Removal of peptidoglycan and inhibition of active cellular processes leads to daptomycin tolerance in Enterococcus faecalis

Daptomycin is a cyclic lipopeptide antibiotic used in the clinic for treatment of severe enterococcal infections. Recent reports indicate that daptomycin targets active cellular processes, specifically, peptidoglycan biosynthesis. Within, we examined the efficacy of daptomycin against Enterococcus faecalis under a range of environmental growth conditions including inhibitors that target active cellular processes. Daptomycin was far less effective against cells in late stationary phase compared to cells in exponential phase, and this was independent of cellular ATP levels. Further, the addition of either the de novo protein synthesis inhibitor chloramphenicol or the fatty acid biosynthesis inhibitor cerulenin induced survival against daptomycin far better than controls. Alterations in metabolites associated with peptidoglycan synthesis correlated with protection against daptomycin. This was further supported as removal of peptidoglycan induced physiological daptomycin tolerance, a synergistic relation between daptomycin and fosfomycin, an inhibitor of the fist committed step peptidoglycan synthesis, was observed, as well as an additive effect when daptomycin was combined with ampicillin, which targets crosslinking of peptidoglycan strands. Removal of the peptidoglycan of Enterococcus faecium, Staphylococcus aureus, and Bacillus subtilis also resulted in significant protection against daptomycin in comparison to whole cells with intact cell walls. Based on these observations, we conclude that bacterial growth phase and metabolic activity, as well as the presence/absence of peptidoglycan are major contributors to the efficacy of daptomycin.

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Determinants of kinin release in isolated rat hindquarters

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1998.274.1.r120 ◽

1998 ◽

Vol 274 (1) ◽

pp. R120-R125 ◽

Cited By ~ 2

Author(s):

Tohru Sakakibara ◽

Thomas H. Hintze ◽

Alberto Nasjletti

Keyword(s):

Protein Synthesis ◽

Trypsin Inhibitor ◽

De Novo ◽

Soybean Trypsin Inhibitor ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Bicarbonate Buffer ◽

Kallikrein Inhibitor ◽

Isolated Rat

We studied the determinants of kinin release into the venous effluent of rat hindquarters perfused with Krebs bicarbonate buffer. Kinin release in preparations perfused with control media (14.6 ± 2.5–20.7 ± 6.7 pg/15 min) was surpassed by that in preparations perfused with media containing kininase inhibitors (243 ± 53 to 276 ± 78 pg/15 min). Kinin release increased when purified kininogen (from 242 ± 43 to 3,365 ± 725 pg/15 min) or kallikrein (from 270 ± 49 to 30,649 ± 8,040 pg/15 min) was added to the perfusate. Conversely, kinin release fell when the kallikrein inhibitor aprotinin (from 272 ± 58 to 122 ± 27 pg/15 min) or soybean trypsin inhibitor (from 273 ± 52 to 195 ± 25 pg/15 min) was added. Both basal and kininogen-induced kinin release were attenuated in preparations perfused with media containing cycloheximide, a protein synthesis inhibitor, but kallikrein-induced kinin release was not. These data suggest that kinin release from perfused rat hindquarters reflects the activity of both the kinin-degrading and kinin-generating pathways and that the latter is sustained by a kallikrein manufactured de novo and by preexistent kininogen(s).

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Natural and environmental oestrogens induce TGFB1 synthesis in oviduct cells

Reproduction ◽

10.1530/rep-17-0425 ◽

2018 ◽

Vol 155 (3) ◽

pp. 233-244 ◽

Cited By ~ 3

Author(s):

Barbara P S Cometti ◽

Raghvendra K Dubey ◽

Bruno Imthurn ◽

Marinella Rosselli

Keyword(s):

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

De Novo ◽

Embryo Implantation ◽

Early Embryo ◽

Biochanin A ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Paracrine Factors ◽

