Sugar shock: Probing Streptococcus pyogenes metabolism through bioluminescence imaging

Streptococcus pyogenes (S. pyogenes) can thrive in its host during an infection, and, as a result, it must be able to respond to external stimuli and available carbon sources. The pre-clinical use of engineered pathogens capable of constitutive light production may provide real-time information on microbial-specific metabolic processes. Here we mapped the central metabolism of a luxABCDE-modified S. pyogenes Xen20 (Strep. Xen20) to its de novo synthesis of luciferase substrates as assessed by the rate of light production in response to different environmental triggers. Previous characterization predicted that the lux operon was under the myo-inositol iolE promotor. Here we show that supplementation with myo-inositol generated increased Xen20 luminescence. Surprisingly, when supplemented with infection-relevant carbon sources, such as glucose or glycine, light production was diminished. This was presumably due to the scavenging of pyruvate by L-lactate dehydrogenase (LDH). Inhibition of LDH by its inhibitor, oxamate, partially restored luminescent signal in the presence of glucose, presumably by allowing the resulting pyruvate to proceed to acetyl-coenzyme A (CoA). This phenomenon appeared specific to the lactic acid bacterial metabolism as glucose or glycine did not reduce signal in an analogous luxABCDE-modified Gram-positive pathogen, Staph. Xen29. The Strep. Xen20 cells produced light in a concentration-dependent manner, inversely related to the amount of glucose present. Taken together, our measures of microbial response could provide new information regarding the responsiveness of S. pyogenes metabolism to acute changes in its local environments and cellular health.

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Influence of ammonia concentration on [15N]ammonia uptake andde novosynthesis of different amino acids by mixed rumen microorganisms from the sheep rumenin vitro

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200003677 ◽

1999 ◽

Vol 1999 ◽

pp. 212-212 ◽

Cited By ~ 1

Author(s):

C. Atasoglu ◽

C.J. Newbold ◽

R.J. Wallace

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

De Novo ◽

Ammonia Concentration ◽

Dependent Manner ◽

Microbial Protein ◽

Concentration Dependent Manner ◽

Rumen Microorganisms ◽

Ammonia Uptake

Ammonia is thought to be the main source of nitrogen for protein synthesis by the rumen microorganisms, but peptides and amino acids derived from protein degradation are also incorporated into microbial protein. Recent experiments carried out by Atasogluet al.(1998) demonstrated that preformed amino acids decrease the uptake of ammonia into microbial protein and microbial amino acids in a concentration-dependent manner. However, little is known about how rumen ammonia concentrations affect ammonia uptake into microbial protein. The present study was undertaken to determine the influence of rumen ammonia concentrations on ammonia incorporation andde novosynthesis of individual amino acids by the mixed rumen microorganismsin vitro.

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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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Interaction of Graphene Oxide Modified with Linear and Branched PEG with Monocytes Isolated from Human Blood

Nanomaterials ◽

10.3390/nano12010126 ◽

2021 ◽

Vol 12 (1) ◽

pp. 126

Author(s):

Pavel Khramtsov ◽

Maria Bochkova ◽

Valeria Timganova ◽

Anton Nechaev ◽

Sofya Uzhviyuk ◽

...

Keyword(s):

Graphene Oxide ◽

Immune Cells ◽

Human Peripheral Blood ◽

Oxide Surface ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Composition And Structure ◽

Luminescent Signal ◽

Ros Formation ◽

The Impact

Multiple graphene-based therapeutics have recently been developed, however potential risks related to the interaction between nanomaterials and immune cells are still poorly understood. Therefore, studying the impact of graphene oxide on various populations of immune cells is of importance. In this work, we aimed to investigate the effects of PEGylated graphene oxide on monocytes isolated from human peripheral blood. Graphene oxide nanoparticles with lateral sizes of 100–200 nm and 1–5 μm were modified with linear and branched PEG (GO-PEG). Size, elemental composition, and structure of the resulting nanoparticles were characterized. We confirmed that PEG was successfully attached to the graphene oxide surface. The influence of GO-PEG on the production of reactive oxygen species (ROS), cytokines, phagocytosis, and viability of monocytes was studied. Uptake of GO-PEG by monocytes depends on PEG structure (linear or branched). Branched PEG decreased the number of GO-PEG nanoparticles per monocyte. The viability of monocytes was not altered by co-cultivation with GO-PEG. GO-PEG decreased the phagocytosis of Escherichia coli in a concentration-dependent manner. ROS formation by monocytes was determined by measuring luminol-, lucigenin-, and dichlorodihydrofluorescein-dependent luminescence. GO-PEG decreased luminescent signal probably due to inactivation of ROS, such as hydroxyl and superoxide radicals. Some types of GO-PEG stimulated secretion of IL-10 by monocytes, but this effect did not correlate with their size or PEG structure.

