against the injury of pulmonary artery induced by LPS].

PubMed

Huang, Xin-li; Ling, Yi-ling; Zhu, Tie-nian

2002-11-01

To investigate the alleviating effect of N-acetylcysteine (NAC) on lung injury induced by lipopolysaccharides (LPS) and its mechanism. The effects of NAC on changes of the pulmonary arterial reactivity and the ultrastructure of pulmonary arterial endothelium induced by LPS were observed with the isolated artery ring technique and scanning electron microscope (SEM). Malondialdehyde (MDA), nitric oxide (NO) contents and superoxide dismutase (SOD) activity of pulmonary artery tissues were detected. The exposure of pulmonary artery to LPS (4 microg/ml, 7 h) led to reduction of endothelium-dependent relaxation response to acetylcholine (ACh), which was reversed by the concomitant exposure to NAC (0.5 mmol/L, 7 h), whereas NAC itself had no effect on the response. Significant structural injury were observed under SEM in LPS group and alleviated the changes in LPS + NAC group. The MDA, NO contents increased but SOD activity decreased in LPS group, which were reversed by the concomitant exposure to NAC. NAC protects pulmonary artery endothelium and enhances endothelium-dependent relaxation response of pulmonary artery by antioxidation effect, which may be one of the mechanisms of its reversing pulmonary artery hypertension and following lung injury induced by LPS.

Molecular Characterization of Lipopolysaccharide Binding to Human α-1-Acid Glycoprotein

PubMed Central

Huang, Johnny X.; Azad, Mohammad A. K.; Yuriev, Elizabeth; Baker, Mark A.; Nation, Roger L.; Li, Jian; Cooper, Matthew A.; Velkov, Tony

2012-01-01

The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Serratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, Ra, Rd, and Re rough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria. PMID:23316371

γ-Oryzanol reduces adhesion molecule expression in vascular endothelial cells via suppression of nuclear factor-κB activation.

PubMed

Sakai, Satoshi; Murata, Takahisa; Tsubosaka, Yoshiki; Ushio, Hideki; Hori, Masatoshi; Ozaki, Hiroshi

2012-04-04

γ-Oryzanol (γ-ORZ) is a mixture of phytosteryl ferulates purified from rice bran oil. In this study, we examined whether γ-ORZ represents a suppressive effect on the lipopolysaccharide (LPS)-induced adhesion molecule expression on vascular endothelium. Treatment with LPS elevated the mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in bovine aortic endothelial cells (BAECs). Pretreatment with γ-ORZ dose-dependently decreased the LPS-mediated expression of these genes. Western blotting also revealed that pretreatment with γ-ORZ dose-dependently inhibited LPS-induced VCAM-1 expression in human umbilical vein endothelial cells. Consistently, pretreatment with γ-ORZ dose-dependently reduced LPS-induced U937 monocyte adhesion to BAECs. In immunofluorescence, LPS caused nuclear factor-κB (NF-κB) nuclear translocation in 40% of BAECs, which indicates NF-κB activation. Pretreatment with γ-ORZ, as well as its components (cycloartenyl ferulate, ferulic acid, or cycloartenol), dose-dependently inhibited LPS-mediated NF-κB activation. Collectively, our results suggested that γ-ORZ reduced LPS-mediated adhesion molecule expression through NF-κB inhibition in vascular endothelium.

Esculin Inhibits the Inflammation of LPS-Induced Acute Lung Injury in Mice Via Regulation of TLR/NF-κB Pathways.

PubMed

Tianzhu, Zhang; Shumin, Wang

2015-08-01

In this study, we investigated anti-inflammatory effects of esculin (ESC) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS, and ESC (20 and 40 mg/kg) was given orally 1 h prior to LPS administration. After 6 h, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. ESC pretreatment decreased LPS-induced evident lung histopathological changes, lung wet-to-dry weight ratio, and lung myeloperoxidase activity. In addition, pretreatment with ESC inhibited inflammatory cells and proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β, and interleukin-6 in BALF. Furthermore, we demonstrated that ESC inhibited the Toll-like receptor-2 (TLR2), Toll-like receptor-4 (TLR4), myeloid differentiation primary response gene-88 (MyD88), and nuclear factor-κB (NF-κB) p65 in LPS-induced ALI. The results indicated that the ESC had a protective effect on LPS-induced ALI in mice.

Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response.

PubMed

Paramo, Teresa; Tomasio, Susana M; Irvine, Kate L; Bryant, Clare E; Bond, Peter J

2015-12-09

Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the "membrane-like" nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics.

Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response

PubMed Central

Paramo, Teresa; Tomasio, Susana M.; Irvine, Kate L.; Bryant, Clare E.; Bond, Peter J.

2015-01-01

Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the “membrane-like” nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics. PMID:26647780

Joint study of lipopolysaccharide suspensions with thermal lensing and optoacoustic methods

NASA Astrophysics Data System (ADS)

Orlova, Nataliya V.; Brusnichkin, Anton V.; Proskurnin, Mikhail A.; Fokin, Andrey V.; Ovchinnikov, Oleg B.; Egerev, Sergey V.

2004-07-01

Pyrogens being introduced intravenously increase body temperature that leads to hazardous consequences and even to lethal outcome. One of the widespread pyrogen systems is presented by suspensions composed of bacterial endotoxins (or lypopolysaccharides, LPS). The aim of the work is to compare experimentally two methods for the determination of LPS at the submicrogram level and below. Both methods suppose that the LPS suspension is irradiated by a laser pulse. The thermal lens (TL) method (microsecond to millisecond irradiation cycle) detects LPS by a direct pick-up of the transient thermal field. The optoacoustic (OA) method (nanosecond laser pulses) has a potential to use non-thermal constitutents of the LPS response and to provide some selectivity of LPS detection with respect to optically uniform contaminants in the sample. In experiments, the selectivity was enhanced by means of analytical reagents, methylene blue and Stains All dyes. It was shown that both methods are mutually complementary. Then, their detectability potential increases and reaches 10 ppb if there occur ion pairs of LPS and cationic dye.

Modulation of lipopolysaccharide-induced chorioamnionitis by Ureaplasma parvum in sheep

PubMed Central

SNYDER, Candice C.; WOLFE, Katherine B.; GISSLEN, Tate; KNOX, Christine L.; KEMP, Matthew W.; KRAMER, Boris W.; NEWNHAM, John P.; JOBE, Alan H.; KALLAPUR, Suhas G.

2013-01-01

Objective Ureaplasma colonization in the setting of polymicrobial flora is common in women with chorioamnionitis, and is a risk factor for preterm delivery and neonatal morbidity. We hypothesized that ureaplasma colonization of amniotic fluid will modulate chorioamnionitis induced by E. coli lipopolysaccharide (LPS). Methods Sheep received intra-amniotic (IA) injections of media (control) or live ureaplasma either 7 or 70d before delivery. Another group received IA LPS 2d before delivery. To test for interactions, U. parvum exposed animals were challenged with IA LPS, and delivered 2d later. All animals were delivered preterm at 125±1 day gestation. Results Both IA ureaplasmas and LPS induced leukocyte infiltration of chorioamnion. LPS greatly increased the expression of pro-inflammatory cytokines and myeloperoxidase in leukocytes, while ureaplasmas alone caused modest responses. Interestingly, 7d but not 70d ureaplasma exposure significantly downregulated LPS induced pro-inflammatory cytokines and myeloperoxidase expression in the chorioamnion. Conclusion Acute U. parvum exposure (7d) can suppress LPS induced chorioamnionitis. PMID:23410690

Participation of α2‐adrenoceptors in sodium appetite inhibition during sickness behaviour following administration of lipopolysaccharide

PubMed Central

Almeida, Roberto L.; David, Richard B.; de Paula, Patricia M.; Andrade, Carina A. F.; Menani, José V.

2015-01-01

Abstract Sickness behaviour, a syndrome characterized by a general reduction in animal activity, is part of the active‐phase response to fight infection. Lipopolysaccharide (LPS), an effective endotoxin to model sickness behaviour, reduces thirst and sodium excretion, and increases neurohypophysial secretion. Here we review the effects of LPS on thirst and sodium appetite. Altered renal function and hydromineral fluid intake in response to LPS occur in the context of behavioural reorganization, which manifests itself as part of the syndrome. Recent data show that, in addition to its classical effect on thirst, non‐septic doses of LPS injected intraperitoneally produce a preferential inhibition of intracellular thirst versus extracellular thirst. Moreover, LPS also reduced hypertonic NaCl intake in sodium‐depleted rats that entered a sodium appetite test. Antagonism of α2‐adrenoceptors abolished the effect of LPS on sodium appetite. LPS and cytokine transduction potentially recruit brain noradrenaline and α2‐adrenoceptors to control sodium appetite and sickness behaviour. PMID:26036817

Naringin protects against lipopolysaccharide-induced cardiac injury in mice.

PubMed

Xianchu, Liu; Lan, Professor Zheng; Qiufang, Li; Yi, Liu; Xiangcheng, Ruan; Wenqi, Hou; Yang, Ding

2016-12-01

Previous research has demonstrated that lipopolysaccharide (LPS) can induce sepsis and lead to myocardial dysfunction. Naringin has various biological activities in LPS-induced sepsis. In this study, our aim was to investigate the effects of Naringin on LPS-induced cardiac injury and clarify its potential mechanism. We found that in vivo treatment with Naringin significantly ameliorated body weight loss, and attenuated cardiac histopathological changes after LPS challenge. Furthermore, Naringin inhibited LPS-induced increase of TNF-α, IL-1β and IL-6 activities to alleviate inflammatory response in heart. Moreover, Naringin supplement dramatically increased SOD levels, and prevented MDA levels to ameliorate oxidative stress compared with the LPS group in heart. Lastly, treatment with Naringin also significantly decreased the ratio of BAX to BCL-2 to resist apoptosis in heart. It is concluded that Naringin may be a promising therapeutic agent on LPS-induced cardiac injury by anti-inflammatory, anti-oxidant and anti-apoptotic effects. Copyright © 2016 Elsevier B.V. All rights reserved.

Modulation of lipopolysaccharide-induced chorioamnionitis by Ureaplasma parvum in sheep.

PubMed

Snyder, Candice C; Wolfe, Katherine B; Gisslen, Tate; Knox, Christine L; Kemp, Matthew W; Kramer, Boris W; Newnham, John P; Jobe, Alan H; Kallapur, Suhas G

2013-05-01

Ureaplasma colonization in the setting of polymicrobial flora is common in women with chorioamnionitis, and is a risk factor for preterm delivery and neonatal morbidity. We hypothesized that Ureaplasma colonization of amniotic fluid would modulate chorioamnionitis induced by Escherichia coli lipopolysaccharide (LPS). Sheep received intraamniotic (IA) injections of media (control) or live Ureaplasma either 7 or 70 days before delivery. Another group received IA LPS 2 days before delivery. To test for interactions, U parvum-exposed animals were challenged with IA LPS, and delivered 2 days later. All animals were delivered preterm at 125 ± 1 day of gestation. Both IA Ureaplasma and LPS induced leukocyte infiltration of chorioamnion. LPS greatly increased the expression of proinflammatory cytokines and myeloperoxidase in leukocytes, while Ureaplasma alone caused modest responses. Interestingly, 7-day but not 70-day Ureaplasma exposure significantly down-regulated LPS-induced proinflammatory cytokines and myeloperoxidase expression in the chorioamnion. Acute (7-day) U parvum exposure can suppress LPS-induced chorioamnionitis. Copyright © 2013 Mosby, Inc. All rights reserved.

Effect of Lipopolysaccharide on Progesterone Production during Luteinization of Granulosa and Theca cells In Vitro.

PubMed

Shimizu, Takashi; Echizenya, Riku; Miyamoto, Akio

2016-04-01

The aim of this study is to examine the effect of lipopolysaccharide (LPS) on progesterone production during luteinization of granulosa and theca cells isolated from bovine large follicles. Granulosa and theca cells isolated from large follicles of bovine ovaries were exposed to LPS under appropriate hormone conditions in vitro. Progesterone (P4) production in theca cells, but not granulosa cells, was decreased by long-term exposure of LPS. Long-term exposure of LPS suppressed the gene expression of luteinizing hormone receptor in theca cells. Although long-term exposure of LPS did not affect the expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxy-steroid dehydrogenase (3β-HSD) genes, it did inhibit the protein expression of StAR and 3β-HSD in theca cells. These findings suggest that theca cells, rather than granulosa cells, are susceptible to LPS during luteinization and that LPS inhibits P4 production by decreasing protein levels of StAR during luteinization of theca cells. © 2016 Wiley Periodicals, Inc.

[Chromogranin A derived peptide CGA47-66 inhibits hyper-permeability of blood brain barrier in mice with sepsis].

PubMed

Zeng, Yan; Zhang, Dan; Jiang, Liping; Wei, Fu; Xu, Shan

2016-02-01

To explore the effect of chromofungin (CHR), a chromogranin A (CGA) derived peptide CGA47-66, on hyper-permeability of blood brain barrier in septic mice. 120 healthy male C57BL/6 mice were randomly divided into groups, with 12 mice in each group. Seventy-two mice were used for dynamic observation of the contents of water and Evan blue (EB) in brain tissue after being treated with lipopolysaccharide (LPS). Another 48 mice were divided into normal saline control group (NS group), LPS induced sepsis model group (LPS group), low-dose CHR pretreatment group (CL+LPS group), and high-dose CHR pretreatment group (CH+LPS group). The septic model was reproduced by intraperitoneal injection of 10 mg/kg LPS 0.1 mL, and the mice in NS group was given equal volume of normal saline. The mice in CL+LPS group and CH+LPS group were intraperitoneally injected with 15.5 μg/kg and 77.5 μg/kg CHR 10 minutes before LPS injection. Six hours after LPS injection, 4 mL/kg of 2% EB was injected via caudal vein, the contents of water and EB in brain tissue were determined, and EB immune fluorescence in brain tissue was determined to assess the changes in permeability of blood brain barrier. Brain pathology was observed with hematoxylin and eosin (HE) staining. With the extension of time after LPS injection, the contents of water and EB in brain tissue were gradually increased, and the time of difference with statistical significance appeared earlier when compared with that of control group in the contents of water than that in EB contents (3 hours and 6 hours, respectively). The contents of water and EB in brain tissue in LPS group were significantly increased as compared with NS group [water content: (79.77±0.62)% vs. (78.28±0.44)%, P < 0.01; EB content (μg/g): 13.87±4.50 vs. 7.13±1.76, P < 0.05]. CHR pretreatment with either of two dosages could reverse the increase in water and EB contents in brain tissue induced by LPS, and the effect was more significant in CH+LPS group [water content: (78.15±0.73)% vs. (79.77±0.62)%, EB (μg/g): 7.09±2.59 vs. 13.87±4.50, both P < 0.05]. It was shown by EB fluorescence observation that the fluorescence signal displayed only in the meninges in NS group, and EB fluorescence was widely distributed in brain parenchyma in LPS group, indicating that the EB leakage in LPS group was more marked than that of NS group. In CHR pretreatment groups, EB fluorescence was decreased in brain parenchyma, indicating that EB leakage was significantly less marked, while it was more obvious in high dose CHR group. It was shown by HE staining that cerebral blood vessel structure was intact in NS group, and the gap around blood vessel was not significant increased. On the other hand, brain structure in LPS group appeared loose, with widening of small perivascular spaces and obvious edema. Brain edema in CHR pretreatment groups was improved as compared with that of the LPS group, and it was more apparent in high dose CHR group. LPS induced change in blood brain barrier permeability in mice in a time-dependent manner. Exogenous CGA derived peptides CHR can inhibit LPS induced hyper-permeability of blood brain barrier in septic mice, thus reduces brain edema, protects the brain tissue, and the effect is more obvious with a high dose of CHR (77.5 μg/kg).

Correlation of 16S Ribosomal DNA Signature Sequences with Temperature-Dependent Growth Rates of Mesophilic and Psychrotolerant Strains of the Bacillus cereus Group

PubMed Central

Prüß, Birgit M.; Francis, Kevin P.; von Stetten, Felix; Scherer, Siegfried

1999-01-01

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance. PMID:10198030

CO from enhanced HO activity or from CORM-2 inhibits both O2- and NO production and downregulates HO-1 expression in LPS-stimulated macrophages.

PubMed

Srisook, Klaokwan; Han, Shan-Shu; Choi, Hyung-Sim; Li, Mei-Hua; Ueda, Hideo; Kim, Chaekyun; Cha, Young-Nam

2006-01-12

Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by the stress-inducible heme oxygenase-1 (HO-1), has recently been demonstrated to provide cytoprotection against cell death in macrophages stimulated with bacterial lipopolysaccharide (LPS). In the present study, we determined the effects of CO on the production of reactive oxygen species (ROS) and nitric oxide (NO) by the LPS-stimulated RAW 264.7 macrophages. In addition, effect of CO-exposure on the production of superoxide (O(2)(-)) in the phorbol myristate acetate (PMA)-stimulated PLB-985 neutrophils was determined. Production of ROS by the LPS-stimulated macrophages pretreated with 50microM [Ru(CO)(3)Cl(2)](2), a CO-releasing molecule (CORM-2), was abolished and the production of O(2)(-) by the PMA-stimulated neutrophils pretreated with the CORM-2 was decreased markedly. The CORM-2 (50microM) was not cytotoxic to both the unstimulated and LPS-stimulated macrophages when determined by employing mitochondrial reductase function test (MTT assay). In macrophages pretreated with increasing doses of CORM-2, both the LPS-derived upregulations of iNOS (NO production) and HO-1 expression (CO production) were suppressed in a dose-dependent manner. Alternatively, when the macrophages were treated with LPS and CO-donor together, the LPS-derived increase in NO production was decreased. Conversely, when the control and LPS-stimulated macrophages were treated with zinc protoporphyrin IX (ZnPP) to inhibit the HO activity blocking endogenous production of CO (basal and enhanced), macrophages died extensively. Interestingly, production of NO in the LPS-stimulated macrophages increased significantly following the ZnPP treatment. Addition of CORM-2 to the LPS-treated cells that were being treated additionally with ZnPP did not prevent the cell death. However, endogenous overproduction of CO by super-induction of HO-1 (obtained by pretreatment of macrophages with either buthionine sulfoximine or hemin) decreased the LPS-derived iNOS expression without affecting cell survival. Combined, these results indicated that enhanced HO activity is essential for the survival of LPS-stimulated macrophages. Thus, upregulation of HO-1 and overproduction of CO may allow the survival of LPS-stimulated macrophages; first, by eliminating the free heme to prevent Fenton reaction, second, by limiting the availability of free heme required for induction of NO-producing heme enzyme (i.e., iNOS), third, by limiting additional production of O(2)(-) and NO via CO-derived inhibition on the activities of heme enzymes like NADPH oxidase and iNOS, respectively. CO may allow the LPS-activated macrophages to return back to the normal quiet state insensitive to additional stimuli causing oxidative stress.