Aryl Hydrocarbon

Autocrine/paracrine factors generated in response to 17β-oestradiol (E2), within the oviduct, facilitate early embryo development for implantation. Since transforming growth factor beta 1 (TGFB1) plays a key role in embryo implantation, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Here, we investigated whether oviduct cells synthesize TGFB1 and whether E2 and environmental oestrogens (EOEs; xenoestrogens and phytoestrogens) modulate its synthesis. Under basal conditions, bovine oviduct cells (OCs; oviduct epithelial cells and oviduct fibroblasts; 1:1 ratio) synthesized TGFB1. E2 concentration-dependent induced TGFB1 levels in OCs and these effects were mimicked by some, but not all EOEs (genistein, biochanin A and 4-hydroxy-2′,4′,6′-trichlorobiphenyl, 4-hydroxy-2′,4′,6′-dichlorobiphenyl); moreover, EOEs enhanced (P < 0.05) the stimulatory effects of E2 on TGFB1 synthesis. The OCs expressed oestrogen receptors alpha and beta and aryl hydrocarbon; moreover, co-treatment with ER antagonist ICI182780 blocked the stimulatory effects of E2 and EOEs on TGFB1 synthesis. Treatment with non-permeable E2-BSA failed to induce TGFB1, thereby ruling out the involvement of membrane ERs. Cycloheximide (protein synthesis inhibitor) blocked E2-induced TGFB1 synthesis providing evidence forde novosynthesis. The stimulatory effects of E2 and EOEs, were inhibited (P < 0.05) by MAPK inhibitor (PD98059), whereas intracellular-Ca2+chelator (BAPTA-AM) and adenylyl cyclase inhibitor (SQ22536) abrogated the effects of E2, but not EOEs, suggesting that post-ER effects of E2 and EOEs involve different pathways. Our results provide the first evidence that in OCs, E2 and EOEs stimulate TGFB1 synthesis via an ER-dependent pathway. Exposure of the oviduct to EOEs may result in continuous/sustained induction of TGFB1 levels in a non-cyclic fashion and may induce deleterious effects on reproduction.

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Social Fear Memory Requires Two Stages of Protein Synthesis in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms21155537 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5537

Author(s):

Johannes Kornhuber ◽

Iulia Zoicas

Keyword(s):

Protein Synthesis ◽

Temporal Dynamics ◽

De Novo ◽

Fear Memory ◽

Protein Synthesis Inhibitor ◽

Dependent Manner ◽

Synthesis Inhibitor ◽

Systemic Injection ◽

Social Fear ◽

Two Stages

It is well known that long-term consolidation of newly acquired information, including information related to social fear, require de novo protein synthesis. However, the temporal dynamics of protein synthesis during the consolidation of social fear memories is unclear. To address this question, mice received a single systemic injection with the protein synthesis inhibitor, anisomycin, at different time-points before or after social fear conditioning (SFC), and memory was assessed 24 h later. We showed that anisomycin impaired the consolidation of social fear memories in a time-point-dependent manner. Mice that received anisomycin 20 min before, immediately after, 6 h, or 8 h after SFC showed reduced expression of social fear, indicating impaired social fear memory, whereas anisomycin caused no effects when administered 4 h after SFC. These results suggest that consolidation of social fear memories requires two stages of protein synthesis: (1) an initial stage starting during or immediately after SFC, and (2) a second stage starting around 6 h after SFC and lasting for at least 5 h.

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Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells.

The Journal of Cell Biology ◽

10.1083/jcb.110.4.1427 ◽

1990 ◽

Vol 110 (4) ◽

pp. 1427-1438 ◽

Cited By ~ 215

Author(s):

S Aznavoorian ◽

M L Stracke ◽

H Krutzsch ◽

E Schiffmann ◽

L A Liotta

Keyword(s):