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Stimulation of Tetrahydrobiopterin Synthesis by Basic Fibroblast Growth Factor in Vascular Endothelial Cells

Pteridines ◽

10.1515/pteridines.2003.14.1.9 ◽

2003 ◽

Vol 14 (1) ◽

pp. 9-12 ◽

Cited By ~ 2

Author(s):

Shunichi Shimizu ◽

Yoshiyuki Miyasaka ◽

Shinichiro Yamamoto ◽

Masakazu Ishii ◽

Yuji Kiuchi

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Basic Fibroblast Growth Factor ◽

De Novo ◽

Mrna Level ◽

Mrna Levels ◽

Dependent Manner ◽

Fibroblast Growth ◽

Concentration Dependent Manner

Abstract The purpose of this study was to examine whether basic fibroblast growth factor (bFGF) stimulates tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells. BH4 content was determined by oxidation under acidic conditions as biopterin and analysed with reversed-phase high Performance liquid chromatography. Measurement of the mRNA level of QTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of the de novo pathway of BH4 synthesis. The addition of bFGF to endothelial cells increased the BH4 content and GTPCH mRNA levels in an incubation period- and a concentration-dependent manner. 2,4-Diamino-6- hydroxypyrimidine, an inhibitor of GTPCH, strongly reduced the bFGF-induced increase in BH4 content. These findings suggest that bFGF stimulates BH4 synthesis via a de novo pathway with the induction of GTPCH.

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Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.266.4.l455 ◽

1994 ◽

Vol 266 (4) ◽

pp. L455-L460 ◽

Cited By ~ 1

Author(s):

D. K. Nakayama ◽

D. A. Geller ◽

M. Di Silvio ◽

G. Bloomgarden ◽

P. Davies ◽

...

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Pulmonary Artery ◽

De Novo ◽

Interleukin 1 ◽

No Production ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Pulmonary Artery Smooth Muscle ◽

Inducible Nitric Oxide

We recently reported (Am. J. Respir. Cell Mol. Biol. 7: 471-476, 1992) that a mixture of lipopolysaccharide (LPS) and cytokines produced a time-dependent increase in mRNA and protein expression of inducible nitric oxide synthase (iNOS) in cultured rat pulmonary artery smooth muscle cells (RPASM). In the current study we extend observations on regulation of iNOS in RPASM by showing that de novo synthesis of tetrahydrobiopterin (BH4) is critical for LPS and cytokine-induced NO production. A mixture of LPS and the cytokines gamma-interferon, interleukin-1 beta, and tumor necrosis factor-alpha increased steady-state levels of mRNA of GTP-cyclohydrolase-I (GTP-CH), the rate-limiting enzyme in BH4 biosynthesis. Levels of mRNA to GTP-CH became detectable by 4 h, with further increases at 24 h by Northern blot analysis and reverse-transcriptase polymerase chain reaction. Total intracellular biopterin levels, undetectable under basal conditions, increased after 24 h exposure to LPS and cytokines (to 32.3 +/- 0.8 pmol/mg protein). LPS and cytokine-induced NO production, determined by nitrite concentrations in the medium, was decreased in a concentration-dependent manner by the GTP-CH inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP) at 24 h. DAHP also inhibited completely the LPS- and cytokine-induced accumulation of intracellular biopterins. Sepiapterin, which supplies BH4 through a salvage pathway independent of GTP-CH, reversed the effect of DAHP on LPS and cytokine-induced NO production.(ABSTRACT TRUNCATED AT 250 WORDS)

