scholarly journals Transcription and redox enzyme activities: comparison of equilibrium and disequilibrium levels in the three-spined stickleback

2013 ◽  
Vol 280 (1755) ◽  
pp. 20122974 ◽  
Author(s):  
M. Nikinmaa ◽  
R. J. S. McCairns ◽  
M. W. Nikinmaa ◽  
K. A. Vuori ◽  
M. Kanerva ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
High Throughput ◽  
Population Differentiation ◽  
Gasterosteus Aculeatus ◽  
De Novo ◽  
Time Lag ◽  
Redox Enzyme ◽  
Activity Data ◽  
Acute Changes

Evolutionary and acclimatory responses require functional variability, but in contrast with mRNA and protein abundance data, most physiological measurements cannot be obtained in a high-throughput manner. Consequently, one must either rely on high-throughput transcriptomic or proteomic data with only predicted functional information, or accept the limitation that most physiological measurements can give fewer data than those provided by transcriptomics or proteomics. We evaluated how transcriptional and redox enzyme activity data agreed with regard to population differentiation (i.e. a system in steady state in which any time lag between transcription, translation and post-translational effects would be irrelevant) and in response to an acute 6°C increase in temperature (i.e. a disequilibrium state wherein translation could not have caught up with transcription) in the three-spined stickleback ( Gasterosteus aculeatus ). Transcriptional and enzyme activity data corresponded well with regard to population differentiation, but less so with regard to acute temperature increase. The data thus suggest that transcriptional and functional measurements can lead to similar conclusions when a biological system is in a steady state. The responses to acute changes must, as has been demonstrated earlier, be based on changes in cellular conditions or properties of existing proteins without significant de novo synthesis of new gene products.

scholarly journals Genomic structure of murine methylmalonyl-CoA mutase: evidence for genetic and epigenetic mechanisms determining enzyme activity

1993 ◽  
Vol 296 (3) ◽  
pp. 663-670 ◽  
Author(s):  
M F Wilkemeyer ◽  
E R Andrews ◽  
F D Ledley
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
Transcription Initiation ◽  
Cultured Cells ◽  
Genomic Structure ◽  
Genetic Regulation ◽  
Regulatory Elements ◽  
Transcription Unit ◽  
Mrna Levels ◽  
Sequence Motifs

Methylmalonyl-CoA mutase (MCM) is a nuclear-encoded mitochondrial matrix enzyme. We have reported characterization of murine MCM and cloning of a murine MCM cDNA and now describe the murine Mut locus, its promoter and evidence for tissue-specific variation in MCM mRNA, enzyme and holo-enzyme levels. The Mut locus spans 30 kb and contains 13 exons constituting a unique transcription unit. A B1 repeat element was found in the 3′ untranslated region (exon 13). The transcription initiation site was identified and upstream sequences were shown to direct expression of a reporter gene in cultured cells. The promoter contains sequence motifs characteristic of: (1) TATA-less housekeeping promoters; (2) enhancer elements purportedly involved in co-ordinating expression of nuclear-encoded mitochondrial proteins; and (3) regulatory elements including CCAAT boxes, cyclic AMP-response elements and potential AP-2-binding sites. Northern blots demonstrate a greater than 10-fold variation in steady-state mRNA levels, which correlate with tissue levels of enzyme activity. However, the ratio of holoenzyme to total enzyme varies among different tissues, and there is no correlation between steady-state mRNA levels and holoenzyme activity. These results suggest that, although there may be regulation of MCM activity at the level of mRNA, the significance of genetic regulation is unclear owning to the presence of epigenetic regulation of holoenzyme formation.

scholarly journals Induction of carnitine acetyltransferase by clofibrate in rat liver

1981 ◽  
Vol 194 (1) ◽  
pp. 249-255 ◽  
Author(s):  
B Mittal ◽  
C K R Kurup
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Time Lag ◽  
Mitochondrial Protein ◽  
Carnitine Acetyltransferase ◽  
Enzyme Protein ◽  
General Protein Synthesis ◽  
Liver And Kidney ◽  
General Protein

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine O-acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.

scholarly journals Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 753
Author(s):  
Miroslav Glasa ◽  
Richard Hančinský ◽  
Katarína Šoltys ◽  
Lukáš Predajňa ◽  
Jana Tomašechová ◽  
...  
Keyword(s):  
High Throughput ◽  
Potato Virus ◽  
Mixed Infection ◽  
High Throughput Sequencing ◽  
Potato Virus Y ◽  
De Novo ◽  
Sweet Pepper ◽  
Complete Sequence ◽  
Genome Region ◽  
Mapping Parameters

In recent years, high throughput sequencing (HTS) has brought new possibilities to the study of the diversity and complexity of plant viromes. Mixed infection of a single plant with several viruses is frequently observed in such studies. We analyzed the virome of 10 tomato and sweet pepper samples from Slovakia, all showing the presence of potato virus Y (PVY) infection. Most datasets allow the determination of the nearly complete sequence of a single-variant PVY genome, belonging to one of the PVY recombinant strains (N-Wi, NTNa, or NTNb). However, in three to-mato samples (T1, T40, and T62) the presence of N-type and O-type sequences spanning the same genome region was documented, indicative of mixed infections involving different PVY strains variants, hampering the automated assembly of PVY genomes present in the sample. The N- and O-type in silico data were further confirmed by specific RT-PCR assays targeting UTR-P1 and NIa genomic parts. Although full genomes could not be de novo assembled directly in this situation, their deep coverage by relatively long paired reads allowed their manual re-assembly using very stringent mapping parameters. These results highlight the complexity of PVY infection of some host plants and the challenges that can be met when trying to precisely identify the PVY isolates involved in mixed infection.