Protein Synthesis ◽

Matrix Protein ◽

Type Iv Collagen ◽

De Novo ◽

Protein Synthesis Inhibitor ◽

Dependent Manner ◽

Recognition Sequence ◽

Synthesis Inhibitor ◽

Concentration Dependent Manner ◽

Type Iv

Transduction of signals initiating motility by extracellular matrix (ECM) molecules differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Laminin, fibronectin, and type IV collagen stimulated both chemotaxis and haptotaxis of the A2058 human melanoma cell line. Peak chemotactic responses were reached at 50-200 nM for laminin, 50-100 nM for fibronectin, and 200-370 nM for type IV collagen. Checkerboard analysis of each attractant in solution demonstrated a predominantly directional (chemotactic) response, with a minor chemokinetic component. The cells also migrated in a concentration-dependent manner to insoluble step gradients of substratum-bound attractant (haptotaxis). The haptotactic responses reached maximal levels at coating concentrations of 20 nM for laminin and type IV collagen, and from 30 to 45 nM for fibronectin. Pretreatment of cells with the protein synthesis inhibitor, cycloheximide (5 micrograms/ml), resulted in a 5-30% inhibition of both chemotactic and haptotactic responses to each matrix protein, indicating that de novo protein synthesis was not required for a significant motility response. Pretreatment of cells with 50-500 micrograms/ml of synthetic peptides containing the fibronectin cell-recognition sequence GRGDS resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis and haptotaxis (70-80% inhibition compared to control motility); negative control peptide GRGES had only a minimal effect. Neither GRGDS nor GRGES significantly inhibited motility to laminin or type IV collagen. Therefore, these results support a role for the RGD-directed integrin receptor in both types of motility response to fibronectin. After pretreatment with pertussis toxin (PT), chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to laminin was intermediate in sensitivity; chemotaxis to fibronectin was completely insensitive; and chemotaxis to type IV collagen was profoundly inhibited by PT. In marked contrast to the inhibition of chemotaxis, the hepatotactic responses to all three ligands were unaffected by any of the tested concentrations of PT. High concentrations of cholera toxin (CT; 10 micrograms/ml) or the cAMP analogue, 8-Br-cAMP (0.5 mM), did not significantly affect chemotactic or haptotactic motility to any of the attractant proteins, ruling out the involvement of cAMP in the biochemical pathway initiating motility in these cells. The sensitivity of chemotaxis induced by laminin and type IV collagen, but not fibronectin, to PT indicates the involvement of a PT-sensitive G protein in transduction of the signals initiating motility to soluble laminin and type IV collagen.(ABSTRACT TRUNCATED AT 400 WORDS)

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Effect of Cerophyl growth medium on exocytosis in Tetrahymena thermophila

Journal of Cell Science ◽

10.1242/jcs.78.1.23 ◽

1985 ◽

Vol 78 (1) ◽

pp. 23-48

Author(s):

D.M. Pesciotta ◽

B.H. Satir

Keyword(s):

Cell Division ◽

De Novo ◽

Tetrahymena Thermophila ◽

Freeze Fracture ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Particle Array ◽

Proteose Peptone ◽

Section Electron ◽

Cell Changes

Culturing the ciliate Tetrahymena thermophila in Cerophyl has provided an opportunity for studying the assembly and/or synthesis of the intramembrane particle array, the rosette, which marks the site of exocytosis in these cells. Cultures grown in this medium cease cell division after only 12h and enter ‘stationary phase’ earlier (12h of growth) relative to growth in standard medium (proteose peptone). In addition, the cell changes from the normally observed pear-shaped body to a thinner more ellipsoid form. Despite the initial similarities to starving cells, several differences are observed in the Cerophyl-grown cells. One is that cell size remains constant for at least 72h in contrast to starved cells. Secondly, in spite of this block in cell division, results from freeze-fracture replicas of the cell membrane of these cells show that they continue to assemble rosettes, the number of which increases approximately six times, from 0.34 rosette/microgram2 to 2.1 rosettes/microgram2. Addition of the protein synthesis inhibitor, cycloheximide (6h exposure), during growth in Cerophyl shows that 70% of rosettes can be assembled, despite the blockage of translation, by using pre-existing component(s) from a pool. The remaining 30% must involve de novo synthesis of one or more components; this percentage can be increased with longer exposure to the drug. Thirdly, an apparent increase in the number of mucocysts is observed by thin-section electron microscopy. At first (12–24h) only docked mucocysts seem to accumulate in the cell. However, by 36h a considerable increase seems to have taken place, particularly in the number of mucocysts located in the cytoplasm. In the cycloheximide-treated cells this increase in mucocysts begins to be blocked after 6h of exposure to the drug. These observations are in agreement with the results obtained from the freeze-fracture data on the concomitant increase in number of rosettes. This system therefore offers the first possibility of exploring the biosynthesis of these components.

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Granulocyte/macrophage colony-stimulating factor stimulates the expression of the 5-lipoxygenase-activating protein (FLAP) in human neutrophils.