Lidocaine Prevents Oxidative Stress-Induced Endothelial Dysfunction of the Systemic Artery in Rats With Intermittent Periodontal Inflammation.

PubMed

Saito, Takumi; Yamamoto, Yasuhiro; Feng, Guo-Gang; Kazaoka, Yoshiaki; Fujiwara, Yoshihiro; Kinoshita, Hiroyuki

2017-06-01

Periodontal inflammation causes endothelial dysfunction of the systemic artery. However, it is unknown whether the use of local anesthetics during painful dental procedures alleviates periodontal inflammation and systemic endothelial function. This study was designed to examine whether the gingival or systemic injection of lidocaine prevents oxidative stress-induced endothelial dysfunction of the systemic artery in rats with intermittent periodontal inflammation caused by lipopolysaccharides (LPS). Some rats received 1500 µg LPS injections to the gingiva during a week interval from the age of 8 to 11 weeks (LPS group). Lidocaine (3 mg/kg), LPS + lidocaine (3 mg/kg), LPS + lidocaine (1.5 mg/kg), and LPS + lidocaine (3 mg/kg, IP) groups simultaneously received gingival 1.5 or 3 mg/kg or IP 3 mg/kg injection of lidocaine on the same schedule as the gingival LPS. Isolated aortas or mandibles were subjected to the evaluation of histopathologic change, isometric force recording, reactive oxygen species, and Western immunoblotting. Mean blood pressure and heart rate did not differ among the control, LPS, LPS + lidocaine (3 mg/kg), and lidocaine (3 mg/kg) groups. LPS application reduced acetylcholine (ACh, 10 to 10 mol/L)-induced relaxation (29% difference at ACh 3 × 10 mol/L, P = .01), which was restored by catalase. Gingival lidocaine (1.5 and 3 mg/kg) dose dependently prevented the endothelial dysfunction caused by LPS application (24.5%-31.1% difference at ACh 3 × 10 mol/L, P = .006 or .001, respectively). Similar to the gingival application, the IP injection of lidocaine (3 mg/kg) restored the ACh-induced dilation of isolated aortas from rats with the LPS application (27.5% difference at ACh 3 × 10 mol/L, P < .001). Levels of reactive oxygen species were double in aortas from the LPS group (P < .001), whereas the increment was abolished by polyethylene glycol-catalase, gingival lidocaine (3 mg/kg), or the combination. The LPS induced a 4-fold increase in the protein expression of tumor necrosis factor-α in the periodontal tissue (P < .001), whereas the lidocaine (3 mg/kg) coadministration partly reduced the levels. Lidocaine application also decreased the protein expression of the nicotinamide adenine dinucleotide phosphate oxidase subunit p47phox, which was enhanced by the gingival LPS (5.6-fold increase; P < .001). Lidocaine preserved the aortic endothelial function through a decrease in arterial reactive oxygen species produced by nicotinamide adenine dinucleotide phosphate oxidase and periodontal tumor necrosis factor-α levels in rats with periodontal inflammation. These results suggest the beneficial effect of the gingival application of local anesthetics on the treatment of periodontal diseases on endothelial function of systemic arteries.

Metalloproteinase inhibition prevents acute respiratory distress syndrome.

PubMed

Carney, D E; McCann, U G; Schiller, H J; Gatto, L A; Steinberg, J; Picone, A L; Nieman, G F

2001-08-01

The acute respiratory distress syndrome (ARDS) occurs in patients with clearly identifiable risk factors, and its treatment remains merely supportive. We postulated that patients at risk for ARDS can be protected against lung injury by a prophylactic treatment strategy that targets neutrophil-derived proteases. We hypothesized that a chemically modified tetracycline 3 (COL-3), a potent inhibitor of neutrophil matrix metalloproteinases (MMPs) and neutrophil elastase (NE) with minimal toxicity, would prevent ARDS in our porcine endotoxin-induced ARDS model. Yorkshire pigs were anesthetized, intubated, surgically instrumented for hemodynamic monitoring, and randomized into three groups: (1) control (n = 4), surgical instrumentation only; (2) lipopolysaccharide (LPS) (n = 4), infusion of Escherichia coli lipopolysaccharide at 100 microg/kg; and (3) COL-3 + LPS (n = 5), ingestion of COL-3 (100 mg/kg) 12 h before LPS infusion. All animals were monitored for 6 h following LPS or sham LPS infusion. Serial bronchoalveolar lavage (BAL) samples were analyzed for MMP concentration by gelatin zymography. Lung tissue was fixed for morphometric assessment at necropsy. LPS infusion was marked by significant (P < 0.05) physiological deterioration as compared with the control group, including increased plateau airway pressure (P(plat)) (control = 15.7 +/- 0.4 mm Hg, LPS = 23.0 +/- 1.5 mm Hg) and a decrement in arterial oxygen partial pressure (P(a)O(2)) (LPS = 66 +/- 15 mm Hg, Control = 263 +/- 25 mm Hg) 6 h following LPS or sham LPS infusion, respectively. Pretreatment with COL-3 reduced the above pathophysiological changes 6 h following LPS infusion (P(plat) = 18.5 +/- 1.7 mm Hg, P(a)O(2) = 199 +/- 35 mm Hg; P = NS vs control). MMP-9 and MMP-2 concentration in BAL fluid was significantly increased between 2 and 4 h post-LPS infusion; COL-3 reduced the increase in MMP-9 and MMP-2 concentration at all time periods. Morphometrically LPS caused a significant sequestration of neutrophils and monocytes into pulmonary tissue. Pretreatment with COL-3 ameliorated this response. The wet/dry lung weight ratio was significantly greater (P < 0.05) in the LPS group (10.1 +/- 1.0 ratio) than in either the control (6.4 +/- 0.5 ratio) or LPS+COL-3 (7.4 +/- 0.6 ratio) group. A single prophylactic treatment with COL-3 prevented lung injury in our model of endotoxin-induced ARDS. The proposed mechanism of COL-3 is a synergistic inhibition of the terminal neutrophil effectors MMPs and NE. Similar to the universal practice of prophylaxis against gastric stress ulceration and deep venous thromboses in trauma patients, chemically modified tetracyclines may likewise be administered to prevent acute lung injury in critically injured patients at risk of developing ARDS. Copyright 2001 Academic Press.

Dependence of endotoxin-induced vascular hyporeactivity on extracellular L-arginine.

PubMed

Schott, C A; Gray, G A; Stoclet, J C

1993-01-01

1. The dependence on extracellular L-arginine of vascular hyporeactivity induced by bacterial lipopolysaccharide (LPS) was studied in vivo in rats infused with LPS and in vitro in endothelium-denuded rat thoracic aortic rings exposed to LPS. 2. Infusion of LPS during 50 min at a dose of 10 mg kg-1 h-1 produced a significant impairment of the pressor effect of noradrenaline, while in tissues collected 60 min after the start of LPS infusion, no significant alteration in either plasma arginine concentration or aortic arginine content was found compared to saline-infused controls (where plasma arginine was 78.5 +/- 7 microM and aortic arginine 394 +/- 124 nmol g-1 tissue). 3. Incubation of isolated, endothelium-denuded aortic rings with LPS (10 micrograms ml-1) in the absence of L-arginine for 4 h at 37 degrees C produced a 6 fold (P < 0.01) rightward shift in the noradrenaline concentration-effect curve compared to polymyxin B (1 micrograms ml-1, a LPS neutralizing agent) and reduced by 15% the maximum observed tension. 4. The presence of L-arginine (100 microM) during the incubation with LPS and throughout the following contraction experiments caused a 15 fold (P < 0.01) increase in the EC50 of noradrenaline and greater depression (45%) of the maximum observed tension compared to polymyxin B-treated controls. Responses in control, non LPS-treated rings were unaffected by the presence of L-arginine. 5. The addition of L-arginine to rings incubated with LPS in the absence of L-arginine and maximally precontracted with noradrenaline (10 microM) induced a dose-dependent relaxation. The EC50 of L-arginine was 8.0+/-0.3mu.6. The reactivity of LPS-treated rings to noradrenaline both in the absence and presence of L-arginine was restored to control levels by N0-nitro-L-arginine methyl ester (L-NAME, 300 mu), an inhibitor of NO production and by methylene blue (3 JAM), an inhibitor of guanylate cyclase.7. Incubation of isolated aortae in the absence of L-arginine did not significantly decrease the tissue arginine content, whether LPS (10 fg ml-') was present or not. Similarly, the presence of L-arginine(100 mu) in the incubation medium did not modify the tissue arginine content.8. These results show that the LPS-induced impairment of vasoconstriction elicited by noradrenaline is dependent on extracellular L-arginine, although the tissue arginine content is not depleted after LPS pretreatment, and that circulating L-arginine is sufficient to activate maximally the vascular L-arginine/NO pathway in endotoxaemic rats.

Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sabatino, María Eugenia; Sosa, Liliana del Valle; Petiti, Juan Pablo

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophsmore » taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signaling pathways. - Highlights: • In hyperplastic pituitaries, LPS triggered the lactotroph cell proliferation and IL-6 release. • Functional Toll-like receptor 4 (TLR4) is expressed at the plasma membrane of tumoral lactotrophs. • Increases in TLR4 and CD14 intracellular expression levels were detected after an LPS challenge. • The proliferative stimulation and IL-6 release involved the PI3K-Akt pathway and NF-κB activation. • 17β-estradiol attenuated the LPS-evoked tumoral lactotroph proliferation and IL-6 secretion.« less

Hypothesis Formation and Qualitative Reasoning in Molecular Biology

DTIC Science & Technology

1989-06-01

presents studies of the trp operon in the bacterium S . Marcescens . In vitro transcription studies showed that transcription termination does occur in...observed was that there are two 4.4. ANNOTATED CHRONOLOGY OF THE RESEARCH 135 translation-start codons in the S . marcescens leader region. The authors...of leader-region mRNA secondary structures in attenuation in the S . marcescens trp operon. A different bac- terium was used because it included

Transcriptional Activation of Pyoluteorin Operon Mediated by the LysR-Type Regulator PltR Bound at a 22 bp lys Box in Pseudomonas aeruginosa M18

PubMed Central

Wang, Guohao; Xu, Yuquan

2012-01-01

Pseudomonas aeruginosa M18, a rhizosphere-isolated bacterial strain showing strong antifungal activity, can produce secondary metabolites such as phenazine-1-carboxylic acid and pyoluteorin (Plt). The LysR-type transcriptional regulator PltR activates the Plt biosynthesis operon pltLABCDEFG, the expression of which is induced by Plt. Here, we identified and characterized the non-conserved pltL promoter (pltLp) specifically activated by PltR and its upstream neighboring lys box from the complicated pltR–pltL intergenic sequence. The 22 bp palindromic lys box, which consists of two 9 bp complementary inverted repeats interrupted by 4 bp, was found to contain the conserved, GC-rich LysR-binding motif (T-N11-A). Evidence obtained in vivo from mutational and lacZ report analyses and in vitro from electrophoretic mobility shift assays reveals that the PltR protein directly bound to the pltLp region as the indispensable binding motif “lys box”, thereby transcriptionally activating the pltLp-driven plt operon expression. Plt, as a potential non-essential coinducer of PltR, specifically induced the pltLp expression and thus strengthened its biosynthetic plt operon expression. PMID:22761817

Role of the Mce1 transporter in the lipid homeostasis of Mycobacterium tuberculosis.

PubMed

Forrellad, Marina Andrea; McNeil, Michael; Santangelo, María de la Paz; Blanco, Federico Carlos; García, Elizabeth; Klepp, Laura Inés; Huff, Jason; Niederweis, Michael; Jackson, Mary; Bigi, Fabiana

2014-03-01

Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the causative agent of human tuberculosis, has developed several strategies involving proteins and other compounds known collectively as virulence factors to subvert human host defences and invade the human host. The Mce proteins are among these virulence-related proteins and are encoded by the mce1, mce2, mce3 and mce4 operons in the genome of M. tuberculosis. It has been proposed that these operons encode ABC-like lipid transporters; however, the nature of their substrates has only been revealed in the case of the Mce4 proteins. Here we found that the knockout of the mce1 operon alters the lipid profile of M. tuberculosis H37Rv and the uptake of palmitic acid. Thin layer chromatography and liquid chromatography-mass spectrometry analysis showed that the mce1 mutant accumulates more mycolic acids than the wild type and complemented strains. Interestingly, this accumulation of mycolic acid is exacerbated when bacteria are cultured in the presence of palmitic acid or arachidonic acid. These results suggest that the mce1 operon may serve as a mycolic acid re-importer. Copyright © 2013 Elsevier Ltd. All rights reserved.

Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon.

PubMed

Calvio, Cinzia; Celandroni, Francesco; Ghelardi, Emilia; Amati, Giuseppe; Salvetti, Sara; Ceciliani, Fabrizio; Galizzi, Alessandro; Senesi, Sonia

2005-08-01

The number and disposition of flagella harbored by eubacteria are regulated by a specific trait successfully maintained over generations. The genes governing the number of flagella in Bacillus subtilis have never been identified, although the ifm locus has long been recognized to influence the motility phenotype of this microorganism. The characterization of a spontaneous ifm mutant of B. subtilis, displaying diverse degrees of cell flagellation in both liquid and solid media, raised the question of how the ifm locus governs the number and assembly of functional flagella. The major finding of this investigation is the characterization of a newly identified dicistronic operon, named swrA, that controls both swimming motility and swarming differentiation in B. subtilis. Functional analysis of the swrA operon allowed swrAA (previously named swrA [D. B. Kearns, F. Chu, R. Rudner, and R. Losick, Mol. Microbiol. 52:357-369, 2004]) to be the first gene identified in B. subtilis that controls the number of flagella in liquid environments and the assembly of flagella in response to cell contact with solid surfaces. Evidence is given that the second gene of the operon, swrAB, is essential for enabling the surface-adhering cells to undergo swarming differentiation. Preliminary data point to a molecular interaction between the two gene products.

Regulation of nrf operon expression in pathogenic enteric bacteria: sequence divergence reveals new regulatory complexity

PubMed Central

Godfrey, Rita E.; Lee, David J.; Busby, Stephen J. W.

2017-01-01

Summary The Escherichia coli K‐12 nrf operon encodes a periplasmic nitrite reductase, the expression of which is driven from a single promoter, pnrf. Expression from pnrf is activated by the FNR transcription factor in response to anaerobiosis and further increased in response to nitrite by the response regulator proteins, NarL and NarP. FNR‐dependent transcription is suppressed by the binding of two nucleoid associated proteins, IHF and Fis. As Fis levels increase in cells grown in rich medium, the positioning of its binding site, overlapping the promoter −10 element, ensures that pnrf is sharply repressed. Here, we investigate the expression of the nrf operon promoter from various pathogenic enteric bacteria. We show that pnrf from enterohaemorrhagic E. coli is more active than its K‐12 counterpart, exhibits substantial FNR‐independent activity and is insensitive to nutrient quality, due to an improved −10 element. We also demonstrate that the Salmonella enterica serovar Typhimurium core promoter is more active than previously thought, due to differences around the transcription start site, and that its expression is repressed by downstream sequences. We identify the CsrA RNA binding protein as being responsible for this, and show that CsrA differentially regulates the E. coli K‐12 and Salmonella nrf operons. PMID:28211111

Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation

PubMed Central

Dueholm, Morten S; Søndergaard, Mads T; Nilsson, Martin; Christiansen, Gunna; Stensballe, Allan; Overgaard, Michael T; Givskov, Michael; Tolker-Nielsen, Tim; Otzen, Daniel E; Nielsen, Per H

2013-01-01

The fap operon, encoding functional amyloids in Pseudomonas (Fap), is present in most pseudomonads, but so far the expression and importance for biofilm formation has only been investigated for P. fluorescens strain UK4. In this study, we demonstrate the capacity of P. aeruginosa PAO1, P. fluorescens Pf-5, and P. putida F1 to express Fap fibrils, and investigated the effect of Fap expression on aggregation and biofilm formation. The fap operon in all three Pseudomonas species conferred the ability to express Fap fibrils as shown using a recombinant approach. This Fap overexpression consistently resulted in highly aggregative phenotypes and in increased biofilm formation. Detailed biophysical investigations of purified fibrils confirmed FapC as the main fibril monomer and supported the role of FapB as a minor, nucleating constituent as also indicated by bioinformatic analysis. Bioinformatics analysis suggested FapF and FapD as a potential β-barrel membrane pore and protease, respectively. Manipulation of the fap operon showed that FapA affects monomer composition of the final amyloid fibril, and that FapB is an amyloid protein, probably a nucleator for FapC polymerization. Our study highlights the fap operon as a molecular machine for functional amyloid formation. PMID:23504942

The sps Gene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

PubMed Central

Cangiano, Giuseppina; Sirec, Teja; Panarella, Cristina; Isticato, Rachele; Baccigalupi, Loredana; De Felice, Maurilio

2014-01-01

The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform. PMID:25239894

Hematologic interactions of endotoxin, tumor necrosis factor alpha (TNF alpha), interleukin 1, and adrenal hormones and the hematologic effects of TNF alpha in Corynebacterium parvum-primed rats.

