scholarly journals Screening for Mevalonate Biosynthetic Pathway Inhibitors Using Sensitized Bacterial Strains

2011 ◽  
Vol 16 (6) ◽  
pp. 637-646 ◽  
Author(s):  
Sandrine Ferrand ◽  
Jianshi Tao ◽  
Xiaoyu Shen ◽  
Dorothy McGuire ◽  
Andres Schmid ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Mevalonate Pathway ◽  
Inducible Promoter ◽  
High Density ◽  
Bacterial Strains ◽  
Differential Growth ◽  
Low Concentrations ◽  
Mevalonate Diphosphate Decarboxylase ◽  
A Cell ◽  
Dependent Pathway

A simple, optical density-based assay for inhibitors of the mevalonate-dependent pathway for isoprenoid biosynthesis was developed. The assay uses pathway-sensitized Staphylococcus aureus strains and is fully compatible with high-density screening in a 1536-well format. S. aureus strains were constructed in which genes required for mevalonate-dependent isopentenyl pyrophosphate (IPP) synthesis were regulated by an isopropyl-β-D-thiogalactopyranoside (IPTG)–inducible promoter. Inhibitors of the target enzymes displayed greater antibacterial potency in media containing low concentrations of IPTG, and therefore less induction of mevalonate pathway genes, than in media with high IPTG conditions. This differential growth phenotype was exploited to bias the cell-based screening hits toward specific inhibitors of mevalonate-dependent IPP biosynthesis. Screens were run against strains engineered for regulation of the enzymes HMG-CoA synthase (MvaS) and mevalonate kinase (mvaK1), mevalonate diphosphate decarboxylase (mvaD), and phosphomevalonate kinase (mvaK2). The latter three enzymes are regulated as an operon. These assays resulted in the discovery of potent antibacterial hits that were progressed to an active hit-to-lead program. The example presented here demonstrates that a cell sensitization strategy can be successfully applied to a 1.3-million compound high-throughput screen in a high-density 1536-well format.

scholarly journals Cholesterol biosynthetic pathway induces cellular senescence through ERRa

2021 ◽  
Author(s):  
Dorian V Ziegler ◽  
Mathieu Vernier ◽  
Joanna Czarnecka-Herok ◽  
Charlotte Scholtes ◽  
Christelle Machon ◽  
...  
Keyword(s):  
Cellular Senescence ◽  
Biosynthetic Pathway ◽  
Mevalonate Pathway ◽  
Cholesterol Biosynthesis ◽  
Age Related ◽  
Diet Regimen ◽  
Cholesterol Biosynthetic Pathway ◽  
A Cell ◽  
Normal Human

Cellular senescence is a cell program induced by various stresses that leads to a stable proliferation arrest and to a senescence-associated secretory phenotype. Accumulation of senescent cells during age-related diseases participates in these pathologies and regulates healthy lifespan. Recent evidences point out a global dysregulated intracellular metabolism associated to senescence phenotype. Nonetheless, the functional contribution of metabolic homeostasis in regulating senescence is barely understood. In this work, we describe how the mevalonate pathway, an anabolic pathway leading to the endogenous biosynthesis of poly-isoprenoids, such as cholesterol, acts as a positive regulator of cellular senescence in normal human cells. Mechanistically, this mevalonate-induced senescence is partly mediated by the downstream cholesterol biosynthetic pathway. This pathway promotes transcriptional activity of ERRα leading to dysfunctional mitochondria, ROS production, DNA damage and a p53-dependent senescence. Supporting the relevance of these observations, increase of senescence in liver due to a high-fat diet regimen is abrogated in ERRα knockout mouse. Overall, this work unravels the role of cholesterol biosynthesis in the induction of an ERRα-dependent mitochondrial program leading to cellular senescence and related pathological alterations.

scholarly journals A cell-penetrating whole molecule antibody targeting intracellular HBx suppresses hepatitis B virus via TRIM21-dependent pathway

Theranostics ◽  
2018 ◽  
Vol 8 (2) ◽  
pp. 549-562 ◽  
Author(s):  
Jun-Fang Zhang ◽  
Hua-Long Xiong ◽  
Jia-Li Cao ◽  
Shao-Juan Wang ◽  
Xue-Ran Guo ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
B Virus ◽  
Cell Penetrating ◽  
A Cell ◽  
Antibody Targeting ◽  
Dependent Pathway

scholarly journals Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

2002 ◽  
Vol 68 (9) ◽  
pp. 4390-4398 ◽  
Author(s):  
S. A. F. T. van Hijum ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Lactobacillus Reuteri ◽  
Bacterial Strains ◽  
Potential Health ◽  
Isolation And Characterization ◽  
A Cell ◽  
Encoding Gene ◽  
First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