Journal of Experimental Medicine ◽

10.1084/jem.179.4.1225 ◽

1994 ◽

Vol 179 (4) ◽

pp. 1225-1232 ◽

Cited By ~ 70

Author(s):

M Pouliot ◽

P P McDonald ◽

P Borgeat ◽

S R McColl

Keyword(s):

De Novo ◽

Human Neutrophils ◽

Protein Synthesis Inhibitor ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

Synthesis Inhibitor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

The synthesis of leukotrienes in human blood neutrophils chiefly relies on the activity of two enzymes, phospholipase A2 and 5-lipoxygenase (5-LO). In turn, the activation of the 5-LO requires the participation of a recently characterized membrane-bound protein, the 5-LO-activating protein (FLAP). In this study, we have investigated conditions under which FLAP expression in neutrophils may be modulated. Of several cytokines tested, only granulocyte/macrophage colony-stimulating factor (GM-CSF) (and to a lesser extent tumor necrosis factor alpha) significantly increased expression of FLAP. GM-CSF increased FLAP mRNA steady-state levels in a time- and dose-dependent manner. The stimulatory effect of GM-CSF on FLAP mRNA was inhibited by prior treatment of the cells with the transcription inhibitor, actinomycin D, and pretreatment of the cells with the protein synthesis inhibitor, cycloheximide, failed to prevent the increase in FLAP mRNA induced by GM-CSF. The accumulation of newly synthesized FLAP, as determined by immunoprecipitation after incorporation of 35S-labeled amino acids, was also increased after incubation of neutrophils with GM-CSF. In addition, the total level of FLAP protein was increased in GM-CSF-treated neutrophils, as determined by two-dimensional gel electrophoresis, followed by Western blot. GM-CSF did not alter the stability of the FLAP protein, indicating that the effect of GM-CSF on FLAP accumulation was the consequence of increased de novo synthesis as opposed to decreased degradation of FLAP. Finally, incubation of neutrophils with the synthetic glucocorticoid dexamethasone directly stimulated the upregulation of FLAP mRNA and protein, and enhanced the effect of GM-CSF. Taken together, these data demonstrate that FLAP expression may be upmodulated after appropriate stimulation of neutrophils. The increase in FLAP expression induced by GM-CSF in inflammatory conditions could confer upon neutrophils a prolonged capacity to synthesize leukotrienes.

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Oxidative and Electrophilic Stresses Activate Nrf2 through Inhibition of Ubiquitination Activity of Keap1

Molecular and Cellular Biology ◽

10.1128/mcb.26.1.221-229.2006 ◽

2006 ◽

Vol 26 (1) ◽

pp. 221-229 ◽

Cited By ~ 544

Author(s):

Akira Kobayashi ◽

Moon-Il Kang ◽

Yoriko Watai ◽

Kit I. Tong ◽

Takahiro Shibata ◽

...

Keyword(s):

Molecular Mechanisms ◽

De Novo ◽

Degradation Mechanism ◽

Nuclear Accumulation ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Nrf2 Activation ◽

Nrf2 Protein

ABSTRACT The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.

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The mitochondrial K-ATP channel opener diazoxide upregulates STIM1 and Orai1 via ROS and the MAPK pathway in adult rat cardiomyocytes

10.21203/rs.3.rs-28577/v2 ◽

2020 ◽

Author(s):

Joice Thomas Gavali ◽

Elba Dolores Carrillo ◽

Maria Carmen Garcia ◽

Jorge Alberto Sanchez

Keyword(s):

Nuclear Translocation ◽

De Novo ◽

Mapk Pathway ◽

Ischemia Reperfusion ◽

Cardiac Cells ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Rat Cardiomyocytes ◽

Adult Rat ◽

Protein Levels

Abstract Background: Openers of mitochondrial adenosine triphosphate-dependent potassium (mKATP) channels like diazoxide increase reactive oxygen species (ROS) production in cardiac cells and reduce Ca2+ elevations produced by ischemia-reperfusion, protecting the heart from damage. In this study we tested the hypothesis that opening mKATP channels regulates expression of the major components of store-operated Ca2+ entry (SOCE) STIM1 and Orai1.Results: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that diazoxide increased expression of STIM1 and Orai1 at the mRNA and protein levels, respectively, in adult rat cardiomyocytes. Immunofluorescence analyses revealed that diazoxide also disrupted the striated distribution pattern of STIM1. These effects were prevented by the ROS scavenger N-acetyl cysteine (NAC), the mKATP channel antagonist 5-hydroxydecanoate (5-HD), or the protein synthesis inhibitor cycloheximide (CHX). Confocal microscopy revealed that diazoxide also led to nuclear translocation of the transcription factors c-Fos and NFkB, which was also blocked by NAC or 5-HD. Finally, the MAPK pathway inhibitor UO126 attenuated diazoxide-induced upregulation of STIM1 and Orai1 expressionConclusions: Our results suggest that opening mitochondrial potassium ATP channels with diazoxide upregulates the expression of STIM1 and Orai1 by de novo synthesis by a mechanism that involves NFkB, c-Fos, and ROS via MAPK/ERK signaling.