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Carbon Sources for Yeast Growth as a Precondition of Hydrogen Peroxide Induced Hormetic Phenotype

International Journal of Microbiology ◽

10.1155/2015/697813 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

Ruslana Vasylkovska ◽

Natalia Petriv ◽

Halyna Semchyshyn

Keyword(s):

Hydrogen Peroxide ◽

Growth Medium ◽

Carbon Sources ◽

Carbon Substrate ◽

Dependent Manner ◽

Yeast Growth ◽

Concentration Dependent Manner ◽

Reproductive Ability ◽

The Relationship ◽

Hormetic Response

Hormesis is a phenomenon of particular interest in biology, medicine, pharmacology, and toxicology. In this study, we investigated the relationship between H2O2-induced hormetic response inS. cerevisiaeand carbon sources in yeast growth medium. In general, our data indicate that (i) hydrogen peroxide induces hormesis in a concentration-dependent manner; (ii) the effect of hydrogen peroxide on yeast reproductive ability depends on the type of carbon substrate in growth medium; and (iii) metabolic and growth rates as well as catalase activity play an important role in H2O2-induced hormetic response in yeast.

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Responsiveness of human neutrophils to interleukin-4: induction of cytoskeletal rearrangements, de novo protein synthesis and delay of apoptosis

Biochemical Journal ◽

10.1042/bj3250147 ◽

1997 ◽

Vol 325 (1) ◽

pp. 147-153 ◽

Cited By ~ 87

Author(s):

Denis GIRARD ◽

Robert PAQUIN ◽

André D. BEAULIEUL

Keyword(s):

Protein Synthesis ◽

De Novo ◽

Biological Activities ◽

Interleukin 4 ◽

Human Neutrophils ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cytoskeletal Rearrangements ◽

First Time ◽

De Novo Protein Synthesis

Interleukin-4 (IL-4) and IL-13 are cytokines that share many biological activities. We have previously demonstrated that IL-13 affects a number of neutrophil responses, and here we extend our observations to IL-4. We present, for the first time, direct evidence for the presence of functional IL-4 receptors on human neutrophils. We report that IL-4 induces RNA synthesis in a concentration-dependent manner and, based on observations of the induction of morphological cell shape changes and spreading onto glass, we demonstrate that IL-4 activates neutrophil cytoskeletal rearrangements. We further show that IL-4 is a potent activator of de novo protein synthesis in neutrophils, and we identify by microsequencing one of these proteins as the cytoskeletal protein actin. We were also able to demonstrate for the first time that actin is cleaved into at least two fragments of ∼ 30 kDa (pI 5.4) and ∼ 25 kDa (pI 5.0) in neutrophils. Finally, we report that IL-4 delays neutrophil apoptosis, as assessed by morphological observations from cytocentrifuge preparations, as well as by measurement of differences in staining by flow cytometry with both propidium iodide and Hoechst reagent. Taken together, we conclude that IL-4 is a more potent neutrophil agonist than previously believed. We discuss the possibility that the induction of the de novo synthesis of actin by IL-4 is related to the mechanism by which this cytokine delays apoptosis; in addition, the cleavage of this protein is likely to contribute to the apoptotic process.

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Enhancement of cortisol-induced SAA1 transcription by SAA1 in the human amnion

Journal of Molecular Endocrinology ◽

10.1530/jme-18-0263 ◽

2019 ◽

Vol 62 (4) ◽

pp. 149-158 ◽

Cited By ~ 3

Author(s):

Yi Lu ◽

Wang-sheng Wang ◽

Yi-kai Lin ◽

Jiang-wen Lu ◽

Wen-jiao Li ◽

...