Detection of Biogenic Amine Producer Bacteria in a Typical Italian Goat Cheese

2008 ◽  
Vol 71 (1) ◽  
pp. 205-209 ◽  
Author(s):  
SILVIA BONETTA ◽  
SARA BONETTA ◽  
ELISABETTA CARRARO ◽  
JEAN DANIEL COÏSSON ◽  
FABIANO TRAVAGLIA ◽  
...  
Keyword(s):  
Biogenic Amines ◽  
Enterococcus Faecalis ◽  
Biogenic Amine ◽  
Pediococcus Acidilactici ◽  
Bacterial Strains ◽  
Goat Cheese ◽  
Low Concentrations ◽  
Amine Content ◽  
High Concentrations ◽  
Almost All

The aim of this study was to research decarboxylating bacterial strains and biogenic amine content in a typical Italian goat cheese (Robiola di Roccaverano). The study was performed on fresh and ripened samples of goat cheese manufactured from industrial and artisanal producers. Sixty-seven bacterial strains isolated showed decarboxylating activity, and Enterococcus faecalis was the most widespread decarboxylating species in all artisanal and industrial products. Pediococcus acidilactici and Enterococcus malodoratus were also identified as biogenic amine producers in Robiola di Roccaverano cheese. All the E. faecalis strains isolated in this study were able to decarboxylate tyrosine. Tyramine was the most abundant biogenic amine in cheese samples, while histamine was the most widespread. High amounts of these two biogenic amines were found in ripened samples (up to 2,067 mg/kg for tyramine and 1,786 mg/kg for histamine), whereas 2-phenylethylamine and tryptamine were present in almost all ripened cheeses at low concentrations. The detection of strains producing biogenic amines and the high concentrations of tyramine and histamine found in ripened Robiola di Roccaverano could represent a potential risk to the consumer.

Bioenergetics Studies of the Cyanobacterium Anabaena variabilis

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1280-1284 ◽  
Author(s):  
Siegfried Scherer ◽  
Heike Sadowski ◽  
Peter Böger
Keyword(s):  
Electron Transport ◽  
Photosystem Ii ◽  
Cytochrome B ◽  
Anabaena Variabilis ◽  
Electron Donors ◽  
Free System ◽  
Low Concentrations ◽  
Cyanobacterium Anabaena ◽  
A Cell ◽  
C Complex

A cell-free system exhibiting both photophosphorylation (P/2e= 1) and oxidative phosphoryltion (P/O up to 0.8) is described for the cyanobacterium Anabaena variabilis. NADH ant NADPH were found to be equally effective as electron donors for oxidative phosphorylation. Low concentrations of UHDBT, an inhibitor of the cytochrome b/c complex of mitochondria ant loroplasts, were found to inhibit photosystem-II electron transport reactions, but did not affet the cytochrome b6/f-complex of Anabaena. The inhibition by myxothiazol, antimycin and heptyihydroxyquinoline corroborates the hypothesis that both respiration and photosynthesis share the cytochrome b6/f-complex.

scholarly journals THE TIME OF SYNTHESIS AND THE CONSERVATION OF MITOSIS-RELATED PROTEINS IN CULTURED HUMAN AMNION CELLS

1967 ◽  
Vol 34 (1) ◽  
pp. 97-110 ◽  
Author(s):  
Jesse E. Sisken ◽  
Elaina Wilkes
Keyword(s):  
Time Lapse ◽  
Major Effect ◽  
Human Amnion ◽  
Daughter Cells ◽  
Low Concentrations ◽  
Associated Proteins ◽  
A Cell ◽  
Quantitative Analyses ◽  
Related Proteins ◽  
Kinetics Of

p-Fluorophenylalanine (PFPA), an analogue of phenylalanine which may be incorporated into proteins, increases the duration of mitosis. In the present experiments, based upon quantitative analyses of time-lapse cinemicrographic films, brief treatments of cells with PFPA are shown to affect the duration of metaphase in only those cells which enter division during or shortly after treatment. The offspring of cells with prolonged metaphases also tend to have prolonged metaphases. Analyses of the kinetics of the appearance of prolonged metaphases indicate that some protein specifically associated with mitosis is synthesized primarily during a period which corresponds closely to G2. The manner in which the defect is passed on to daughter cells indicates that the protein involved is conserved and reutilized by daughter cells for their subsequent divisions. Comparable experiments performed with low concentrations of puromycin indicate that the major effect of PFPA is due to its incorporation into protein rather than its ability to inhibit protein synthesis. The fact that puromycin-induced effects can also be passed on to daughter cells is interpreted to mean that cells make only specific amounts of some mitosis-associated proteins and that if a cell "inherits" a deficiency in such protein it is not able to compensate for the deficiency.