PubMed

Ulich, T R; del Castillo, J; Ni, R X; Bikhazi, N

1989-06-01

Endotoxin reduces the release among other cytokines of tumor necrosis factor (TNF) and interleukin 1 (IL-1) and causes peripheral lymphopenia and a dose-response-dependent initial neutropenia followed by a monophasic neutrophilia. TNF alone induces lymphopenia and an initial neutropenia followed by a biphasic neutrophilia. IL-1 alone induces lymphopenia and a monophasic neutrophilia. TNF-plus-IL-1 caused a greater lymphopenia than either monokine alone, suggesting that both monokines contribute to LPS-induced lymphopenia. TNF-plus-IL-1 induced neutropenia similar in magnitude to that induced by TNF alone and induced a neutrophilia significantly greater than that induced by either monokine alone, suggesting that LPS-induced neutropenia is caused by TNF, while LPS-induced neutrophilia is due to the combined effects of TNF and II-1. TNF and IL-1 were administered together with LPS to simulate the in vivo condition of endogenous monokine release during gram-negative bacteremia. TNF combined with LPS increased both the duration and magnitude of LPS-induced lymphopenia, LPS-induced neutropenia, and LPS-induced neutrophilia. TNF-plus-LPS treated rats at 2 hours after injection exhibited a striking 93% decrease in bone marrow neutrophils even though no peripheral neutrophilia was yet apparent, suggesting that the subsequent neutrophilia was due to demargination and recirculation of neutrophils sequestered in the peripheral vasculature immediately after their release from the bone marrow. Epinephrine, which causes neutrophilia by demargination but not by release of marrow neutrophils, reversed the initial neutropenia in TNF-plus-LPS-treated rats and increased the neutrophilia. IL-1 combined with LPS increased LPS-induced neutrophilia, suggesting that endogenous IL-1 also contributed to LPS-induced neutrophilia. Corynebacterium parvum-primed rats with hyperplasia of the monocyte-macrophage system and treated with TNF differed from naive rats treated with TNF in that the second peak was as great as the initial peak of neutrophilia, supporting the hypothesis that the second peak of TNF-induced neutrophilia is due to the release of endogenous monokines. In conclusion, exogenous TNF, IL-1, and adrenal hormones affect circulating numbers of lymphocytes and neutrophils in a fashion consistent with their postulated endogenous role in the regulation of leukocyte trafficking during bacterial infection.

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

PubMed

Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

2016-10-18

The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB 2 FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB 2 FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the cytoplasmic membrane prior to transport to the cell surface. How ATP hydrolysis is coupled with LPS release from the membrane is not understood. We have identified residues at the interface between the ATPase and the transmembrane domains of this heteromeric ABC complex that are important for LPS transport, some of which coordinate ATPase activity with LPS release. Copyright © 2016 Simpson et al.

Buthionine sulfoximine, a glutathione depletor, attenuates endotoxic fever and reduces IL-1β and IL-6 level in rats.

PubMed

Wrotek, Sylwia; Domagalski, Krzysztof; Jędrzejewski, Tomasz; Dec, Eliza; Kozak, Wiesław

2017-02-01

The aim of our study was to investigate the effect of buthionine sulfoximine (BSO) - a glutathione depletor - on a course of endotoxic fever and IL-1β and IL-6 production. Male Wistar rats were subjected to intraperitoneal injection of lipopolysaccharide (LPS) from E. coli (50μg/kg, ip) to provoke fever. The level of spleen glutathione, plasma interleukin (IL)-1β, IL-6, and deep body temperature (Tb) were measured. The LPS administration provoked fever (the average Tb was 38.14±0.05°C in NaCl/LPS-treated rats vs 37.10±0.03°C in control, not-treated rats; p<0.001). We observed that LPS injection induced a decrease in spleen glutathione level (7.67±0.92nM/g vs 13.27±0.47nM/g in not-treated rats; p<0.001). Furthermore, the injection of LPS provoked an elevation of plasma IL-1β and IL-6 concentration (from values below the lowest detectable standard in not-treated animals to 199.99±34.89pg/mL and 7500±542.21pg/mL, respectively; p<0.001). Pretreatment with BSO enhanced glutathione decrease in LPS-treated rats (5.05±0.49nM/g), and significantly affected fever (maximal Tb was 37.81±0.07°C in BSO/LPS-treated rats vs 38.76±0.11°C in NaCl/LPS-treated rats). BSO 4h after LPS injection decreased IL-1β and IL-6 gene expression (about 1.5 fold, and 2 fold, respectively). In a consequence we observed a decrease in plasma IL-6 concentration (4h after LPS injection plasma IL-6 was 4167.17±956.54pg/mL in BSO/LPS-treated rats vs 7500±542.21pg/mL in NaCl/LPS-treated rats; p<0.001), and later IL-1β (7h after LPS injection the IL-1β concentration was not detected). Based on these data, we conclude that BSO, in addition to well-known application as an inhibitor of glutathione synthesis, is an antipyretic agent which reduces both IL-1β and IL-6 concentration. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enhancement of OVA-induced murine lung eosinophilia by co-exposure to contamination levels of LPS in Asian sand dust and heated dust

PubMed Central

2014-01-01

Background A previous study has shown that the aggravation of Asian sand dust (ASD) on ovalbumin (OVA)-induced lung eosinphilia was more severe in lipopolysaccharide (LPS)-rich ASD than in SiO2-rich ASD. Therefore, the effects of different LPS contamination levels in ASD on the aggravation of OVA-induced lung eosinophilia were investigated in the present study. Methods Before beginning the in vivo experiment, we investigated whether the ultra-pure LPS would act only on TLR4 or not using bone marrow-derived macrophages (BMDMs) of wild–type, TLR2-/-, TLR4-/- and MyD88-/- BALB/c mice. ASD collected from the desert was heated to remove toxic organic substances (H-ASD). BALB/c mice were instilled intratracheally with 12 different testing samples prepared with LPS (1 ng and 10 ng), H-ASD, and OVA in a normal saline solution. The lung pathology, cytological profiles in the bronchoalveolar lavage fluid (BALF), the levels of inflammatory cytokines/chemokines in BALF and OVA-specific immunoglobulin in serum were investigated. Results The LPS exhibited no response to the production of TNF-α and IL-6 in BMDMs from TLR4-/-, but did from TLR2-/-. H-ASD aggravated the LPS-induced neutrophilic lung inflammation. In the presence of OVA, LPS increased the level of eosinophils slightly and induced trace levels of Th2 cytokines IL-5 and IL-13 at the levels of 1 ng and 10 ng. In the presence of OVA and H-ASD, LPS induced severe eosinophil infiltration and proliferation of goblet cells in the airways as well as remarkable increases in Th2 cytokines IL-5 and IL-13 in BALF. The mixture containing LPS (1 ng) showed adjuvant activity on OVA-specific IgE and IgG1 production. Conclusions The results suggest that H-ASD with naturally-occurring levels of LPS enhances OVA-induced lung eosinophilia via increases in Th2-mediated cytokines and antigen-specific immunoglobulin. These results indicate that LPS is a strong candidate for being a major aggravating substance in ASD. PMID:24982682

Enhancement of OVA-induced murine lung eosinophilia by co-exposure to contamination levels of LPS in Asian sand dust and heated dust.

PubMed

Ren, Yahao; Ichinose, Takamichi; He, Miao; Song, Yuan; Yoshida, Yasuhiro; Yoshida, Seiichi; Nishikawa, Masataka; Takano, Hirohisa; Sun, Guifan; Shibamoto, Takayuki

2014-01-01

A previous study has shown that the aggravation of Asian sand dust (ASD) on ovalbumin (OVA)-induced lung eosinphilia was more severe in lipopolysaccharide (LPS)-rich ASD than in SiO2-rich ASD. Therefore, the effects of different LPS contamination levels in ASD on the aggravation of OVA-induced lung eosinophilia were investigated in the present study. Before beginning the in vivo experiment, we investigated whether the ultra-pure LPS would act only on TLR4 or not using bone marrow-derived macrophages (BMDMs) of wild-type, TLR2-/-, TLR4-/- and MyD88-/- BALB/c mice. ASD collected from the desert was heated to remove toxic organic substances (H-ASD). BALB/c mice were instilled intratracheally with 12 different testing samples prepared with LPS (1 ng and 10 ng), H-ASD, and OVA in a normal saline solution. The lung pathology, cytological profiles in the bronchoalveolar lavage fluid (BALF), the levels of inflammatory cytokines/chemokines in BALF and OVA-specific immunoglobulin in serum were investigated. The LPS exhibited no response to the production of TNF-α and IL-6 in BMDMs from TLR4-/-, but did from TLR2-/-. H-ASD aggravated the LPS-induced neutrophilic lung inflammation. In the presence of OVA, LPS increased the level of eosinophils slightly and induced trace levels of Th2 cytokines IL-5 and IL-13 at the levels of 1 ng and 10 ng. In the presence of OVA and H-ASD, LPS induced severe eosinophil infiltration and proliferation of goblet cells in the airways as well as remarkable increases in Th2 cytokines IL-5 and IL-13 in BALF. The mixture containing LPS (1 ng) showed adjuvant activity on OVA-specific IgE and IgG1 production. The results suggest that H-ASD with naturally-occurring levels of LPS enhances OVA-induced lung eosinophilia via increases in Th2-mediated cytokines and antigen-specific immunoglobulin. These results indicate that LPS is a strong candidate for being a major aggravating substance in ASD.

Signal-transducing mechanisms of ketamine-caused inhibition of interleukin-1{beta} gene expression in lipopolysaccharide-stimulated murine macrophage-like Raw 264.7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, T.-L.; Chang, C.-C.; Lin, Y.-L.

2009-10-01

Ketamine may affect the host immunity. Interleukin-1{beta} (IL-1{beta}), IL-6, and tumor necrosis factor-{alpha} (TNF-{alpha}) are pivotal cytokines produced by macrophages. This study aimed to evaluate the effects of ketamine on the regulation of inflammatory cytokine gene expression, especially IL-1{beta}, in lipopolysaccharide (LPS)-activated murine macrophage-like Raw 264.7 cells and its possible signal-transducing mechanisms. Administration of Raw 264.7 cells with a therapeutic concentration of ketamine (100 {mu}M), LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. Exposure to 100 {mu}M ketamine decreased the binding affinity of LPS and LPS-binding protein but didmore » not affect LPS-induced RNA and protein synthesis of TLR4. Treatment with LPS significantly increased IL-1{beta}, IL-6, and TNF-{alpha} gene expressions in Raw 264.7 cells. Ketamine at a clinically relevant concentration did not affect the synthesis of these inflammatory cytokines, but significantly decreased LPS-caused increases in these cytokines. Immunoblot analyses, an electrophoretic mobility shift assay, and a reporter luciferase activity assay revealed that ketamine significantly decreased LPS-induced translocation and DNA binding activity of nuclear factor-kappa B (NF{kappa}B). Administration of LPS sequentially increased the phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK. However, a therapeutic concentration of ketamine alleviated such augmentations. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA reduced cellular TLR4 amounts and ameliorated LPS-induced RAS activation and IL-1{beta} synthesis. Co-treatment with ketamine and TLR4 siRNA synergistically ameliorated LPS-caused enhancement of IL-1{beta} production. Results of this study show that a therapeutic concentration of ketamine can inhibit gene expression of IL-1{beta} possibly through suppressing TLR4-mediated signal-transducing phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK and subsequent translocation and transactivation of NF{kappa}B.« less

Transcriptome analysis reveals key roles of AtLBR-2 in LPS-induced defense responses in plants.

PubMed

Iizasa, Sayaka; Iizasa, Ei'ichi; Watanabe, Keiichi; Nagano, Yukio

2017-12-29

Lipopolysaccharide (LPS) from Gram-negative bacteria cause innate immune responses in animals and plants. The molecules involved in LPS signaling in animals are well studied, whereas those in plants are not yet as well documented. Recently, we identified Arabidopsis AtLBR-2, which binds to LPS from Pseudomonas aeruginosa (pLPS) directly and regulates pLPS-induced defense responses, such as pathogenesis-related 1 (PR1) expression and reactive oxygen species (ROS) production. In this study, we investigated the pLPS-induced transcriptomic changes in wild-type (WT) and the atlbr-2 mutant Arabidopsis plants using RNA-Seq technology. RNA-Seq data analysis revealed that pLPS treatment significantly altered the expression of 2139 genes, with 605 up-regulated and 1534 down-regulated genes in WT. Gene ontology (GO) analysis on these genes showed that GO terms, "response to bacterium", "response to salicylic acid (SA) stimulus", and "response to abscisic acid (ABA) stimulus" were enriched amongst only in up-regulated genes, as compared to the genes that were down-regulated. Comparative analysis of differentially expressed genes between WT and the atlbr-2 mutant revealed that 65 genes were up-regulated in WT but not in the atlbr-2 after pLPS treatment. Furthermore, GO analysis on these 65 genes demonstrated their importance for the enrichment of several defense-related GO terms, including "response to bacterium", "response to SA stimulus", and "response to ABA stimulus". We also found reduced levels of pLPS-induced conjugated SA glucoside (SAG) accumulation in atlbr-2 mutants, and no differences were observed in the gene expression levels in SA-treated WT and the atlbr-2 mutants. These 65 AtLBR-2-dependent up-regulated genes appear to be important for the enrichment of some defense-related GO terms. Moreover, AtLBR-2 might be a key molecule that is indispensable for the up-regulation of defense-related genes and for SA signaling pathway, which is involved in defense against pathogens containing LPS.

Porphyromonas endodontalis lipopolysaccharides induce RANKL by mouse osteoblast in a way different from that of Escherichia coli lipopolysaccharide.

PubMed

Tang, Yin; Sun, Feifei; Li, Xiaoting; Zhou, Yuan; Yin, Shihai; Zhou, Xuedong

2011-12-01

Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-κB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts. LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Toll-like receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. Both of the anti-TLR2 and anti-TLR4 antibodies significantly (P < .05) inhibited the expression of RANKL from osteoblasts stimulated with P. endodontalis LPS; only anti-TLR2 antibody had a significant (P < .05) inhibitory effect on E. coli LPS signaling. SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) prevented the up-regulation of RANKL expression in P. endodontalis LPS-infected osteoblasts (P < .05). The inhibitory effect of wortmannin (phosphatidylinositol 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase-1/2 [MEK 1/2] inhibitor) were observed in E. coli LPS-treated mouse osteoblasts (P < .05). Results from this study showed that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4-JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin (E. coli LPS). Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression.

PubMed

Gee, Katrina; Lim, Wilfred; Ma, Wei; Nandan, Devki; Diaz-Mitoma, Francisco; Kozlowski, Maya; Kumar, Ashok

2002-11-15

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.

Josamycin suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1β in murine macrophages.

PubMed

Choi, Eun-Young; Choe, So-Hui; Hyeon, Jin-Yi; Park, Hae Ryoun; Choi, In Soon; Kim, Sung-Jo

2018-06-05

Josamycin has immunomodulatory properties independent of its antibacterial actions. This study was designed to explore the influences and associated mechanisms of josamycin upon the generation of proinflammatory mediators in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogenic bacterium associated with periodontal disease. LPS was purified by employing phenol-water extraction protocol. Culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-1β. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. The expression levels of IL-1β were analyzed by confocal laser scanning microscopy. NF-κB-dependent SEAP levels were estimated by reporter assay. Josamycin significantly attenuated NO production elicited by P. intermedia LPS as well as induction of iNOS protein and mRNA in RAW264.7 cells. While the release of IL-1β from cells stimulated by LPS was suppressed in the presence of josamycin, josamycin failed to reduce the degree of IL-1β mRNA expression. Josamycin did not reduce the stability of IL-1β mRNA induced by P. intermedia LPS, at the same time josamycin also failed to suppress the LPS-induced intracellular IL-1β expression. Josamycin augmented HO-1 induction in cells exposed to P. intermedia LPS, and SnPP, an inhibitor of HO-1 activity, reversed the suppressive impact of josamycin upon NO generation induced by LPS. Josamycin diminished NF-κB transcriptional activity induced by P. intermedia LPS. Further, josamycin enhanced SOCS1 mRNA level in cells activated with LPS. Josamycin suppressed P. intermedia LPS-induced generation of NO and IL-1β in RAW264.7 macrophages via the inhibition of NF-κB activation as well as HO-1 and SOCS1 induction. Josamycin may have benefits as a host modulatory agent in treating periodontal disease. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Therapeutic Effect of Intravenous Infusion of Perfluorocarbon Emulsion on LPS-Induced Acute Lung Injury in Rats

PubMed Central

Lv, Qi; Yin, Xiaofeng; Song, Jianqi; Landén, Ning Xu; Fan, Haojun

2014-01-01

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are the leading causes of death in critical care. Despite extensive efforts in research and clinical medicine, mortality remains high in these diseases. Perfluorocarbon (PFC), a chemical compound known as liquid ventilation medium, is capable of dissolving large amounts of physiologically important gases (mainly oxygen and carbon dioxide). In this study we aimed to investigate the effect of intravenous infusion of PFC emulsion on lipopolysaccharide (LPS) induced ALI in rats and elucidate its mechanism of action. Forty two Wistar rats were randomly divided into three groups: 6 rats were treated with saline solution by intratracheal instillation (control group), 18 rats were treated with LPS by intratracheal instillation (LPS group) and the other 18 rats received PFC through femoral vein prior to LPS instillation (LPS+PFC group). The rats in the control group were sacrificed 6 hours later after saline instillation. At 2, 4 and 6 hours of exposure to LPS, 6 rats in the LPS group and 6 rats in LPS+PFC group were sacrificed at each time point. By analyzing pulmonary pathology, partial pressure of oxygen in the blood (PaO2) and lung wet-dry weight ratio (W/D) of each rat, we found that intravenous infusion of PFC significantly alleviated acute lung injury induced by LPS. Moreover, we showed that the expression of pulmonary myeloperoxidase (MPO), intercellular adhesion molecule-1 (ICAM-1) of endothelial cells and CD11b of polymorphonuclear neutrophils (PMN) induced by LPS were significantly decreased by PFC treatment in vivo. Our results indicate that intravenous infusion of PFC inhibits the infiltration of PMNs into lung tissue, which has been shown as the core pathogenesis of ALI/ARDS. Thus, our study provides a theoretical foundation for using intravenous infusion of PFC to prevent and treat ALI/ARDS in clinical practice. PMID:24489970

LPS-Induced Low-Grade Inflammation Increases Hypothalamic JNK Expression and Causes Central Insulin Resistance Irrespective of Body Weight Changes.

