Kinetics of the cellular intake of a gene expression inducer at high concentrations

2015 ◽  
Vol 11 (9) ◽  
pp. 2579-2587 ◽  
Author(s):  
Huy Tran ◽  
Samuel M. D. Oliveira ◽  
Nadia Goncalves ◽  
Andre S. Ribeiro
Keyword(s):  
Gene Expression ◽  
Single Event ◽  
Transcription Activity ◽  
High Concentrations ◽  
Kinetics Of

Characterization of the cellular intake kinetics of a lactose analogue fromin vivosingle-event measurements of transcription activity.

scholarly journals In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells

2016 ◽  
Vol 31 (6) ◽  
pp. 540-550 ◽  
Author(s):  
Kevin A. Feeney ◽  
Marrit Putker ◽  
Marco Brancaccio ◽  
John S. O’Neill
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Circadian Gene ◽  
Cultured Fibroblasts ◽  
High Concentrations ◽  
Lower Temperature ◽  
Kinetics Of

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.

scholarly journals Characterization of the Binding of Adenosine Diphosphate to Human Platelet Membranes

1977 ◽  
Author(s):  
C. Legrand ◽  
B. Bauvois ◽  
J. P. Caen
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Adenosine Diphosphate ◽  
Affinity Constant ◽  
Membrane Bound ◽  
High Concentrations ◽  
Kinetics Of

ADP-mediated platelet aggregation is a routinely employed test but its mechanism is poorly understood. The aim of this study was to compare the binding of ADP to plasma membranes isolated from normal platelets and thrombasthenic platelets (which do not aggregate with ADP). Binding of ADP to isolated membranes was assayed by incubation with 14C-ADP followed by Mill i pore filtration. In standard conditions, 14C-ADP was not transformed and non specific binding represented lessthan 3 % of the total binding. Using 1 μM 14C-ADP, the binding has been shown to be a rapid (t 1/2 = 2 mn 30 sec), saturable and reversible phenomenon at 37° C. The existence of a major population of binding sites, with an affinity constant Ka = 0.43 (+ 0.1) χ 106M-1, has been demonstrated. The kinetics of the binding was normal with membranes Tsolated from the platelets of 4 thrombasthenic patients and the affinity constant, when determined, was in the normal range. Dissociation of the membrane-bound 14C-ADP occurred rapidly at 37° C (t l/2c≃3mn) when samples were diluted enough (dilution 1 : 100 was currently employed) to avoid rebinding of the radioligand. Accelerated dissociation (t 1/2 ≃ 1 mn) was observed when the dilution was performed in the presence of an excess of unlabeled ADP, suggesting the existence of negatively cooperative site-site interactions among the ADP binding sites. This effect was only observed at high concentrations of ADP (> 10–5M) and its eventual role in vivo remains to be established. Two thrombasthenic membrane preparations studied in the same way dissociated as did the control membranes.

scholarly journals Proteases: History, discovery, and roles in health and disease

2019 ◽  
Vol 294 (5) ◽  
pp. 1643-1651 ◽  
Author(s):  
Judith S. Bond
Keyword(s):  
Proteolytic Enzymes ◽  
Structural Characteristics ◽  
Research Area ◽  
History Of ◽  
Health And Disease ◽  
Regulation Mechanisms ◽  
American Society ◽  
Kinetics Of

The Journal of Biological Chemistry (JBC) has been a major vehicle for disseminating and recording the discovery and characterization of proteolytic enzymes. The pace of discovery in the protease field accelerated during the 1971–2010 period that Dr. Herb Tabor served as the JBC's editor-in-chief. When he began his tenure, the fine structure and kinetics of only a few proteases were known; now thousands of proteases have been characterized, and over 600 genes for proteases have been identified in the human genome. In this review, besides reflecting on Dr. Tabor's invaluable contributions to the JBC and the American Society for Biochemistry and Molecular Biology (ASBMB), I endeavor to provide an overview of the extensive history of protease research, highlighting a few discoveries and roles of proteases in vivo. In addition, metalloproteinases, particularly meprins of the astacin family, will be discussed with regard to structural characteristics, regulation, mechanisms of action, and roles in health and disease. Proteases and protein degradation play crucial roles in living systems, and I briefly address future directions in this highly diverse and thriving research area.

In vivo imaging of herpes simplex virus type 1 thymidine kinase gene expression: early kinetics of radiolabelled FIAU

2000 ◽  
Vol 27 (3) ◽  
pp. 283-291 ◽  
Author(s):  
Roland Haubner ◽  
Norbert Avril ◽  
Petros A. Hantzopoulos ◽  
Bernd Gansbacher ◽  
Markus Schwaiger
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
In Vivo Imaging ◽  
Thymidine Kinase Gene ◽  
Kinase Gene ◽  
Simplex Virus ◽  
Kinetics Of

scholarly journals Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases

2015 ◽  
Vol 59 (11) ◽  
pp. 6741-6748 ◽  
Author(s):  
Meha P. Patel ◽  
Bartlomiej G. Fryszczyn ◽  
Timothy Palzkill
Keyword(s):  
Antibiotic Resistance ◽  
Adaptive Mutation ◽  
Common Mechanism ◽  
Antibiotic Hydrolysis ◽  
The Stability ◽  
Kinetics Of ◽  
M 14