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The CRISPR-Cas9 crATIC HeLa transcriptome: Characterization of a novel cellular model of ATIC deficiency and ZMP accumulation

10.1101/2020.04.28.067066 ◽

2020 ◽

Cited By ~ 1

Author(s):

Randall C Mazzarino ◽

Veronika Baresova ◽

Marie Zikánová ◽

Nathan Duval ◽

Terry G. Wilkinson ◽

...

Keyword(s):

De Novo ◽

Adenosine Monophosphate ◽

Acid Synthesis ◽

Growth Conditions ◽

Cellular Model ◽

Inosine Monophosphate ◽

Cellular Processes ◽

Exogenous Compounds ◽

Inosine Monophosphate Cyclohydrolase ◽

Amp Activated Protein Kinase

ABSTRACTIn de novo purine biosynthesis (DNPS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (2.1.2.3) / inosine monophosphate cyclohydrolase (3.5.4.10) (ATIC) catalyzes the last two reactions of the pathway: conversion of 5-aminoimidazole-4-carboxamide ribonucleotide [aka Z-nucleotide monophosphate (ZMP)] to 5-formamido-4-imidazolecarboxamide ribonucleotide (FAICAR) then to inosine monophosphate (IMP). ZMP is an adenosine monophosphate (AMP) mimetic and a known activator of AMP-activated protein kinase (AMPK). Recently, a HeLa cell line null mutant for ATIC was constructed via CRISPR-Cas9 mutagenesis. This mutant, crATIC, accumulates ZMP during purine starvation. Given that the mutant can accumulate ZMP in the absence of treatment with exogenous compounds, crATIC is likely an important cellular model of DNPS inactivation and ZMP accumulation. In the current study, we characterize the crATIC transcriptome versus the HeLa transcriptome in purine-supplemented and purine-depleted growth conditions. We report and discuss transcriptome changes with particular relevance to Alzheimer’s disease and in genes relevant to lipid and fatty acid synthesis, neurodevelopment, embryogenesis, cell cycle maintenance and progression, extracellular matrix, immune function, TGFβ and other cellular processes.

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The mitochondrial K-ATP channel opener diazoxide upregulates STIM1 and Orai1 via ROS and the MAPK pathway in adult rat cardiomyocytes

10.21203/rs.3.rs-28577/v1 ◽

2020 ◽

Author(s):

Joice Thomas Gavali ◽

Elba Dolores Carrillo ◽

Maria Carmen Garcia ◽

Jorge Alberto Sanchez

Keyword(s):

Transcription Factors ◽

Nuclear Translocation ◽

De Novo ◽

Mapk Pathway ◽

Ischemia Reperfusion ◽

Cardiac Cells ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Rat Cardiomyocytes ◽

Adult Rat

Abstract Background: Openers of mitochondrial adenosine triphosphate-dependent potassium (mKATP) channels like diazoxide increase reactive oxygen species (ROS) production in cardiac cells and reduce Ca2+ elevations produced by ischemia-reperfusion protecting the heart from damage. In this study we tested the hypothesis that opening mKATP channels regulates expression of the major components of store-operated Ca2+ entry (SOCE) STIM1 and Orai1. Methods: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot techniques were used to assess mRNA expression and protein abundance of STIM1 and Orai1. Confocal immunofluorescence imaging was used to visualize the distribution and localization of STIM1 and Orai1 and translocation of transcription factors NFkB and c-Fos into the nucleus.Results: Diazoxide increased expression of STIM1 and Orai1 at the mRNA and protein levels, respectively, in adult rat cardiomyocytes. Diazoxide also disrupted the striated distribution pattern of STIM1. These effects were prevented by the ROS scavenger N-acetyl cysteine (NAC), the mKATP channel antagonist 5-hydroxydecanoate (5-HD), or the protein synthesis inhibitor cycloheximide (CHX). Diazoxide also led to nuclear translocation of the transcription factors c-Fos and NFkB, which was also blocked by NAC or 5-HD. Finally, the MAPK pathway inhibitor UO126 attenuated diazoxide-induced upregulation of STIM1 and Orai1 expression Conclusions: Our results suggest that opening mitochondrial potassium ATP channels with diazoxide upregulates the expression of STIM1 and Orai1 by de novo synthesis by a mechanism that involves NFkB, c-Fos, and ROS via MAPK/ERK signaling.

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