PubMed

Rorato, Rodrigo; Borges, Beatriz de Carvalho; Uchoa, Ernane Torres; Antunes-Rodrigues, José; Elias, Carol Fuzeti; Elias, Lucila Leico Kagohara

2017-07-04

Metabolic endotoxemia contributes to low-grade inflammation in obesity, which causes insulin resistance due to the activation of intracellular proinflammatory pathways, such as the c-Jun N-terminal Kinase (JNK) cascade in the hypothalamus and other tissues. However, it remains unclear whether the proinflammatory process precedes insulin resistance or it appears because of the development of obesity. Hypothalamic low-grade inflammation was induced by prolonged lipopolysaccharide (LPS) exposure to investigate if central insulin resistance is induced by an inflammatory stimulus regardless of obesity. Male Wistar rats were treated with single (1 LPS) or repeated injections (6 LPS) of LPS (100 μg/kg, IP) to evaluate the phosphorylation of the insulin receptor substrate-1 (IRS1), Protein kinase B (AKT), and JNK in the hypothalamus. Single LPS increased the expression of pIRS1, pAKT, and pJNK, whereas the repeated LPS treatment failed to recruit pIRS1 and pAKT. The 6 LPS treated rats showed increased total JNK and pJNK. The 6 LPS rats became unresponsive to the hypophagic effect induced by central insulin administration (12 μM/5 μL, ICV). Prolonged exposure to LPS (24 h) impaired the insulin-induced AKT phosphorylation and the translocation of the transcription factor forkhead box protein O1 (FoxO1) from the nucleus to the cytoplasm of the cultured hypothalamic GT1-7 cells. Central administration of the JNK inhibitor (20 μM/5 μL, ICV) restored the ability of insulin to phosphorylate IRS1 and AKT in 6 LPS rats. The present data suggest that an increased JNK activity in the hypothalamus underlies the development of insulin resistance during prolonged exposure to endotoxins. Our study reveals that weight gain is not mandatory for the development of hypothalamic insulin resistance and the blockade of proinflammatory pathways could be useful for restoring the insulin signaling during prolonged low-grade inflammation as seen in obesity.

Multiple mechanisms involved in diabetes protection by lipopolysaccharide in non-obese diabetic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jun; Department of Pharmacology, College of Medicine, Wuhan University of Science and Technology, Wuhan; Cao, Hui

Toll-like receptor 4 (TLR4) activation has been proposed to be important for islet cell inflammation and eventually β cell loss in the course of type 1 diabetes (T1D) development. However, according to the “hygiene hypothesis”, bacterial endotoxin lipopolysaccharide (LPS), an agonist on TLR4, inhibits T1D progression. Here we investigated possible mechanisms for the protective effect of LPS on T1D development in non-obese diabetic (NOD) mice. We found that LPS administration to NOD mice during the prediabetic state neither prevented nor reversed insulitis, but delayed the onset and decreased the incidence of diabetes, and that a multiple-injection protocol is more effectivemore » than a single LPS intervention. Further, LPS administration suppressed spleen T lymphocyte proliferation, increased the generation of CD4{sup +}CD25{sup +}Foxp3{sup +} regulatory T cells (Tregs), reduced the synthesis of strong Th1 proinflammatory cytokines, and downregulated TLR4 and its downstream MyD88-dependent signaling pathway. Most importantly, multiple injections of LPS induced a potential tolerogenic dendritic cell (DC) subset with low TLR4 expression without influencing the DC phenotype. Explanting DCs from repeated LPS-treated NOD mice into NOD/SCID diabetic mice conferred sustained protective effects against the progression of diabetes in the recipients. Overall, these results suggest that multiple mechanisms are involved in the protective effects of LPS against the development of diabetes in NOD diabetic mice. These include Treg induction, down-regulation of TLR4 and its downstream MyD88-dependent signaling pathway, and the emergence of a potential tolerogenic DC subset. - Highlights: • Administration of lipopolysaccharide (LPS) prevented type 1 diabetes in NOD mice. • Downregulating TLR4 level and MyD88-dependent pathway contributed to protection of LPS. • LPS administration also hampered DC maturation and promoted Treg differentiation.« less

Low-dose lipopolysaccharide (LPS) inhibits aggressive and augments depressive behaviours in a chronic mild stress model in mice.

PubMed

Couch, Yvonne; Trofimov, Alexander; Markova, Natalyia; Nikolenko, Vladimir; Steinbusch, Harry W; Chekhonin, Vladimir; Schroeter, Careen; Lesch, Klaus-Peter; Anthony, Daniel C; Strekalova, Tatyana

2016-05-16

Aggression, hyperactivity, impulsivity, helplessness and anhedonia are all signs of depressive-like disorders in humans and are often reported to be present in animal models of depression induced by stress or by inflammatory challenges. However, chronic mild stress (CMS) and clinically silent inflammation, during the recovery period after an infection, for example, are often coincident, but comparison of the behavioural and molecular changes that underpin CMS vs a mild inflammatory challenge and impact of the combined challenge is largely unexplored. Here, we examined whether stress-induced behavioural and molecular responses are analogous to lipopolysaccharide (LPS)-induced behavioural and molecular effects and whether their combination is adaptive or maladaptive. Changes in measures of hedonic sensitivity, helplessness, aggression, impulsivity and CNS and systemic cytokine and 5-HT-system-related gene expression were investigated in C57BL/6J male mice exposed to chronic stress alone, low-dose LPS alone or a combination of LPS and stress. When combined with a low dose of LPS, chronic stress resulted in an enhanced depressive-like phenotype but significantly reduced manifestations of aggression and hyperactivity. At the molecular level, LPS was a strong inducer of TNFα, IL-1β and region-specific 5-HT2A mRNA expression in the brain. There was also increased serum corticosterone as well as increased TNFα expression in the liver. Stress did not induce comparable levels of cytokine expression to an LPS challenge, but the combination of stress with LPS reduced the stress-induced changes in 5-HT genes and the LPS-induced elevated IL-1β levels. It is evident that when administered independently, both stress and LPS challenges induced distinct molecular and behavioural changes. However, at a time when LPS alone does not induce any overt behavioural changes per se, the combination with stress exacerbates depressive and inhibits aggressive behaviours.

Effects of voluntary wheel running on LPS-induced sickness behavior in aged mice.

PubMed

Martin, Stephen A; Pence, Brandt D; Greene, Ryan M; Johnson, Stephanie J; Dantzer, Robert; Kelley, Keith W; Woods, Jeffrey A

2013-03-01

Peripheral stimulation of the innate immune system with LPS causes exaggerated neuroinflammation and prolonged sickness behavior in aged mice. Regular moderate intensity exercise has been shown to exert anti-inflammatory effects that may protect against inappropriate neuroinflammation and sickness in aged mice. The purpose of this study was to test the hypothesis that voluntary wheel running would attenuate LPS-induced sickness behavior and proinflammatory cytokine gene expression in ~22-month-old C57BL/6J mice. Mice were housed with a running wheel (VWR), locked-wheel (Locked), or no wheel (Standard) for 10 weeks, after which they were intraperitoneally injected with LPS across a range of doses (0.02, 0.08, 0.16, 0.33 mg/kg). VWR mice ran on average 3.5 km/day and lost significantly more body weight and body fat, and increased their forced exercise tolerance compared to Locked and Shoebox mice. VWR had no effect on LPS-induced anorexia, adipsia, weight-loss, or reductions in locomotor activity at any LPS dose when compared to Locked and Shoebox groups. LPS induced sickness behavior in a dose-dependent fashion (0.33>0.02 mg/kg). Twenty-four hours post-injection (0.33 mg/kg LPS or Saline) we found a LPS-induced upregulation of whole brain TNFα, IL-1β, and IL-10 mRNA, and increased IL-1β and IL-6 in the spleen and liver; these effects were not attenuated by VWR. We conclude that VWR does not reduce LPS-induced exaggerated or prolonged sickness behavior in aged animals, or 24h post-injection (0.33 mg/kg LPS or Saline) brain and peripheral proinflammatory cytokine gene expression. The necessity of the sickness response is critical for survival and may outweigh the subtle benefits of exercise training in aged animals. Copyright © 2012 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Department of Infectious Diseases, Peking University Third Hospital, Beijing; Zhang, Yuan

The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β{sub 2}-adrenergic receptor (β{sub 2}-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β{sub 2}-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1βmore » (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β{sub 2}-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β{sub 2}-AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production.« less

Carbon monoxide-releasing molecule-3 suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1β in murine macrophages.

PubMed

Choi, Eun-Young; Choe, So-Hui; Hyeon, Jin-Yi; Choi, Jeom-Il; Choi, In Soon; Kim, Sung-Jo

2015-10-05

This study was performed to analyze the effect of carbon monoxide (CO)-releasing molecule-3 (CORM-3) in alleviating the production of proinflammatory mediators in macrophages treated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated with periodontal disease, and its possible mechanisms of action. LPS was isolated using the hot phenol-water method. Culture supernatants were assayed for nitric oxide (NO) and interleukin-1β (IL-1β). Gene expression was quantified by real-time PCR, and protein expression by immunoblotting. DNA-binding activities of NF-κB subunits were determined using an ELISA-based kit. CORM-3 suppressed the production of inducible NO synthase (iNOS)-derived NO and IL-1β at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. CORM-3 enhanced heme oxygenase-1 (HO-1) expression in cells stimulated with P. intermedia LPS, and inhibition of HO-1 activity by SnPP notably reversed the suppressive effect of CORM-3 on LPS-induced production of NO. LPS-induced phosphorylation of p38 and JNK was not affected by CORM-3. CORM-3 did not influence P. intermedia LPS-induced degradation of IκB-α. Instead, nuclear translocation of NF-κB p65 and p50 subunits was blocked by CORM-3 in LPS-treated cells. In addition, CORM-3 reduced LPS-induced p65 and p50 binding to DNA. Besides, CORM-3 significantly suppressed P. intermedia LPS-induced phosphorylation of STAT1. Overall, this study indicates that CORM-3 suppresses the production of NO and IL-1β in P. intermedia LPS-activated murine macrophages via HO-1 induction and inhibition of NF-κB and STAT1 pathways. The modulation of host inflammatory response by CORM-3 would be an attractive therapeutic approach to attenuate the progression of periodontal disease. Copyright © 2015 Elsevier B.V. All rights reserved.

NF-κB p65 Subunit Mediates Lipopolysaccharide-Induced Na+/I− Symporter Gene Expression by Involving Functional Interaction with the Paired Domain Transcription Factor Pax8

PubMed Central

Nicola, Juan Pablo; Nazar, Magalí; Mascanfroni, Iván Darío; Pellizas, Claudia Gabriela; Masini-Repiso, Ana María

2010-01-01

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) elicits a variety of biological responses. Na+/I− symporter (NIS)-mediated iodide uptake is the main rate-limiting step in thyroid hormonogenesis. We have recently reported that LPS stimulates TSH-induced iodide uptake. Here, we further analyzed the molecular mechanism involved in the LPS-induced NIS expression in Fisher rat thyroid cell line 5 (FRTL-5) thyroid cells. We observed an increase in TSH-induced NIS mRNA expression in a dose-dependent manner upon LPS treatment. LPS enhanced the TSH-stimulated NIS promoter activity denoting the NIS-upstream enhancer region (NUE) as responsible for the stimulatory effects. We characterized a novel putative conserved κB site for the transcription factor nuclear factor-κB (NF-κB) within the NUE region. NUE contains two binding sites for the transcription factor paired box 8 (Pax8), main regulator of NIS transcription. A physical interaction was observed between the NF-κB p65 subunit and paired box 8 (Pax8), which appears to be responsible for the synergic effect displayed by these transcription factors on NIS gene transcription. Moreover, functional blockage of NF-κB signaling and site-directed mutagenesis of the κB cis-acting element abrogated LPS stimulation. Silencing expression of p65 confirmed its participation as an effector of LPS-induced NIS stimulation. Furthermore, chromatin immunoprecipitation corroborated that NIS is a novel target gene for p65 transactivation in response to LPS. Moreover, we were able to corroborate the LPS-stimulatory effect on thyroid cells in vivo in LPS-treated rats, supporting that thyrocytes are capable of responding to systemic infections. In conclusion, our results reveal a new mechanism involving p65 in the LPS-induced NIS expression, denoting a novel aspect in thyroid cell differentiation. PMID:20667985

The ocular endothelin system: a novel target for the treatment of endotoxin-induced uveitis with bosentan.

PubMed

Keles, Sadullah; Halici, Zekai; Atmaca, Hasan Tarik; Yayla, Muhammed; Yildirim, Kenan; Ekinci, Metin; Akpinar, Erol; Altuner, Durdu; Cakici, Ozgur; Bayraktutan, Zafer

2014-05-15

We compared the anti-inflammatory effects of bosentan and dexamethasone in endotoxin-induced uveitis (EIU). Endotoxin-induced uveitis was induced by subcutaneous injection of lipopolysaccharide (LPS, 200 μg) in Wistar rats. Rats were divided randomly into 10 groups (n = 6). Bosentan at doses of 50 and 100 mg/kg were administered orally 1 hour before and 12 hours after LPS injection, and dexamethasone was administered by intraperitoneally 30 minutes before and 30 minutes after LPS injection at a dose of 1 mg/kg. Data were collected at two time points for each control and treatment; animals were killed at either 3 or 24 hours after LPS injection. Histopathologic evaluation and aqueous humour measurements of TNF-α level were performed, and endothelin-1 (ET-1), inducible nitric oxide synthase (iNOS), and endothelin receptor A and B (EDNRA and B) expression were analyzed. The group treated with 100 mg/kg bosentan at 24 hours displayed significantly milder uveitis and fewer inflammatory cells compared to LPS-injected animals, and there were similar findings in the dexamethasone-treated group at 24 hours. The TNF-α levels in the dexamethasone treatment group were lower than those in the LPS-induced uveitis control group (P < 0.05); however, there was no difference between the dexamethasone and bosentan treatment groups at 3 and 24 hours after LPS administration. Bosentan treatment at doses of 50 and 100 mg/kg significantly decreased iNOS expression compared to LPS-injected animals (P < 0.001). The ET-1 expression was suppressed significantly by bosentan and dexamethasone at 3 and 24 hours after LPS administration (P < 0.001). The EDNRA expression in the bosentan treatment groups was statistically significantly lower than that in the LPS-induced uveitis control group at 3 and 24 hours after LPS administration (P < 0.05). Bosentan reduces intraocular inflammation and has similar effects as dexamethasone in a rat model of EIU. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells.

PubMed

Paik, Yong-Han; Schwabe, Robert F; Bataller, Ramón; Russo, Maria P; Jobin, Christian; Brenner, David A

2003-05-01

Bacterial lipopolysaccharide (LPS) stimulates Kupffer cells and participates in the pathogenesis of alcohol-induced liver injury. However, it is unknown whether LPS directly affects hepatic stellate cells (HSCs), the main fibrogenic cell type in the injured liver. This study characterizes LPS-induced signal transduction and proinflammatory gene expression in activated human HSCs. Culture-activated HSCs and HSCs isolated from patients with hepatitis C virus-induced cirrhosis express LPS-associated signaling molecules, including CD14, toll-like receptor (TLR) 4, and MD2. Stimulation of culture-activated HSCs with LPS results in a rapid and marked activation of NF-kappaB, as assessed by in vitro kinase assays for IkappaB kinase (IKK), IkappaBalpha steady-state levels, p65 nuclear translocation, NF-kappaB-dependent luciferase reporter gene assays, and electrophoretic mobility shift assays. Lipid A induces NF-kappaB activation in a similar manner. Both LPS- and lipid A-induced NF-kappaB activation is blocked by preincubation with either anti-TLR4 blocking antibody (HTA125) or Polymyxin B. Lipid A induces NF-kappaB activation in HSCs from TLR4-sufficient (C3H/OuJ) mice but not from TLR4-deficient (C3H/HeJ) mice. LPS also activates c-Jun N-terminal kinase (JNK), as assessed by in vitro kinase assays. LPS up-regulates IL-8 and MCP-1 gene expression and secretion. LPS-induced IL-8 secretion is completely inhibited by the IkappaB super repressor (Ad5IkappaB) and partially inhibited by a specific JNK inhibitor, SP600125. LPS also up-regulates cell surface expression of ICAM-1 and VCAM-1. In conclusion, human activated HSCs utilize components of TLR4 signal transduction cascade to stimulate NF-kappaB and JNK and up-regulate chemokines and adhesion molecules. Thus, HSCs are a potential mediator of LPS-induced liver injury.