ABSTRACTThe widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of β-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active site-associated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 β-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extended-spectrum β-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of β-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levelsin vivoand protein stabilityin vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in β-lactamase evolution and a common mechanism in protein evolution.

scholarly journals Characterization of the Human Artemis Promoter by Heterologous Gene Expression In Vitro and In Vivo

2011 ◽  
Vol 30 (10) ◽  
pp. 751-761 ◽  
Author(s):  
Megan M. Multhaup ◽  
Sweta Gurram ◽  
Kelly M. Podetz-Pedersen ◽  
Andrea D. Karlen ◽  
Debra L. Swanson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Heterologous Gene

scholarly journals Characterization of the Catalytic Activities of the PhoQ Histidine Protein Kinase of Salmonella entericaSerovar Typhimurium

2001 ◽  
Vol 183 (5) ◽  
pp. 1787-1791 ◽  
Author(s):  
Martin Montagne ◽  
Alexandre Martel ◽  
Hervé Le Moual
Keyword(s):  
Escherichia Coli ◽  
Phosphatase Activity ◽  
Divalent Cations ◽  
Histidine Protein Kinase ◽  
Catalytic Activities ◽  
Serovar Typhimurium ◽  
High Concentrations ◽  
Kinetics Of ◽  
Autokinase Activity

ABSTRACT Studies of Escherichia coli membranes that were highly enriched in the Salmonella enterica serovar Typhimurium PhoQ protein showed that the presence of ATP and divalent cations such as Mg2+, Mn2+, Ca2+, or Ba2+ resulted in PhoQ autophosphorylation. However, when Mg2+ or Mn2+was present at concentrations higher than 0.1 mM, the kinetics of PhoQ autophosphorylation were strongly biphasic, with a rapid autophosphorylation phase followed by a slower dephosphorylation phase. A fusion protein lacking the sensory and transmembrane domains retained the autokinase activity but could not be dephosphosphorylated when Mg2+ or Mn2+ was present at high concentrations. The instability of purified [32P]phospho-PhoP in the presence of PhoQ-containing membranes indicated that PhoQ also possesses a phosphatase activity. The PhoQ phosphatase activity was stimulated by increasing the Mg2+ concentration. These data are consistent with a model in which Mg2+ binding to the sensory domain of PhoQ coordinately regulates autokinase and phosphatase activities.

scholarly journals Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

2002 ◽  
Vol 70 (6) ◽  
pp. 3080-3084 ◽  
Author(s):  
Bhavna G. Gordhan ◽  
Debbie A. Smith ◽  
Heidi Alderton ◽  
Ruth A. McAdam ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Auxotrophic Mutant ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

scholarly journals c-Myc inhibition is critical for HSC expansion and Rad51 expression

2018 ◽  
Author(s):  
Merve Aksoz ◽  
Esra Albayrak ◽  
Galip Servet Aslan ◽  
Raife Dilek Turan ◽  
Lamia Yazgi Alyazici ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Nos Inhibitor ◽  
Kinetics Of

c-Myc plays a major role in the maintenance of glycolytic metabolism and hematopoietic stem cell (HSC) quiescence. Targeting modulators of HSC quiescence and metabolism could lead to HSC cell cycle entry with concomitant expansion. Here we show that c-Myc inhibitor 10074-G5 treatment leads to 2-fold increase in murine LSKCD34low HSC compartment post 7 days. In addition, c-Myc inhibition increases CD34+ and CD133+ human HSC number. c-Myc inhibition leads to downregulation of glycolytic and cyclin-dependent kinase inhibitor (CDKI) gene expression ex vivo and in vivo. In addition, c-Myc inhibition upregulates major HDR modulator Rad51 expression in hematopoietic cells. Besides, c-Myc inhibition does not alter proliferation kinetics of endothelial cells, fibroblasts or adipose derived mesenchymal stem cells, however; it limits bone marrow derived mesenchymal stem cell proliferation. We further demonstrate that a cocktail of c-Myc inhibitor 10074-G5 along with tauroursodeoxycholic acid (TUDCA) and i-NOS inhibitor L-NIL provides a robust HSC maintenance and expansion ex vivo as evident by induction of all stem cell antigens analyzed. Intriguingly, the cocktail of c-Myc inhibitor 10074-G5, TUDCA and L-NIL improves HDR related gene expression. These findings provide tools to improve ex vivo HSC maintenance and expansion, autologous HSC transplantation and gene editing through modulation of HSC glycolytic and HDR pathways.

scholarly journals Transcriptional signatures of somatic neoblasts and germline cells in Macrostomum lignano

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Magda Grudniewska ◽  
Stijn Mouton ◽  
Daniil Simanov ◽  
Frank Beltman ◽  
Margriet Grelling ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Model Organism ◽  
Proliferating Cells ◽  
Macrostomum Lignano ◽  
Germline Cells

The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea. However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39, Mlig-rrm1, Mlig-rpa3, Mlig-cdk1, and Mlig-h2a, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.

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