scholarly journals Fermentative production of Vitamin E tocotrienols in Saccharomyces cerevisiae under cold-shock-triggered temperature control

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Bin Shen ◽  
Pingping Zhou ◽  
Xue Jiao ◽  
Zhen Yao ◽  
Lidan Ye ◽  
...  
Keyword(s):  
Vitamin E ◽  
Temperature Control ◽  
Cold Shock ◽  
Mevalonate Pathway ◽  
Transit Peptide ◽  
High Density ◽  
Temperature Control System ◽  
Fermentative Production ◽  
Dry Cell Weight ◽  
Engineered Yeast

Abstract The diverse physiological functions of tocotrienols have listed them as valuable supplementations to α-tocopherol-dominated Vitamin E products. To make tocotrienols more readily available, tocotrienols-producing S. cerevisiae has been constructed by combining the heterologous genes from photosynthetic organisms with the endogenous shikimate pathway and mevalonate pathway. After identification and elimination of metabolic bottlenecks and enhancement of precursors supply, the engineered yeast can produce tocotrienols at yield of up to 7.6 mg/g dry cell weight (DCW). In particular, proper truncation of the N-terminal transit peptide from the plant-sourced enzymes is crucial. To further solve the conflict between cell growth and tocotrienols accumulation so as to enable high-density fermentation, a cold-shock-triggered temperature control system is designed for efficient control of two-stage fermentation, leading to production of 320 mg/L tocotrienols. The success in high-density fermentation of tocotrienols by engineered yeast sheds light on the potential of fermentative production of vitamin E tocochromanols.

scholarly journals Inhibition of Transferrin Recycling and Endosome Tubulation by Phospholipase A2Antagonists

2001 ◽  
Vol 276 (50) ◽  
pp. 47361-47370 ◽  
Author(s):  
Paul de Figueiredo ◽  
Anne Doody ◽  
Renée S. Polizotto ◽  
Daniel Drecktrah ◽  
Salli Wood ◽  
...  
Keyword(s):  
Cell Surface ◽  
Molecular Mechanism ◽  
Golgi Complex ◽  
Broad Spectrum ◽  
Tubule Formation ◽  
Recycling Endosomes ◽  
Low Concentrations ◽  
A Cell ◽  
Recycling Pathway ◽  
Lines Of Evidence

We report here that a broad spectrum of phospholipase A2(PLA2) antagonists produce a concentration-dependent, differential block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but have no acute affect on Tf uptake from the cell surface. At low concentrations of antagonists (∼1 μm), Tf and TfR accumulated in centrally located recycling endosomes, whereas at higher concentrations (∼10 μm), Tf-TfR accumulated in peripheral sorting endosomes. Several independent lines of evidence suggest that this inhibition of recycling may result from the inhibition of tubule formation. First, BFA-stimulated endosome tubule formation was similarly inhibited by PLA2antagonists. Second, endocytosed tracers were found in larger spherical endosomes in the presence of PLA2antagonists. And third, endosome tubule formation in a cell-free, cytosol-dependent reconstitution system was equally sensitive PLA2antagonists. These results are consistent with the conclusion that endosome membrane tubules are formed by the action of a cytoplasmic PLA2and that PLA2-dependent tubules are involved in intracellular recycling of Tf and TfR. When taken together with previous studies on the Golgi complex, these results also indicate that an intracellular PLA2activity provides a novel molecular mechanism for inducing tubule formation from multiple organelles.

scholarly journals Intra-S-Phase Checkpoint Activation by Direct CDK2 Inhibition

2004 ◽  
Vol 24 (14) ◽  
pp. 6268-6277 ◽  
Author(s):  
Yonghong Zhu ◽  
Carmen Alvarez ◽  
Ronald Doll ◽  
Hirokazu Kurata ◽  
Xiao Min Schebye ◽  
...  
Keyword(s):  
Genomic Stability ◽  
S Phase ◽  
Cell Cycle Checkpoints ◽  
Minichromosome Maintenance ◽  
Checkpoint Activation ◽  
Cdk2 Activity ◽  
A Cell ◽  
Dependent Pathway ◽  
Number Of Cells ◽  
S Phase Checkpoint

ABSTRACT To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.

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