Arctigenin ameliorates inflammation in vitro and in vivo by inhibiting the PI3K/AKT pathway and polarizing M1 macrophages to M2-like macrophages.

PubMed

Hyam, Supriya R; Lee, In-Ah; Gu, Wan; Kim, Kyung-Ah; Jeong, Jin-Ju; Jang, Se-Eun; Han, Myung Joo; Kim, Dong-Hyun

2013-05-15

Seeds of Arctium lappa, containing arctigenin and its glycoside arctiin as main constituents, have been used as a diuretic, anti-inflammatory and detoxifying agent in Chinese traditional medicine. In our preliminary study, arctigenin inhibited IKKβ and NF-κB activation in peptidoglycan (PGN)- or lipopolysaccharide (LPS)-induced peritoneal macrophages. To understand the anti-inflammatory effect of arctigenin, we investigated its anti-inflammatory effect in LPS-stimulated peritoneal macrophages and on LPS-induced systemic inflammation as well as 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. Arctigenin inhibited LPS-increased IL-1β, IL-6 and TNF-α expression in LPS-stimulated peritoneal macrophages, but increased LPS-reduced IL-10 and CD204 expression. Arctigenin inhibited LPS-induced PI3K, AKT and IKKβ phosphorylation, but did not suppress LPS-induced IRAK-1 phosphorylation. However, arctigenin did not inhibit NF-κB activation in LPS-stimulated PI3K siRNA-treated peritoneal macrophages. Arctigenin suppressed the binding of p-PI3K antibody and the nucleus translocation of NF-κB p65 in LPS-stimulated peritoneal macrophages. Arctigenin suppressed blood IL-1β and TNF-α level in mice systemically inflamed by intraperitoneal injection of LPS. Arctigenin also inhibited colon shortening, macroscopic scores and myeloperoxidase activity in TNBS-induced colitic mice. Arctigenin inhibited TNBS-induced IL-1β, TNF-α and IL-6 expression, as well as PI3K, AKT and IKKβ phosphorylation and NF-κB activation in mice, but increased IL-10 and CD204 expression. However, it did not affect IRAK-1 phosphorylation. Based on these findings, arctigenin may ameliorate inflammatory diseases, such as colitis, by inhibiting PI3K and polarizing M1 macrophages to M2-like macrophages. Copyright © 2013 Elsevier B.V. All rights reserved.

Lipopolysaccharide effects on the proliferation of NRK52E cells via alternations in gap-junction function.

PubMed

Hei, Ziqing; Zhang, Ailan; Wei, Jing; Gan, Xiaoliang; Wang, Yanling; Luo, Gangjian; Li, Xiaoyun

2012-07-01

Gap junctions regulate proper kidney function by facilitating intercellular communication, vascular conduction, and tubular purinergic signaling. However, no clear relationship has been described between gap-junction function and acute kidney injury induced by the endotoxin lipopolysaccharide (LPS). Normal rat kidney epithelial cells (NRK52E cells) were seeded at high and low densities to promote or impede gap-junction formation, respectively, and establish distinctive levels of intercellular communication in culture. Cells were then challenged with LPS at various concentrations (10-1,000 ng/mL). LPS-induced formation and function of gap junctions were assessed by measuring changes in cell proliferation and colony-forming rates, fluorescent dye transmission to adjacent cells, expression levels of connexin43, and repositioning of confluent cells in response to the gap junction inhibitor oleamide or agonist retinoic acid. The cell proliferation rate and colony-forming rate of high- and low-density NRK52E cells were decreased upon LPS challenge, in a dose-dependent manner. The colony-forming rate of confluent high-density cells was significantly lower than that of low-density cells. Oleamide treatment raised the LPS-induced colony-forming rate of high-density cells, whereas retinoic acid decreased the rate. Neither oleamide nor retinoic acid significantly affected the LPS-induced colony-forming rate of low-density cells. Fluorescence transmission of high-density cells was reduced by LPS challenge, in a dose-dependent manner, but inclusion of retinoic acid increased the LPS-induced transmission of fluorescence. LPS challenge of either high- or low-density NRK52E cells resulted in down-regulated connexin43 expression. Gap-junction function plays an important role in concentration-dependent cytotoxic effect of LPS on normal rat kidney cells in vitro.

Magnolol attenuates sepsis-induced gastrointestinal dysmotility in rats by modulating inflammatory mediators

PubMed Central

Yang, Tie-Cheng; Zhang, Shu-Wen; Sun, Li-Na; Wang, Hong; Ren, Ai-Min

2008-01-01

AIM: To investigate the protective effects of magnolol on sepsis-induced inflammation and intestinal dysmotility. METHODS: Sepsis was induced by a single intraperitoneal injection of lipopolysaccharide (LPS). Male Wistar rats were randomly assigned to one of three treatment groups: magnolol prior to LPS injection (LPS/Mag group); vehicle prior to LPS injection (LPS/Veh group); vehicle prior to injection of saline (Control/Veh). Intestinal transit and circular muscle mechanical activity were assessed 12 h after LPS injection. Tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), monocyte chemoattractant protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS) mRNA in rat ileum were studied by RT-PCR 2 h after LPS injection. Nuclear factor-κB (NF-κB) activity in the intestine was also investigated at this time using electrophoretic mobility shift assay. In addition, antioxidant activity was determined by measuring malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity in the intestine 2 h after LPS injection. RESULTS: Magnolol significantly increased intestinal transit and circular muscle mechanical activity in LPS-treated animals. TNF-α, MCP-1 and iNOS mRNA expression in the small intestine were significantly reduced after magnolol treatment in LPS-induced septic animals, compared with untreated septic animals. Additionally, magnolol significantly increased IL-10 mRNA expression in septic rat ileum. Magnolol also significantly suppressed NF-κB activity in septic rat intestine. In addition, magnolol significantly decreased MDA concentration and increased SOD activity in rat ileum. CONCLUSION: Magnolol prevents sepsis-induced suppression of intestinal motility in rats. The potential mechanism of this benefit of magnolol appears to be modulation of self-amplified inflammatory events and block of oxidative stress in the intestine. PMID:19109869

Hypoxia preconditioning increases survival and decreases expression of Toll-like receptor 4 in pulmonary artery endothelial cells exposed to lipopolysaccharide.

PubMed

Ali, Irshad; Nanchal, Rahul; Husnain, Fouad; Audi, Said; Konduri, G Ganesh; Densmore, John C; Medhora, Meetha; Jacobs, Elizabeth R

2013-09-01

Abstract Pulmonary or systemic infections and hypoxemic respiratory failure are among the leading causes of admission to intensive care units, and these conditions frequently exist in sequence or in tandem. Inflammatory responses to infections are reproduced by lipopolysaccharide (LPS) engaging Toll-like receptor 4 (TLR4). Apoptosis is a hallmark of lung injury in sepsis. This study was conducted to determine whether preexposure to LPS or hypoxia modulated the survival of pulmonary artery endothelial cells (PAECs). We also investigated the role TLR4 receptor expression plays in apoptosis due to these conditions. Bovine PAECs were cultured in hypoxic or normoxic environments and treated with LPS. TLR4 antagonist TAK-242 was used to probe the role played by TLR4 receptors in cell survival. Cell apoptosis and survival were measured by caspase 3 activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) incorporation. TLR4 expression and tumor necrosis factor α (TNF-α) production were also determined. LPS increased caspase 3 activity in a TAK-242-sensitive manner and decreased MTT incorporation. Apoptosis was decreased in PAECs preconditioned with hypoxia prior to LPS exposure. LPS increased TNF-α production, and hypoxic preconditioning blunted it. Hypoxic preconditioning reduced LPS-induced TLR4 messenger RNA and TLR4 protein. TAK-242 decreased to baseline the LPS-stimulated expression of TLR4 messenger RNA regardless of environmental conditions. In contrast, LPS followed by hypoxia substantially increased apoptosis and cell death. In conclusion, protection from LPS-stimulated PAEC apoptosis by hypoxic preconditioning is attributable in part to reduction in TLR4 expression. If these signaling pathways apply to septic patients, they may account for differing sensitivities of individuals to acute lung injury depending on oxygen tensions in PAECs in vivo.

TAK-242, a small-molecule inhibitor of Toll-like receptor 4 signalling, unveils similarities and differences in lipopolysaccharide- and lipidinduced inflammation and insulin resistance in muscle cells

PubMed Central

Hussey, Sophie E.; Liang, Hanyu; Costford, Sheila R.; Klip, Amira; DeFronzo, Ralph A.; Sanchez-Avila, Alicia; Ely, Brian; Musi, Nicolas

2012-01-01

Emerging evidence suggests that TLR (Toll-like receptor) 4 and downstream pathways [MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor κB)] play an important role in the pathogenesis of insulin resistance. LPS (lipopolysaccharide) and saturated NEFA (non-esterified fatty acids) activate TLR4, and plasma concentrations of these TLR4 ligands are elevated in obesity and Type 2 diabetes. Our goals were to define the role of TLR4 on the insulin resistance caused by LPS and saturated NEFA, and to dissect the independent contribution of LPS and NEFA to the activation of TLR4-driven pathways by employing TAK-242, a specific inhibitor of TLR4. LPS caused robust activation of the MAPK and NF-κB pathways in L6 myotubes, along with impaired insulin signalling and glucose transport. TAK-242 completely prevented the inflammatory response (MAPK and NF-κB activation) caused by LPS, and, in turn, improved LPS-induced insulin resistance. Similar to LPS, stearate strongly activated MAPKs, although stimulation of the NF-κB axis was modest. As seen with LPS, the inflammatory response caused by stearate was accompanied by impaired insulin action. TAK-242 also blunted stearate-induced inflammation; yet, the protective effect conferred by TAK-242 was partial and observed only on MAPKs. Consequently, the insulin resistance caused by stearate was only partially improved by TAK-242. In summary, TAK-242 provides complete and partial protection against LPS- and NEFA-induced inflammation and insulin resistance, respectively. Thus, LPS-induced insulin resistance depends entirely on TLR4, whereas NEFA works through TLR4-dependent and -independent mechanisms to impair insulin action. PMID:23050932

TAK-242, a small-molecule inhibitor of Toll-like receptor 4 signalling, unveils similarities and differences in lipopolysaccharide- and lipid-induced inflammation and insulin resistance in muscle cells.

PubMed

Hussey, Sophie E; Liang, Hanyu; Costford, Sheila R; Klip, Amira; DeFronzo, Ralph A; Sanchez-Avila, Alicia; Ely, Brian; Musi, Nicolas

2012-11-30

Emerging evidence suggests that TLR (Toll-like receptor) 4 and downstream pathways [MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor κB)] play an important role in the pathogenesis of insulin resistance. LPS (lipopolysaccharide) and saturated NEFA (non-esterified fatty acids) activate TLR4, and plasma concentrations of these TLR4 ligands are elevated in obesity and Type 2 diabetes. Our goals were to define the role of TLR4 on the insulin resistance caused by LPS and saturated NEFA, and to dissect the independent contribution of LPS and NEFA to the activation of TLR4-driven pathways by employing TAK-242, a specific inhibitor of TLR4. LPS caused robust activation of the MAPK and NF-κB pathways in L6 myotubes, along with impaired insulin signalling and glucose transport. TAK-242 completely prevented the inflammatory response (MAPK and NF-κB activation) caused by LPS, and, in turn, improved LPS-induced insulin resistance. Similar to LPS, stearate strongly activated MAPKs, although stimulation of the NF-κB axis was modest. As seen with LPS, the inflammatory response caused by stearate was accompanied by impaired insulin action. TAK-242 also blunted stearate-induced inflammation; yet, the protective effect conferred by TAK-242 was partial and observed only on MAPKs. Consequently, the insulin resistance caused by stearate was only partially improved by TAK-242. In summary, TAK-242 provides complete and partial protection against LPS- and NEFA-induced inflammation and insulin resistance, respectively. Thus, LPS-induced insulin resistance depends entirely on TLR4, whereas NEFA works through TLR4-dependent and -independent mechanisms to impair insulin action.

Neutrophil-cytokine interactions in a rat model of sulindac-induced idiosyncratic liver injury.

PubMed

Zou, Wei; Roth, Robert A; Younis, Husam S; Malle, Ernst; Ganey, Patricia E

2011-12-18

Previous studies indicated that lipopolysaccharide (LPS) interacts with the nonsteroidal anti-inflammatory drug sulindac (SLD) to produce liver injury in rats. In the present study, the mechanism of SLD/LPS-induced liver injury was further investigated. Accumulation of polymorphonuclear neutrophils (PMNs) in the liver was greater in SLD/LPS-cotreated rats compared to those treated with SLD or LPS alone. In addition, PMN activation occurred specifically in livers of rats cotreated with SLD/LPS. The hypothesis that PMNs and proteases released from them play critical roles in the hepatotoxicity was tested. SLD/LPS-induced liver injury was attenuated by prior depletion of PMNs or by treatment with the PMN protease inhibitor, eglin C. Previous studies suggested that tumor necrosis factor-α (TNF) and the hemostatic system play critical roles in the pathogenesis of liver injury induced by SLD/LPS. TNF and plasminogen activator inhibitor-1 (PAI-1) can contribute to hepatotoxicity by affecting PMN activation and fibrin deposition. Therefore, the role of TNF and PAI-1 in PMN activation and fibrin deposition in the SLD/LPS-induced liver injury model was tested. Neutralization of TNF or inhibition of PAI-1 attenuated PMN activation. TNF had no effect on PAI-1 production or fibrin deposition. In contrast, PAI-1 contributed to fibrin deposition in livers of rats treated with SLD/LPS. In summary, PMNs, TNF and PAI-1 contribute to the liver injury induced by SLD/LPS cotreatment. TNF and PAI-1 independently contributed to PMN activation, which is critical to the pathogenesis of liver injury. Moreover, PAI-1 contributed to liver injury by promoting fibrin deposition. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhongshan; College of Life Sciences, Sichuan University, Chengdu 610065; Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST

2014-09-26

Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane bymore » seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.« less

Lipopolysaccharide (LPS) stimulation of fungal secondary metabolism

PubMed Central

Khalil, Zeinab G.; Kalansuriya, Pabasara; Capon, Robert J.

2014-01-01

We report on a preliminary investigation of the use the Gram-negative bacterial cell wall constituent lipopolysaccharide (LPS) as a natural chemical cue to stimulate and alter the expression of fungal secondary metabolism. Integrated high-throughput micro-cultivation and micro-analysis methods determined that 6 of 40 (15%) of fungi tested responded to an optimal exposure to LPS (0.6 ng/mL) by activating, enhancing or accelerating secondary metabolite production. To explore the possible mechanisms behind this effect, we employed light and fluorescent microscopy in conjunction with a nitric oxide (NO)-sensitive fluorescent dye and an NO scavenger to provide evidence that LPS stimulation of fungal secondary metabolism coincided with LPS activation of NO. Several case studies demonstrated that LPS stimulation can be scaled from single microplate well (1.5 mL) to preparative (>400 mL) scale cultures. For example, LPS treatment of Penicillium sp. (ACM-4616) enhanced pseurotin A and activated pseurotin A1 and pseurotin A2 biosynthesis, whereas LPS treatment of Aspergillus sp. (CMB-M81F) substantially accelerated and enhanced the biosynthesis of shornephine A and a series of biosynthetically related ardeemins and activated production of neoasterriquinone. As an indication of broader potential, we provide evidence that cultures of Penicillium sp. (CMB-TF0411), Aspergillus niger (ACM-4993F), Rhizopus oryzae (ACM-165F) and Thanatephorus cucumeris (ACM-194F) were responsive to LPS stimulation, the latter two examples being particular noteworthy as neither are known to produce secondary metabolites. Our results encourage the view that LPS stimulation can be used as a valuable tool to expand the molecular discovery potential of fungal strains that either have been exhaustively studied by or are unresponsive to traditional culture methodology. PMID:25379339

Protective effects of hydrogen-rich medium on lipopolysaccharide-induced monocytic adhesion and vascular endothelial permeability through regulation of vascular endothelial cadherin.

PubMed

Yu, Y; Wang, W N; Han, H Z; Xie, K L; Wang, G L; Yu, Y H

2015-06-11

We observed the effect of hydrogen-rich medium on lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs), hyaline leukocyte conglutination, and permeability of the endothelium. Endotheliocytes were inoculated on 6-well plates and randomly divided into 4 groups: control, H2, LPS, LPS+H2, H2, and LPS+H2 in saturated hydrogen-rich medium. We applied Wright's stain-ing to observe conglutination of hyaline leukocytes and HUVECs, flow cytometry to determine the content of vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), enzyme-linked immunosorbent assay to measure the E-selectin concentration in the cell liquor, the transendothelial electrical resistance (TEER) to test the permeability of endothelial cells, and Western blot and immunofluorescence to test the expression and distribution of vascular endothelial (VE)-cadherin. Compared with control cells, there was an increase in endothelium-hyaline leukocyte conglutination, a reduction in VCAM-1, ICAM-1, and E-selectin, and the TEER value increased obviously. Compared with LPS, there was an obvious reduction in the conglutination of LPS+H2 cells, a reduction in VCAM-1, ICAM-1, and E-selectin levels, and a reduction in the TEER-resistance value, while the expression of VE-cadherin increased. Fluorescence results showed that, compared with control cells, the VE-cadherin in LPS cells was in-complete at the cell joints. Compared with LPS cells, the VE-cadherin in LPS+H2 cells was even and complete at the cell joints. Liquid rich in hydrogen could reduce LPS-induced production of adhesion molecules and endothelium-hyaline leukocyte conglutination, and influence the expression and distribution of VE-cadherin to regulate the permeability of the endothelium.