Keyword(s):

De Novo ◽

Acute Phase Protein ◽

Fetal Membranes ◽

Dependent Manner ◽

Forward Induction ◽

Concentration Dependent Manner ◽

Human Amnion ◽

Stat3 Phosphorylation ◽

Phase Protein ◽

Serum Amyloid A1

Our previous studies have demonstrated that human fetal membranes are capable of de novo synthesis of serum amyloid A1 (SAA1), an acute phase protein of inflammation, wherein SAA1 may participate in parturition by inducing a number of inflammation mediators including interleukine-1β, interleukine-6 and prostaglandin E2. However, the regulation of SAA1 expression in the fetal membranes remains largely unknown. In the current study, we examined the regulation of SAA1 expression by cortisol, a crucial steroid produced locally in the fetal membranes at parturition, and the interaction between cortisol and SAA1 in the feed-forward induction of SAA1 expression in human amnion fibroblasts. Results showed that cortisol-induced SAA1 expression in a concentration-dependent manner, which was greatly enhanced by SAA1 despite modest induction of SAA1 expression by itself. Mechanism studies revealed that the induction of SAA1 expression by cortisol and SAA1 was blocked by either the transcription factor STAT3 antagonist AZD0530 or siRNA-mediated knockdown of STAT3. Furthermore, cortisol- and SAA1-induced STAT3 phosphorylation in a sequential order with the induction by SAA1 preceding the induction by cortisol. However, combination of cortisol and SAA1 failed to further intensify the phosphorylation of STAT3. Consistently, cortisol and SAA1 increased the enrichment of STAT3 at the SAA1 promoter. Taking together, this study has demonstrated that cortisol and SAA1 can reinforce each other in the induction of SAA1 expression through sequential phosphorylation of STAT3. The enhancement of cortisol-induced SAA1 expression by SAA1 may lead to excessive SAA1 accumulation resulting in parturition-associated inflammation in the fetal membranes.

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Activation of the adhesion GPCR GPR133 (ADGRD1) by antibodies targeting the N-terminus

10.1101/2021.09.13.460139 ◽

2021 ◽

Author(s):

Gabriele Stephan ◽

Joshua D. Frenster ◽

Ines Liebscher ◽

Dimitris G. Placantonakis

Keyword(s):

De Novo ◽

Receptor Activation ◽

Normal Brain ◽

Dependent Manner ◽

Antibody Treatment ◽

Concentration Dependent Manner ◽

N Terminus ◽

Adhesion Gpcr ◽

G Protein Coupled ◽

Camp Levels

We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein-coupled receptor (aGPCR) whose canonical signaling raises cytosolic cAMP, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We showed that dissociation of autoproteolytically generated N-terminal and C-terminal fragments (NTF and CTF) of GPR133 at the plasma membrane promotes receptor activation and increases signaling. Toward developing biologics modulating GPR133 function, we tested antibodies against the N-terminus of GPR133 for effects on receptor signaling. Treatment of HEK293T cells overexpressing GPR133 with such antibodies increased cAMP levels in a concentration-dependent manner. Analysis of supernatants following antibody treatment revealed complexes of the antibodies with the autoproteolytically cleaved NTF of GPR133. Cells expressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. The antibody-mediated stimulation of wild-type GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that NTF-CTF dissociation promotes receptor activation and signaling.

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Inhibition of Rat Platelet Aggregation by Mycalolide-B, a Novel Inhibitor of Actin Polymerization with a Different Mechanism of Action from Cytochalasin-D

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614955 ◽

1998 ◽

Vol 79 (03) ◽

pp. 614-619 ◽

Cited By ~ 6

Author(s):

Fumitoshi Asai ◽

Shinya Saito ◽

Hiroshi Ozaki ◽

Nobuhiro Fusetani ◽

Hideaki Karaki ◽

...

Keyword(s):

Platelet Aggregation ◽

Mechanism Of Action ◽

Actin Polymerization ◽

De Novo ◽

Platelet Rich Plasma ◽

Cytochalasin D ◽

Dependent Manner ◽

Clot Retraction ◽

Concentration Dependent Manner ◽

Induced Aggregation

SummaryIn vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 μM) but was inhibited by higher concentrations (3 and 10 μM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 μM). Potentiation of ADP-induced aggregation by MB (0.3 μM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 μM). In contrast, cytochalasin-D (CD) at 3 μM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 μg/ml) or ADP (10 μM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 μM) and CD (3 μM) abolished both ADP (10 μM)- and collagen (3 μg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 μM) had no effects on intracellular Ca2+ concentrations in ADP (10 μM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 μM) was inhibited by MB (0.3-3 μM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 μM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.

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