Bordetella Pertussis Toxin does not induce the release of pro-inflammatory cytokines in human whole blood.

PubMed

Bache, Christina; Spreitzer, Ingo; Becker, Bjoern; Loeschner, Bettina; Rosskopf, Ute; Hanschmann, Kay-Martin; Schwanig, Michael; Schneider, Christian K; Lieb, Bernhard; Montag, Thomas

2012-08-01

Pertussis Toxin (PTx) is one of the most important virulence factors of Bordetella pertussis, the cause of whooping cough. Therefore, the inactivated toxin is an obligatory constituent of acellular pertussis vaccines. It is described in the literature that both native PTx and recombinant Pertussis Toxin (PTg) activate human monocytes whereas others report an inhibition of mammalian monocytes during pertussis infection. B. pertussis, as a Gram-negative bacterium, harbours naturally lipopolysaccharide (LPS, also known as endotoxin), one of the strongest stimulators of monocytes. The latter is triggered via the interaction of endotoxin with inter alia the surface receptor CD14. Consequently, it is necessary to consider a potential contamination of Pertussis Toxin preparations with LPS. First, we determined the LPS content in different preparations of PTx and PTg. All preparations examined were contaminated with LPS; therefore, possible PTx- and PTg-driven monocyte activation independently of LPS was investigated. To meet these aims, we examined monocyte response to PTx and PTg while blocking the LPS receptor CD14 with a specific monoclonal antibody (anti-CD14 mAb). In addition, all toxin preparations examined underwent an LPS depletion. Our results show that it is contaminating LPS, not Pertussis Toxin, which activates human monocytes. Blocking the CD14 receptor prevents Pertussis Toxin-mediated induction of pro-inflammatory cytokines in human monocytes. The depletion of LPS from Pertussis Toxin leads to the same effect. Additionally, the PTx toxicity after LPS depletion procedure was confirmed by animal tests. In contrast, the original Pertussis Toxin preparations not treated as mentioned above generate strong monocyte activation. The results in this publication allow the conclusion that purified Pertussis Toxin preparations do not induce the release of pro-inflammatory cytokines in human whole blood.

Non-receptor tyrosine kinases ITK and BTK negatively regulate mast cell pro-inflammatory responses to lipopolysaccharide

PubMed Central

Huang, Weishan; Morales, J. Luis; Gozivoda, Victor P.; August, Avery

2015-01-01

Background Mast cells are indispensible for LPS-induced septic hypothermia, in which TNF-α plays an essential role to initiate septic responses. ITK and BTK regulate mast cell responses to allergen, but their roles in mast cell responses in LPS-induced sepsis are unclear. Objectives We sought to investigate the roles of ITK and BTK in mast cell responses during LPS-induced septic inflammation. Methods Mice (genetically modified or BMMC-reconstituted Sash) were given LPS to induce septic hypothermia, in the presence or absence of indicated inhibitors. Flow cytometry was used to determine LPS-induced cell influx and TNF-α production in peritoneal cells. Microarray was used for genome-wide gene expression analysis on BMMCs. Quantitative PCR and multiplex were used to determine transcribed and secreted pro-inflammatory cytokines. Microscopy and western blotting were used to determine activation of signal transduction pathways. Results The absence of ITK and BTK leads to exacerbation of LPS-induced septic hypothermia and neutrophil influx. Itk−/−Btk−/− mast cells exhibit hyperactive preformed and LPS-induced TNF-α production, and lead to more severe LPS-induced septic hypothermia when reconstituted into mast cell deficient Sash mice. LPS-induced NF-κB, Akt and p38 activation is enhanced in Itk−/−Btk−/− mast cells, and blockage of PI3K, Akt or p38 downstream MNK1 activation significantly suppresses TNF-α hyper-production and attenuates septic hypothermia. Conclusions ITK and BTK regulate thermal homeostasis during septic response through mast cell function in mice. They share regulatory function downstream of TLR4/LPS in mast cells, through regulating the activation of canonical NF-κB, PI3K/Akt and p38 signaling pathways. PMID:26581914

Tanshinone IIA Sodium Sulfonate Attenuates LPS-Induced Intestinal Injury in Mice

PubMed Central

Yang, Xin-Jing; Qian, Jin-Xian; Wei, Yao; Guo, Qiang; Jin, Jun; Sun, Xue; Liu, Sheng-Lan

2018-01-01

Background Tanshinone IIA sodium sulfonate (TSS) is known to possess anti-inflammatory effects and has exhibited protective effects in various inflammatory conditions; however, its role in lipopolysaccharide- (LPS-) induced intestinal injury is still unknown. Objective The present study is designed to explore the role and possible mechanism of TSS in LPS-induced intestinal injury. Methods Male C57BL/6J mice, challenged with intraperitoneal LPS injection, were treated with or without TSS 0.5 h prior to LPS exposure. At 1, 6, and 12 h after LPS injection, mice were sacrificed, and the small intestine was excised. The intestinal tissue injury was analyzed by HE staining. Inflammatory factors (TNF-α, IL-1β, and IL-6) in the intestinal tissue were examined by ELISA and RT-PCR. In addition, expressions of autophagy markers (microtubule-associated light chain 3 (LC3) and Beclin-1) were detected by western blot and RT-PCR. A number of autophagosomes were also observed under electron microscopy. Results TSS treatment significantly attenuated small intestinal epithelium injury induced by LPS. LPS-induced release of inflammatory mediators, including TNF-α, IL-1β, and IL-6, were markedly inhibited by TSS. Furthermore, TSS treatment could effectively upregulate LPS-induced decrease of autophagy levels, as evidenced by the increased expression of LC3 and Beclin-1, and more autophagosomes. Conclusion The protective effect of TSS on LPS-induced small intestinal injury may be attributed to the inhibition of inflammatory factors and promotion of autophagy levels. The present study may provide novel insight into the molecular mechanisms of TSS on the treatment of intestinal injury. PMID:29706995

Ketamine potentiates oxidative stress and influences behavior and inflammation in response to lipolysaccharide (LPS) exposure in early life.

PubMed

Réus, Gislaine Z; Simões, Lutiana R; Colpo, Gabriela D; Scaini, Giselli; Oses, Jean P; Generoso, Jaqueline S; Prossin, Alan R; Kaddurah-Daouk, Rima; Quevedo, João; Barichello, Tatiana

2017-06-14

Immune activation (IA) during the early neonatal period is a risk factor for the development of schizophrenia. Lipopolysaccharide (LPS) injected in neonates lead to behavioral and brain changes that persist to adult life. We investigated oxidative stress, levels of cytokines, and the locomotor activity of IA in a schizophrenia animal model in which neonatal male Wistar rats were administered with an injection of LPS (50μg/kg) on postnatal day 3 and different doses of ketamine (5, 15 and 25mg/kg) for 7days during adulthood. Rats LPS-induced did not have locomotor activity alterations. Locomotor activity was elevated in neonatally saline-injected in the higher dose ketamine-treated animals. Carbonyl protein in the prefrontal cortex (PFC), hippocampus and striatum were increased in the LPS- and saline-induced in the ketamine (25mg/kg)-treated animals. Lipid damage occurred in the PFC, striatum and hippocampus in the LPS- and saline-induced in the ketamine (15 and 25mg/kg) -treated animals. In the hippocampus the superoxide dismutase (SOD) was decreased in the LPS- and saline-induced in the ketamine-treated with the dose of 25mg/kg. In the PFC SOD was reduced in the LPS-induced in the ketamine (25mg/kg)-treated animals. Catalase in the PFC and hippocampus was reduced in the LPS- and saline-induced in the ketamine (25mg/kg)-treated animals. Pro- and anti-inflammatory cytokines were lower in the brains of LPS-induced in the higher dose ketamine-treated rats. IA influences the locomotor activity and cytokine levels induced by ketamine, and it has a negative effect in potentiating the oxidative stress by higher doses of ketamine in the brain. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Chronic fetal exposure to Ureaplasma parvum suppresses innate immune responses in sheep

PubMed Central

Kallapur, Suhas G.; Kramer, Boris W.; Knox, Christine L.; Berry, Clare A.; Collins, Jennifer J.P; Kemp, Matthew W.; Nitsos, Ilias; Polglase, Graeme R.; Robinson, James; Hillman, Noah H.; Newnham, John P.; Chougnet, Claire; Jobe, Alan H.

2011-01-01

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different pro-inflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic injections of media (control) or Ureaplasma parvum serovar 3 either 7d or 70d before preterm delivery. Another group received an intraamniotic injection of E.coli lipo-polysaccharide (LPS) 2d prior to delivery. To test for interactions, intraamniotic U. parvum exposed animals were challenged with intraamniotic LPS and delivered 2d later. All animals were delivered at 124±1d gestation (Term=150d). Compared to the 2d LPS exposure group, the U. parvum 70d+LPS group had: 1) decreased lung pro and anti-inflammatory cytokine expression 2) fewer CD3+ T-lymphocytes, CCL2+, myeloperoxidase+, and PU.1+ cells in the lung. Interestingly, exposure to U. parvum for 7d did not change responses to a subsequent intraamniotic LPS challenge, and exposure to intraamniotic U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGFβ1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70d+LPS but not U. parvum 7d+LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of down-regulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis exposed preterm infants may respond to lung injury and postnatal nosocomial infections. PMID:21784974

Age- and gender-dependent impairments of neurobehaviors in mice whose mothers were exposed to lipopolysaccharide during pregnancy.

PubMed

Wang, Hua; Meng, Xiu-Hong; Ning, Huan; Zhao, Xian-Feng; Wang, Qun; Liu, Ping; Zhang, Heng; Zhang, Cheng; Chen, Gui-Hai; Xu, De-Xiang

2010-02-01

Lipopolysaccharide (LPS)-induced intrauterine infection has been associated with neurodevelopmental injury in rodents. The purpose of the present study was to analyze the dynamic changes of neurobehaviors in mice whose mothers were exposed to LPS during pregnancy. The pregnant mice were intraperitoneally (i.p.) injected with LPS (8 microg/kg) daily from gestational day (gd) 8 to gd 15. A battery of neurobehavioral tasks was performed in mice at postnatal day (PND) 70, 200, 400 and 600. Results showed that the spatial learning and memory ability, determined by radial six-arm water maze (RAWM), were obviously impaired in two hundred-day-old female mice and four hundred-day-old male mice whose mothers were exposed to LPS during pregnancy. Open field test showed that the number of squares crossed and peripheral time, a marker of anxiety and exploration activity, were markedly increased in two hundred-day-old female mice following prenatal LPS exposure. In addition, prenatal LPS exposure significantly shortened the latency to the first grid crossing in six hundred-day-old female offspring. Moreover, sensorimotor impairment in the beam walking was observed in two hundred-day-old female mice whose mothers were exposed to LPS during pregnancy. Species-typical behavior examination showed that prenatal LPS exposure markedly increased weight burrowed in seventy-day-old male offspring and six hundred-day-old female offspring. Correspondingly, prenatal LPS exposure significantly reduced weight hoarded in two hundred-day-old female offspring. Taken together, these results suggest that prenatal LPS exposure induces neurobehavioral impairments at adulthood in an age- and gender-dependent manner. 2009 Elsevier Ireland Ltd. All rights reserved.

CELECOXIB ATTENUATES SYSTEMIC LIPOPOLYSACCHARIDE-INDUCED BRAIN INFLAMMATION AND WHITE MATTER INJURY IN THE NEONATAL RATS

PubMed Central

FAN, L.-W.; KAIZAKI, A.; TIEN, L.-T.; PANG, Y.; TANAKA, S.; NUMAZAWA, S.; BHATT, A. J.; CAI, Z.

2013-01-01

Lipopolysaccharide (LPS)-induced white matter injury in the neonatal rat brain is associated with inflammatory processes. Cyclooxygenase-2 (COX-2) can be induced by inflammatory stimuli, such as cytokines and pro-inflammatory molecules, suggesting that COX-2 may be considered as the target for anti-inflammation. The objective of the present study was to examine whether celecoxib, a selective COX-2 inhibitor, can reduce systemic LPS-induced brain inflammation and brain damage. Intraperitoneal (i.p.) injection of LPS (2 mg/kg) was performed in postnatal day 5 (P5) of Sprague-Dawley rat pups and celecoxib (20 mg/kg) or vehicle was administered i.p. 5 min after LPS injection. The body weight and wire hanging maneuver test were performed 24 hr after the LPS exposure, and brain injury was examined after these tests. Systemic LPS exposure resulted in an impairment of behavioral performance and acute brain injury, as indicated by apoptotic death of oligodendrocytes (OLs) and loss of OL immunoreactivity in the neonatal rat brain. Treatments with celecoxib significantly reduced systemic LPS-induced neurobehavioral disturbance and brain damage. Celecoxib administration significantly attenuated systemic LPS-induced increments in the number of activated microglia and astrocytes, concentrations of IL-1β and TNFα, and protein levels of phosphorylated-p38 MAPK in the neonatal rat brain. The protection of celecoxib was also associated with a reduction of systemic LPS-induced COX-2+ cells which were double labeled with GFAP+ (astrocyte) cells. The overall results suggest that celecoxib was capable of attenuating the brain injury and neurobehavioral disturbance induced by systemic LPS exposure, and the protective effects are associated with its anti-inflammatory properties. PMID:23485816

XOR inhibition with febuxostat accelerates pulmonary endothelial barrier recovery and improves survival in lipopolysaccharide-induced murine sepsis.

PubMed

Damarla, Mahendra; Johnston, Laura F; Liu, Gigi; Gao, Li; Wang, Lan; Varela, Lidenys; Kolb, Todd M; Kim, Bo S; Damico, Rachel L; Hassoun, Paul M

2017-08-01

Sepsis is a leading cause of death among patients in the intensive care unit, resulting from multi-organ failure. Activity of xanthine oxidoreductase (XOR), a reactive oxygen species (ROS) producing enzyme, is known to be elevated in nonsurvivors of sepsis compared to survivors. We have previously demonstrated that XOR is critical for ventilator-induced lung injury. Using febuxostat, a novel nonpurine inhibitor of XOR, we sought to determine the role of XOR inhibition in a murine model of sepsis-induced lung injury and mortality. C57BL/6J mice were subjected to intravenous (IV) lipopolysaccharide (LPS) for various time points, and lungs were harvested for analyses. Subsets of mice were treated with febuxostat, pre or post LPS exposure, or vehicle. Separate groups of mice were followed up for mortality after LPS exposure. After 24 hr of IV LPS , mice exhibited an increase in XOR activity in lung tissue and a significant increase in pulmonary endothelial barrier disruption. Pretreatment of animals with febuxostat before exposure to LPS, or treatment 4 h after LPS, resulted in complete abrogation of XOR activity. Inhibition of XOR with febuxostat did not prevent LPS-induced pulmonary vascular permeability at 24 h, however, it accelerated recovery of the pulmonary endothelial barrier integrity in response to LPS exposure. Furthermore, treatment with febuxostat resulted in significant reduction in mortality. Inhibition of XOR with febuxostat accelerates recovery of the pulmonary endothelial barrier and prevents LPS-induced mortality, whether given before or after exposure to LPS. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Intratracheal administration of bacterial lipopolysaccharide elicits pulmonary hypertension in broilers with primed airways.

PubMed

Lorenzoni, A G; Wideman, R F

2008-04-01

Broilers reared under commercial conditions inhale irritant gases and aerosolized particulates contaminated with gram-negative bacteria and bacterial lipopolysaccharide (LPS). Previous studies demonstrated that i.v. injections of LPS can trigger an increase in the pulmonary arterial pressure (PAP); however, the pulmonary hemodynamic response to aerosolized LPS entering via the most common route, the respiratory tract, had not been evaluated in broilers. In experiment 1, broilers reared on new wood shavings litter in clean environmental chambers either were not pretreated (control group) or were pretreated via aerosol inhalation of substances (food color dyes and propylene glycol) known to sensitize the airways. One day later, the broilers were anesthetized, catheterized to record the PAP, and an intratracheal aerosol spray of LPS (1 mL of 2 mg/mL of LPS) was administered. Broilers in the control group as well as broilers pretreated with aerosolized distilled water or yellow and blue food color dyes did not develop pulmonary hypertension (PH; an increase in PAP) after the intratracheal spray of LPS, whereas broilers that had been pretreated with red food color did develop PH in response to intratracheal LPS. In experiment 2, birds raised under commercial conditions on used wood shavings litter developed PH in response to intratracheal LPS regardless of whether they had been pretreated with aerosolized red food color dye. In experiment 3, broilers reared in clean environmental chambers on new wood shavings litter were used to demonstrate that Red Dye #3 and propylene glycol are capable of priming the responsiveness of the airways to a subsequent intratracheal LPS challenge. Common air contaminants such as LPS can result in PH leading to pulmonary hypertension syndrome (ascites) in broilers with appropriately primed airways.

Presumable role of outer membrane proteins of Salmonella containing sialylated lipopolysaccharides serovar Ngozi, sv. Isaszeg and subspecies arizonae in determining susceptibility to human serum.

PubMed

Futoma-Kołoch, Bożena; Godlewska, Urszula; Guz-Regner, Katarzyna; Dorotkiewicz-Jach, Agata; Klausa, Elżbieta; Rybka, Jacek; Bugla-Płoskońska, Gabriela

2015-01-01

The O48 group comprises Salmonella bacteria containing sialic acid in the lipopolysaccharide (LPS). Bacteria with sialylated surface structures are described as pathogens that avoid immunological response of the host by making similar their surface antigens to the host's tissues (molecular mimicry). It is known that the smooth-type LPS of Salmonella enterica and outer membrane proteins (OMP) PgtE, PagC and Rck mediate serum resistant phenotype by affecting complement system (C). The aim of this study was to investigate C3 component activation by Salmonella O48 LPS and OMP. In the present study, we examined C3 component deposition on the three Salmonella O48 strains: S. enterica subspecies enterica serovar Ngozi, S. enterica subsp. enterica sv. Isaszeg, and S. enterica subsp. arizonae containing sialic acid in the O-specific part of LPS. The greatest C3 deposition occurred on Salmonella sv. Isaszeg cells (p < 0.005) as well as on their LPS (low content of sialic acid in LPS) (p < 0.05) after 45 min of incubation in 50% human serum. Weaker C3 deposition ratio on the Salmonella sv. Ngozi (high content of sialic acid in LPS) and Salmonella subsp. arizonae (high content of sialic acid in LPS) cells correlated with the lower C3 activation on their LPS. Immunoblotting revealed that OMP isolated from the tested strains also bound C3 protein fragments. We suggest that activation of C3 serum protein is dependent on the sialic acid contents in the LPS as well as on the presence of OMP in the range of molecular masses of 35-48 kDa.

Dopaminergic neuronal injury in the adult rat brain following neonatal exposure to lipopolysaccharide and the silent neurotoxicity

PubMed Central

Fan, Lir-Wan; Tien, Lu-Tai; Zheng, Baoying; Pang, Yi; Lin, Rick C. S.; Simpson, Kimberly L.; Ma, Tangeng; Rhodes, Philip G.; Cai, Zhengwei

2010-01-01

Our previous studies have shown that neonatal exposure to lipopolysaccharide (LPS) resulted in motor dysfunction and dopaminergic neuronal injury in the juvenile rat brain. To further examine whether neonatal LPS exposure has persisting effects in adult rats, motor behaviors were examined from postnatal day 7 (P7) to P70 and brain injury was determined in P70 rats following an intracerebral injection of LPS (1 mg/kg) in P5 Sprague-Dawley male rats. Although neonatal LPS exposure resulted in hyperactivity in locomotion and stereotyped tasks, and other disturbances of motor behaviors, the impaired motor functions were spontaneously recovered by P70. On the other hand, neonatal LPS-induced injury to the dopaminergic system such as the loss of dendrites and reduced tyrosine hydroxylase immunoreactivity in the substantia nigra persisted in P70 rats. Neonatal LPS exposure also resulted in sustained inflammatory responses in the P70 rat brain, as indicated by an increased number of activated microglia and elevation of interleukin-1β and interleukin-6 content in the rat brain. In addition, when challenged with methamphetamine (METH, 0.5 mg/kg) subcutaneously, rats with neonatal LPS exposure had significantly increased responses in METH-induced locomotion and stereotypy behaviors as compared to those without LPS exposure. These results indicate that although neonatal LPS-induced neurobehavioral impairment is spontaneously recoverable, the LPS exposure-induced persistent injury to the dopaminergic system and the chronic inflammation may represent the existence of silent neurotoxicity. Our data further suggest that the compromised dendritic mitochondrial function might contribute, at least partially, to the silent neurotoxicity. PMID:20875849

[Inhibitory effects of Porphyromonas endodontalis lipopolysaccharides on proliferation and differentiation of mouse osteoblast].

PubMed

Li, Ren; Qiu, Li-hong; Yang, Di; Xue, Ming; Zhong, Ming

2011-03-01

To observe the effect of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on osteoblast cell proliferation and the activity of alkaline phosphatase (ALP) and interleukin (IL)-6 secretion and to investigate the role of Pe-LPS in osteoblast proliferation and differentiation. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (10, 25, 50 mg/L) respectively. The relative growth rate (RGR) was detected by methyl thiazolyl tetrazolium (MTT) at different time point (12, 24, 48, 72 h). MC3T3-E1 cells were also stimulated with 10, 25 or 50 mg/L Pe-LPS for 6, 12, 24 and 48 h. The activity of ALP was detected by enzyme kinetics assay and the secretion of IL-6 was detected by enzyme-linked immunosorbent assay (ELISA). After the stimulation with 25 or 50 mg/L Pe-LPS for 72 h, the RGR of MC3T3-E1 cells descend to 87.46% and 71.12%. The ALP activities of MC3T3-E1 cells were inhibited obviously (P < 0.05) after the stimulation of different concentrations (10, 25, 50 mg/L) Pe-LPS for more than 24 hours. ELISA result showed that IL-6 increased to 32.21 ng/L treated with the 25 mg/L Pe-LPS for 6 h, 25 mg/L Pe-LPS gradually increased the expression of IL-6 from the ELISA results. Pe-LPS can induce the secretion of IL-6 in MC3T3-E1 and decrease the ALP activities of MC3T3-E1, the differentiation of osteoblasts was inhibited. with the long-time toxicity action of Pe-LPS, the proliferation rate of MC3T3-E1 also markedly decreased.

[Effects of lipopolysaccharides from various Porphyromonas on the expression of CD14 and TLRs in mouse osteoblast].

PubMed

Jia, Ge; Xue, Ming; Li, Ren; Lv, You; Qiu, Li-hong

2011-12-01

To observe the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) and Porphyromonas gingivals(P.g) on the expression of CD14 and TLRs in osteoblast. MC3T3-E1 cells were stimulated with 10μg/mL P.e-LPS and P.g-LPS. The change of CD14,TLR2 and TLR4 mRNA was observed at different time point (0,1,3,6,12,24h) using RT-PCR,and the expression of CD14,TLR2 and TLR4 protein was measured by flow cytometry at 24-hour. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS11.0 software package. MC3T3-E1 cells were stimulated with 10μg/mL P.e-LPS for 1h,the expression of CD14 and TLR4 mRNA increased significantly. There was no increase of TLR2 mRNA with stimulation of P.e-LPS. The CD14,TLR2 and TLR4 mRNA expression increased significantly after stimulation with 10μg/mL P.g-LPS. Flow cytometry showed that CD14 and TLR4 protein increased significantly after stimulation with 10μg/mL P.e-LPS. CD14,TLR2 and TLR4 protein increased significantly after treatment with 10μg/mL P.g-LPS. CD14,TLR4 receptors are involved in P.e-LPS effect and CD14,TLR2 and TLR4 receptors are involved in P.g-LPS effect in mouse osteoblast.

HSP-70 mitigates LPS/SKI-induced cell damage by increasing sphingosine kinase 1 (SK1).

PubMed

Ding, Xuan Z; Feng, Xiao R; Borschel, Richard H; Nikolich, Mikeljon P; Feng, Jie; Li, Yan S; Hoover, David L

2010-06-01

Heat shock proteins (HSPs) are potent protectors of cellular integrity against environmental stresses, including toxic microbial products. To investigate the mechanism of HSP-70 cell protection against bacterial lipopolysaccharide (LPS), we established a stable HSP-70 gene-transfected RAW 264.7 murine macrophage model of LPS-induced cell death. Bacterial LPS increases the activity of sphingosine kinase 1 (SK1), which catalyzes formation of sphingosine-1-phosphate (S1P). S1P functions as a critical signal for initiation and maintenance of diverse aspects of immune cell activation and function. When mouse macrophages were incubated with Escherichia coli LPS (1 microg/ml) and sphingosine kinase inhibitor (SKI, 5 microM), 90% of cells died. Neither LPS nor SKI alone at these doses damaged the cells. The LPS/SKI-induced cell death was partially reversed by overexpression of HSP-70 in gene-transfected macrophages. The specificity of HSP-70 in this reversal was demonstrated by transfection of HSP-70-specific siRNA. Down-regulation of HSP-70 expression after transfection of siRNA specific for HSP-70 was associated with increased LPS/SKI-induced cell damage. Overexpression of human or murine HSP-70 (HSPA1A and Hspa1a, respectively) increased both cellular SK1 mRNA and protein levels. Cellular heat shock also increased SK1 protein. These studies confirm the importance of SK1 as a protective moiety in LPS-induced cell injury and demonstrate that HSP-70-mediated protection from cells treated with LPS/SKI is accompanied by upregulating expression of SK1. HSP-70-mediated increases in SK1 and consequent increased levels of S1P may also play a role in protection of cells from other processes that lead to programmed cell death. Published by Elsevier Inc.

Role of poly-(ADP-ribose) synthetase in lipopolysaccharide-induced vascular failure and acute lung injury in pigs.

PubMed

Albertini, M; Clement, M G; Lafortuna, C L; Caniatti, M; Magder, S; Abdulmalek, K; Hussain, S N

2000-06-01

To assess the contribution of poly (adenosine 5'-diphosphate ribose) synthetase (PARS) to the development of bacterial lipopolysaccharide (LPS)-induced acute lung injury and vascular failure in pigs. Four groups of anesthetized, paralyzed, and mechanically ventilated domestic white pigs. Group 1 served as control, whereas Escherichia coli LPS (20 microg/kg/h) was continuously infused in group 2. Group 3 received 20 mg/kg injection of 3-aminobenzamide (a selective inhibitor of PARS activity) 15 minutes before LPS infusion. Only 3-aminobenzamide and not LPS was injected in group 4. All animals were examined for 180 minutes. Systemic and pulmonary hemodynamics and lung mechanics were measured during the experimental period. Lung wet/dry ratio, bronchoalveolar lavage (BAL) protein levels and cell counts and lung nitrotyrosine (footprint of peroxynitrite) immunostaining were also measured in a few animals. LPS infusion evoked a progressive decline in systemic arterial pressure, a small increase in cardiac output, and biphasic elevation of pulmonary arterial pressure. Lung compliance declined progressively, whereas lung and total respiratory resistance rose significantly after LPS infusion. Prominent nitrotyrosine immunostaining was detected around small airways and pulmonary endothelium of LPS-infused animals. No significant changes in lung wet/dry ratio and BAL protein levels and cell counts were produced by LPS infusion. Pretreatment with 3-aminobenzamide did not alter the systemic and pulmonary hemodynamic responses to LPS infusion but eliminated the rise in pulmonary and total respiratory resistance. We concluded that PARS activation plays an important role in the changes of lung mechanics associated with LPS-induced acute lung injury but had no role in vascular failure.

Sodium Tanshinone IIA Sulfonate Improves Hemodynamic Parameters, Cytokine Release, and Multi-Organ Damage in Endotoxemia Rabbits.

PubMed

Ma, Shaolei; Wang, Xian; Wang, Yujie; Zuo, Xiangrong

2018-05-08

BACKGROUND The aim of this study was to evaluate the protective effects of sodium tanshinone IIA sulfonate (STS) on hemodynamic parameters, cytokine release, and multiple organ damage in an animal model of lipopolysaccharide (LPS)-induced endotoxemia. MATERIAL AND METHODS Twenty-four rabbits were randomly divided into 3 groups: control (n=8), LPS (n=8), and STS pretreatment + LPS (n=8) groups. With arterial invasive monitoring, hemodynamic variables were observed at 30 min before and at 0, 10, 20, 30, 60, 120, 180, 240, and 300 min after LPS injection. Circulatory inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10), and relevant biochemical markers, including arterial partial pressure of oxygen (PaO2), plasma cardiac troponin I (cTnI), alanine aminotransferase (ALT), and creatinine (Cr), were measured at each time point. At the end of the experiment, all rabbits were sacrificed; histopathological examination of the heart, lung, liver, and kidney tissue was performed and organ injury was semi-quantitatively scored for each organ. RESULTS Mean arterial pressure (MAP) and heart rate (HR) significantly decreased within 30 min and again after 120 min following LPS injection. However, STS pretreatment gradually normalized MAP and HR after 120 min following LPS injection. In addition, STS ameliorated LPS-induced decrease of PaO2, LPS-induced increase of TNF-α, cTnI, and ALT, and enhanced LPS-induced increase of IL-10. Moreover, STS reduced heart, lung, and liver histopathologic injury. CONCLUSIONS STS can significantly stabilize LPS-induced hemodynamic deterioration, regulate inflammatory cytokine secretion, and protect heart, lung, and liver in rabbits.

Intermittently-induced endotoxaemia has no effect on post-challenge plasma metabolites, but increases body temperature and cortisol concentrations in periparturient dairy cows.

PubMed

Zebeli, Q; Sivaraman, S; Dunn, S M; Ametaj, B N

2013-12-01

This study evaluated the responses of plasma cortisol, metabolites and body temperature to intermittently-induced endotoxaemia in periparturient cows. Sixteen Holstein cows were randomly allocated to one of the two treatment groups. Cows were infused intravenously either with saline solution (control) or with the same solution containing 3 increasing doses of lipopolysaccharide (LPS) for 3 consecutive weeks around parturition as follows: 0.01 μg LPS/kg body weight (BW) on d -14 and -10 prepartum, 0.05 μg LPS/kg BW on d -7 and -3 prepartum, and 0.1 μg LPS/kg BW on d 3 and 7 postpartum. Blood samples were measured shortly before and in 8 time-points after (up to 6h) the challenges on d -14, -7, 3, and 7 to evaluate the post-challenge plasma profile. Results showed greater concentrations of plasma cortisol, in particular after the second and third LPS challenge. An increase in body temperature was recorded after administration of the greatest LPS dose, but this effect diminished during the very last LPS challenge. A biphasic response of glucose was observed; a linear increase up to 60 min after the second LPS challenge followed by a rapid decrease thereafter. Other plasma variables like lactate, cholesterol, non-esterified fatty acids, and beta-hydroxybutyrate were not affected by treatment. In conclusion, LPS administrations did not notably affect post-challenge metabolic responses in periparturient dairy cows but increased the level of plasma cortisol and the body temperature after the highest LPS challenge. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cyclosporin A significantly improves preeclampsia signs and suppresses inflammation in a rat model.

PubMed

Hu, Bihui; Yang, Jinying; Huang, Qian; Bao, Junjie; Brennecke, Shaun Patrick; Liu, Huishu

2016-05-01

Preeclampsia is associated with an increased inflammatory response. Immune suppression might be an effective treatment. The aim of this study was to examine whether Cyclosporin A (CsA), an immunosuppressant, improves clinical characteristics of preeclampsia and suppresses inflammation in a lipopolysaccharide (LPS) induced preeclampsia rat model. Pregnant rats were randomly divided into 4 groups: group 1 (PE) rats each received LPS via tail vein on gestational day (GD) 14; group 2 (PE+CsA5) rats were pretreated with LPS (1.0 μg/kg) on GD 14 and were then treated with CsA (5mg/kg, ip) on GDs 16, 17 and 18; group 3 (PE+CsA10) rats were pretreated with LPS (1.0 μg/kg) on GD 14 and were then treated with CsA (10mg/kg, ip) on GDs 16, 17 and 18; group 4 (pregnant control, PC) rats were treated with the vehicle (saline) used for groups 1, 2 and 3. Systolic blood pressure, urinary albumin, biometric parameters and the levels of serum cytokines were measured on day 20. CsA treatment significantly reduced LPS-induced systolic blood pressure and the mean 24-h urinary albumin excretion. Pro-inflammatory cytokines IL-6, IL-17, IFN-γ and TNF-α were increased in the LPS treatment group but were reduced in (LPS+CsA) group (P<0.05). Anti-inflammatory cytokine IL-4 was decreased in the LPS group but was increased in (LPS+CsA) group (P<0.05). Cyclosporine A improved preeclampsia signs and attenuated inflammatory responses in the LPS induced preeclampsia rat model which suggests that immunosuppressant might be an alternative management option for preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lipopolysaccharide Membrane Building and Simulation

PubMed Central

Jo, Sunhwan; Wu, Emilia L.; Stuhlsatz, Danielle; Klauda, Jeffery B.; Widmalm, Göran; Im, Wonpil

2015-01-01

Summary While membrane simulations are widely employed to study the structure and dynamics of various lipid bilayers and membrane proteins in the bilayers, simulations of lipopolysaccharides (LPS) in membrane environments have been limited due to its structural complexity, difficulties in building LPS-membrane systems, and lack of appropriate molecular force field. In this work, as a first step to extend CHARMM-GUI Membrane Builder to incorporate LPS molecules and to explore their structures and dynamics in membrane environments using molecular dynamics simulations, we describe step-by-step procedures to build LPS bilayer systems using CHARMM and the recently developed CHARMM carbohydrate and lipid force fields. Such procedures are illustrated by building various bilayers of Escherichia coli O6 LPS and their preliminary simulation results are given in terms of per-LPS area and density distributions of various components along the membrane normal. PMID:25753722

Early plant growth and biochemical responses induced by Azospirillum brasilense Sp245 lipopolysaccharides in wheat (Triticum aestivum L.) seedlings are attenuated by procyanidin B2.

PubMed

Vallejo-Ochoa, Juan; López-Marmolejo, Mariel; Hernández-Esquivel, Alma Alejandra; Méndez-Gómez, Manuel; Suárez-Soria, Laura Nicolasa; Castro-Mercado, Elda; García-Pineda, Ernesto

2018-03-01

This study analyzes the effects of procyanidin B2 on early wheat plant growth and plant biochemical responses promoted by lipopolysaccharides (LPS) derived from the rhizobacteria Azospirillum brasilense Sp245. Measurements of leaf, root length, fresh weight, and dry weight showed in vitro plant growth stimulation 4 days after treatment with A. brasilense as well as LPS. Superoxide anion (O 2 ·- ) and hydrogen peroxide (H 2 O 2 ) levels increased in seedling roots treated with LPS (100 μg mL -1 ). The chlorophyll content in leaf decreased while the starch content increased 24 h after treatment in seedling roots. The LPS treatment induced a high increase in total peroxidase (POX) (EC 1.11.1.7) activity and ionically bound cell wall POX content in roots, when compared to respective controls. Early plant growth and biochemical responses observed in wheat seedlings treated with LPS were inhibited by the addition of procyanidin B2 (5 μg mL -1 ), a B type proanthocyanidin (PAC), plant-derived polyphenolic compound with binding properties of LPS. All results suggest first that the ionically bound cell wall POX enzymes could be a molecular target of A. brasilense LPS, and second that the recognition or association of LPS by plant cells is required to activate plant responses. This last event could play a critical role during plant growth regulation by A. brasilense LPS.

Krill Oil-In-Water Emulsion Protects against Lipopolysaccharide-Induced Proinflammatory Activation of Macrophages In Vitro

PubMed Central

Bonaterra, Gabriel A.; Driscoll, David; Schwarzbach, Hans; Kinscherf, Ralf

2017-01-01

Background: Parenteral nutrition is often a mandatory therapeutic strategy for cases of septicemia. Likewise, therapeutic application of anti-oxidants, anti-inflammatory therapy, and endotoxin lowering, by removal or inactivation, might be beneficial to ameliorate the systemic inflammatory response during the acute phases of critical illness. Concerning anti-inflammatory properties in this setting, omega-3 fatty acids of marine origin have been frequently described. This study investigated the anti-inflammatory and LPS-inactivating properties of krill oil (KO)-in-water emulsion in human macrophages in vitro. Materials and Methods: Differentiated THP-1 macrophages were activated using specific ultrapure-LPS that binds only on the toll-like receptor 4 (TLR4) in order to determine the inhibitory properties of the KO emulsion on the LPS-binding capacity, and the subsequent release of TNF-α. Results: KO emulsion inhibited the macrophage binding of LPS to the TLR4 by 50% (at 12.5 µg/mL) and 75% (at 25 µg/mL), whereas, at 50 µg/mL, completely abolished the LPS binding. Moreover, KO (12.5 µg/mL, 25 µg/mL, or 50 µg/mL) also inhibited (30%, 40%, or 75%, respectively) the TNF-α release after activation with 0.01 µg/mL LPS in comparison with LPS treatment alone. Conclusion: KO emulsion influences the LPS-induced pro-inflammatory activation of macrophages, possibly due to inactivation of the LPS binding capacity. PMID:28294970

ILK mediates LPS-induced vascular adhesion receptor expression and subsequent leucocyte trans-endothelial migration.

PubMed

Hortelano, Sonsoles; López-Fontal, Raquel; Través, Paqui G; Villa, Natividad; Grashoff, Carsten; Boscá, Lisardo; Luque, Alfonso

2010-05-01

The inflammatory response to injurious agents is tightly regulated to avoid adverse consequences of inappropriate leucocyte accumulation or failed resolution. Lipopolysaccharide (LPS)-activated endothelium recruits leucocytes to the inflamed tissue through controlled expression of membrane-associated adhesion molecules. LPS responses in macrophages are known to be regulated by integrin-linked kinase (ILK); in this study, we investigated the role of ILK in the regulation of the LPS-elicited inflammatory response in endothelium. This study was performed on immortalized mouse endothelial cells (EC) isolated from lung and coronary vasculature. Cells were thoroughly characterized and the role of ILK in the regulation of the LPS response was investigated by suppressing ILK expression using siRNA and shRNA technologies. Phenotypic and functional analyses confirmed that the immortalized cells behaved as true EC. LPS induced the expression of the inflammatory genes E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). ILK knockdown impaired LPS-mediated endothelial activation by preventing the induction of ICAM-1 and VCAM-1. Blockade of the LPS-induced response inhibited the inflammatory-related processes of firm adhesion and trans-endothelial migration of leucocytes. ILK is involved in the expression of cell adhesion molecules by EC activated with the inflammatory stimulus LPS. This reduced expression modulates leucocyte adhesion to the endothelium and the extravasation process. This finding suggests ILK as a potential anti-inflammatory target for the development of vascular-specific treatments for inflammation-related diseases.

Preservation of renal blood flow by the antioxidant EUK-134 in LPS-treated pigs.

PubMed

Magder, Sheldon; Parthenis, Dimitrios G; Ghouleh, Imad Al

2015-03-25

Sepsis is associated with an increase in reactive oxygen species (ROS), however, the precise role of ROS in the septic process remains unknown. We hypothesized that treatment with EUK-134 (manganese-3-methoxy N,N'-bis(salicyclidene)ethylene-diamine chloride), a compound with superoxide dismutase and catalase activity, attenuates the vascular manifestations of sepsis in vivo. Pigs were instrumented to measure cardiac output and blood flow in renal, superior mesenteric and femoral arteries, and portal vein. Animals were treated with saline (control), lipopolysaccharide (LPS; 10 µg·kg-1·h-1), EUK-134, or EUK-134 plus LPS. Results show that an LPS-induced increase in pulmonary artery pressure (PAP) as well as a trend towards lower blood pressure (BP) were both attenuated by EUK-134. Renal blood flow decreased with LPS whereas superior mesenteric, portal and femoral flows did not change. Importantly, EUK-134 decreased the LPS-induced fall in renal blood flow and this was associated with a corresponding decrease in LPS-induced protein nitrotyrosinylation in the kidney. PO2, pH, base excess and systemic vascular resistance fell with LPS and were unaltered by EUK-134. EUK-134 also had no effect on LPS-associated increase in CO. Interestingly, EUK-134 alone resulted in higher CO, BP, PAP, mean circulatory filling pressure, and portal flow than controls. Taken together, these data support a protective role for EUK-134 in the renal circulation in sepsis.

Matrix metalloproteinase inhibitor attenuates cochlear lateral wall damage induced by intratympanic instillation of endotoxin.

PubMed

Choi, Cheol Hee; Jang, Chul Ho; Cho, Yong Bum; Jo, Si Young; Kim, Min Young; Park, Byung Young

2012-04-01

Oxytetracycline and ilomastat are inhibitors of matrix metalloproteinases (MMPs). Their efficacy in protecting against cochlear damage induced by the intratympanic instillation of lipopolysaccharide (LPS), as a means of inducing labyrinthitis, was investigated. Experiments were performed in 21 young male guinea pigs. Intratympanic instillation of LPS was done in the control group (n=7). Intratympanic instillation of oxytetracycline or ilomastat was done after LPS instillation in the experimental group. Measurements of auditory brainstem response (ABR) and cochlear blood flow (CBF) were performed. The organ of Corti was evaluated by field emission scanning electron microscopy (FE-SEM). The blood-labyrinth barrier (BLB) integrity was evaluated with Evans blue uptake. Gelatin zymography was used to assess the expression of active MMP-2 and MMP-9. Ears treated with MMP inhibitors were significantly protected from hearing loss compared to the LPS group. In LPS group, there was a significant decrease of CBF. However, experimental group displayed a statistically significant recovery of CBF. FE-SEM revealed hair cell damage in the LPS-treated group, but hair cells presented a normal appearance in MMP inhibitors. The LPS group showed a marked increase of Evans blue extravasation in the cochlea. However, MMP inhibitors significantly reduced the BLB opening. Active MMP-9 was expressed in the LPS group. Treatment with MMP inhibitors attenuated active MMP-9 expression. The MMP inhibitors oxytetracycline and ilomastat protect from cochlear lateral wall damage caused by LPS-induced labyrinthitis. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin.

PubMed

Uhlig, S; Nüsing, R; von Bethmann, A; Featherstone, R L; Klein, T; Brasch, F; Müller, K M; Ullrich, V; Wendel, A

1996-05-01

Lipopolysaccharides (LPS), widely used to study the mechanisms of gram-negative sepsis, increase airway resistance by constriction of terminal bronchioles. The role of the cyclooxygenase (COX) isoenzymes and their prostanoid metabolites in this process was studied. Pulmonary resistance, the release of thromboxane (TX) and the expression of COX-2 mRNA were measured in isolated blood-free perfused rat lungs exposed to LPS. LPS induced the release of TX and caused increased airway resistance after about 30 min. Both TX formation and LPS-induced bronchoconstriction were prevented by treatment with the unspecific COX inhibitor acetyl salicylic acid, the specific COX-2 inhibitor CGP-28238, dexamethasone, actinomycin D, or cycloheximide. LPS-induced bronchoconstriction was also inhibited by the TX receptor antagonist BM-13177. The TX-mimetic compound, U-46619, increased airway resistance predominantly by constricting terminal bronchioles. COX-2-specific mRNA in lung tissue was elevated after LPS exposure, and this increase was attenuated by addition of dexamethasone or of actinomycin D. In contrast to LPS, platelet-activating factor (PAF) induced immediate TX release and bronchoconstriction that was prevented by acetyl salicylic acid, but not by CGP-28238. LPS elicits the following biochemical and functional changes in rat lungs: (i) induction of COX-2; (ii) formation of prostaglandins and TX; (iii) activation of the TX receptor on airway smooth muscle cells; (iv) constriction of terminal bronchioles; and (v) increased airway resistance. In contrast to LPS, the PAF-induced TX release is likely to depend on COX-1.

Biophysical Mechanisms of Endotoxin Neutralization by Cationic Amphiphilic Peptides

PubMed Central

Kaconis, Yani; Kowalski, Ina; Howe, Jörg; Brauser, Annemarie; Richter, Walter; Razquin-Olazarán, Iosu; Iñigo-Pestaña, Melania; Garidel, Patrick; Rössle, Manfred; Martinez de Tejada, Guillermo; Gutsmann, Thomas; Brandenburg, Klaus

2011-01-01

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS. PMID:21641310

All-solid state lithium carbon monofluoride batteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Chengdu; Rangasamy, Ezhiylmurugan

A solid state lithium carbon monofluoride battery includes an anode comprising Li, a solid electrolyte, and a cathode including CF.sub.x and LPS. The cathode can also include a carbon compound. The solid electrolyte can include LPS. The LPS can include .beta.-Li.sub.3PS.sub.4. The cathode LPS can include .beta.-Li.sub.3PS.sub.4. A method of making a battery is also disclosed.

Lipopolysaccharide-coated CuS nanoparticles promoted anti-cancer and anti-metastatic effect by immuno-photothermal therapy

PubMed Central

Moorthy, Madhappan S.; Zhang, Wei; Zeng, Ling; Kang, Mingyeong; Kwak, Minseok; Oh, Junghwan; Jin, Jun-O

2017-01-01

To meet the ultimate goal of cancer therapy, which is treating not only the primary tumor but also preventing metastatic cancer, the concept of combining immunotherapy with photothermal therapy (PTT) is gaining great interest. Here, we studied the new material, lipopolysaccharide (LPS) coated copper sulfide nanoparticles (LPS-CuS), for the immuno-photothermal therapy. We evaluated the effect of LPS-CuS for induction of apoptosis of CT26 cells and activation of dendritic cells. Moreover, the LPS-CuS and laser irradiation was examined anti-metastasis effect by liver metastasis model mouse in vivo. Through PTT, LPS-CuS induced elimination of CT26 tumor in BALB/c mice, which produced cancer antigens. In addition, released LPS and cancer antigen by PTT promoted dendritic cell activation in tumor draining lymph node (drLN), and consequently, enhanced the tumor antigen-specific immune responses. Finally, the primary tumor cured mice by LPS-CuS-mediated PTT completely resisted secondary tumor injection in the spleen and also prevented liver metastasis. Our results demonstrated the potential usage of LPS-CuS for the immuno-photothermal therapy against various types of cancer by showing the clear elimination of primary colon carcinoma with complete prevention of spleen and liver metastasis. PMID:29285274

A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells

PubMed Central

Gabriele, Morena; Del Prato, Stefano; Pucci, Laura

2017-01-01

Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS) orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs), bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG), on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER) stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation. PMID:28386305

Gut microbiota-derived lipopolysaccharide uptake and trafficking to adipose tissue: implications for inflammation and obesity.

PubMed

Hersoug, L-G; Møller, P; Loft, S

2016-04-01

The composition of the gut microbiota and excessive ingestion of high-fat diets (HFD) are considered to be important factors for development of obesity. In this review we describe a coherent mechanism of action for the development of obesity, which involves the composition of gut microbiota, HFD, low-grade inflammation, expression of fat translocase and scavenger receptor CD36, and the scavenger receptor class B type 1 (SR-BI). SR-BI binds to both lipids and lipopolysaccharide (LPS) from Gram-negative bacteria, which may promote incorporation of LPS in chylomicrons (CMs). These CMs are transported via lymph to the circulation, where LPS is transferred to other lipoproteins by translocases, preferentially to HDL. LPS increases the SR-BI binding, transcytosis of lipoproteins over the endothelial barrier,and endocytosis in adipocytes. Especially large size adipocytes with high metabolic activity absorb LPS-rich lipoproteins. In addition, macrophages in adipose tissue internalize LPS-lipoproteins. This may contribute to the polarization from M2 to M1 phenotype, which is a consequence of increased LPS delivery into the tissue during hypertrophy. In conclusion, evidence suggests that LPS is involved in the development of obesity as a direct targeting molecule for lipid delivery and storage in adipose tissue. © 2015 World Obesity.

Results after laparoscopic partial splenectomy for children with hereditary spherocytosis: Are outcomes influenced by genetic mutation?

PubMed

Pugi, Jakob; Carcao, Manuel; Drury, Luke J; Langer, Jacob C

2018-05-01

Laparoscopic partial splenectomy (LPS) theoretically maintains long-term splenic immune function for children with hereditary spherocytosis (HS). Our goal was to review our results after LPS and to determine if specific genetic mutations influence outcome. All children with HS undergoing LPS between 2005 and 2016 were reviewed. Thirty-one children underwent LPS (16 male) at a median age of 9 (range 2-18) years. All experienced an increase in hemoglobin and decrease in reticulocyte count early after LPS and at last follow-up. Twenty-two were sent for genetic analysis. Mutations in α-spectrin, β-spectrin, and Ankyrin were identified in 6, 5, and 11 patients, respectively. Gene mutation was not correlated with complications, perioperative transfusion, length of hospital stay, or median hemoglobin, platelet, or reticulocyte counts. Three children required completion splenectomy at 10.9, 6.9, and 3.2years post-LPS, each with a different gene mutation. LPS is effective in reversing anemia and reducing reticulocytosis. So far less than 10% have required completion splenectomy, and those children did benefit from delaying the risks of asplenia. In this preliminary analysis, genetic mutation did not influence outcome after LPS. A larger multicenter study is necessary to further investigate potential correlations with specific genetic mutations. Prognosis Study. IV. Copyright © 2018. Published by Elsevier Inc.

Identification of a ghrelin-like peptide in two species of shark, Sphyrna lewini and Carcharhinus melanopterus.

PubMed

Kawakoshi, Akatsuki; Kaiya, Hiroyuki; Riley, Larry G; Hirano, Tetsuya; Grau, E Gordon; Miyazato, Mikiya; Hosoda, Hiroshi; Kangawa, Kenji

2007-05-01

In this study, we identified a ghrelin-like peptide (ghrelin-LP) in two elasmobranchs. The peptide, isoforms and cDNA encoding its precursor were isolated from the stomach of two sharks, the hammerhead (HH) shark (Sphyrna lewini) and the black-tip reef (BTR) shark (Carcharhinus melanopterus). The ghrelin-LP isolated from each shark was found to be 25 amino acids in length and exhibit high sequence homology with each other; only three amino acids were different. As has been shown in tetrapod and teleost fish ghrelins, shark ghrelin-LPs possess two forms that are distinguished by having the third serine residue (Ser) acylated by either octanoic or decanoic acid. The N-terminal four residues (GVSF), known as the active core of ghrelin, are not identical to those of other species (GSSF). Nevertheless, shark ghrelin-LP elevated Ca(2+) levels in CHO cell line expressing the growth hormone secretagogue receptor (GHS-R). Unlike teleosts ghrelin's, shark ghrelin-LPs are not amidated at the C-terminus. Messenger RNA of ghrelin-LP in the HH shark was predominantly expressed in the stomach as seen in other species, followed by the brain, intestine, gill, heart and liver. The nucleotide sequence of the ghrelin-LP gene in the HH shark was characterized to compare organization of the ghrelin gene with those in other species. The size of the HH ghrelin-LP gene was 8541 bp, two to ten times larger than that of other species studied to date. The HH ghrelin-LP gene is composed of five exons and four introns, which is the same as ghrelin genes in mammals, chicken and rainbow trout. In conclusion, the shark ghrelin-LPs identified in this study exhibit many characteristics for ghrelin in terms of peptide modifications, GHS-R activation, tissue distribution, and gene organization; however, it is necessary to further clarify their biological properties such as growth hormone-releasing or orexigenic activity before designating these peptides as ghrelin.

Magnolol inhibits LPS-induced inflammatory response in uterine epithelial cells : magnolol inhibits LPS-induced inflammatory response.

PubMed

Luo, Jia; Xu, Yanwen; Zhang, Minfang; Gao, Ling; Fang, Cong; Zhou, Canquan

2013-10-01

Endometritis is an inflammation of the uterine lining that is commonly initiated at parturition. The uterine epithelial cells play an important role in defending against invading pathogens. Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been shown to have anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effect of magnolol in modifying lipopolysaccharide (LPS)-induced signal pathways in mouse uterine epithelial cells. We found that magnolol inhibited TNF-α and IL-6 production in LPS-stimulated mouse uterine epithelial cells. We also found that magnolol inhibited LPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK, and P38. Furthermore, magnolol could significantly inhibit the expression of TLR4 stimulating by LPS. These results suggest that magnolol exerts an anti-inflammatory property by downregulating the expression of TLR4 upregulated by LPS, thereby attenuating TLR4-mediated NF-κB and MAPK signaling and the release of pro-inflammatory cytokines. These findings suggest that magnolol may be a therapeutic agent against endometritis.