Divergent effects of intravenous GSH and cysteine on renal and hepatic GSH

1992 ◽  
Vol 263 (2) ◽  
pp. R348-R352 ◽  
Author(s):  
S. Aebi ◽  
B. H. Lauterburg
Keyword(s):  
Steady State ◽  
De Novo ◽  
Feedback Inhibition ◽  
Therapeutic Use ◽  
Steady State Concentration ◽  
De Novo Synthesis ◽  
State Concentration ◽  
Cellular Thiol

There is a growing interest in the therapeutic use of sulfhydryls. To assess the effect of glutathione (GSH) and cysteine on the cellular thiol status, thiols were administered intravenously to rats in doses ranging from 1.67 to 8.35 mmol/kg with and without pretreatment with 4 mmol/kg buthionine-[S,R]-sulfoximine (BSO), an inhibitor of GSH synthesis. One hour after administration of 1.67 mmol/kg GSH, the concentration of GSH rose from 5.2 +/- 1.0 to 8.4 +/- 0.9 mumol/g and from 2.5 +/- 0.5 to 3.7 +/- 0.7 mumol/g in liver and kidneys, respectively. After 8.35 mmol/kg, hepatic GSH did not increase further, but renal GSH rose to 6.7 +/- 1.8 mumol/g. Infusion of cysteine increased hepatic GSH to the same extent as intravenous GSH, but renal GSH did not increase after 1.67 mmol/kg and even significantly decreased to 0.6 +/- 0.2 mumol/g after 8.35 mmol/kg. In the presence of BSO, GSH resulted in a significant increase in renal but not hepatic GSH, suggesting that the kidneys take up intact GSH and indicating that the increment in hepatic GSH was due to de novo synthesis. The present data show that hepatic GSH can be markedly increased in vivo by increasing the supply of cysteine. Measurements of hepatic cysteine indicate that up to a concentration of approximately 0.5 mumol/g cysteine is a key determinant of hepatic GSH, such that the physiological steady-state concentration of GSH in the liver appears to be mainly determined by the availability of cysteine. At higher concentrations GSH does not increase further, possibly due to feedback inhibition of GSH synthesis or increased efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Respiration in man during metabolic alkalosis

1962 ◽  
Vol 17 (1) ◽  
pp. 33-37 ◽  
Author(s):  
Daniel J. Stone
Keyword(s):  
Carbon Dioxide ◽  
Steady State ◽  
Lung Function ◽  
Gas Exchange ◽  
Metabolic Alkalosis ◽  
Time Lag ◽  
Respiratory Compensation ◽  
Arterial Ph ◽  
Oral Sodium ◽  
Ventilation Response

A steady state metabolic alkalosis was induced in two subjects over a period of several days utilizing oral sodium bicarbonate in dosages of 50 g/day. The purpose of inducing steady state metabolic alkalosis was to study the effects of such a state on the respiratory center responses to inspired gas mixtures, containing carbon dioxide, and to contrast these results with the control studies. The experiment was so designed that the arterial pH in both subjects tended to return toward normal in the presence of significant increases in blood bicarbonate. Repeated study of ventilation responses with room air and 4% and 6% carbon dioxide in inspired air revealed a definite and significant decrease in ventilation response to carbon dioxide during the periods of steady state alkalosis as compared to the control periods. Normal responses returned after some time lag. A consistent rise in paCOCO2 occurred with alkalosis, thus demonstrating respiratory compensation. In neither subject was total lung function or gas exchange affected by the alkalosis. The experiment was confirmed on several occasions with reproducible results. Note: (With the Research Assistance of Mary Di Lieto) Submitted on May 22, 1961

scholarly journals Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo

1975 ◽  
Vol 146 (1) ◽  
pp. 121-126 ◽  
Author(s):  
E G Fragoulis ◽  
C E Sekeris
Keyword(s):  
Enzyme Activity ◽  
Steroid Hormones ◽  
De Novo ◽  
Steroid Hormone ◽  
Dopa Decarboxylase ◽  
Double Labelling ◽  
Labelling Technique ◽  
Immune Precipitation ◽  
Enzyme Molecules ◽  
Stimulation Of

The activity of the enzyme dopa (3,4-dihydroxyphenylalanine) decarboxylase, present in the epidermis cells of blowfly larvae, increases during the late third instar under the influence of the steroid hormone, ecdysone. By using the double-labelling technique and immune precipitation with univalent antibody to dopa decarboxylase, we demonstrated that the increase in enzyme activity was due to a stimulation of synthesis of enzyme molecules de novo. In this respect, the action of ecdysone is similar to the action of other steroid hormones.

Semiparametric approach to pharmacokinetic-pharmacodynamic data

1989 ◽  
Vol 256 (4) ◽  
pp. R1005-R1010
Author(s):  
D. Verotta ◽  
S. L. Beal ◽  
L. B. Sheiner
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Drug Concentration ◽  
Venous Blood ◽  
Time Lag ◽  
Elimination Rate ◽  
Hysteresis Loops ◽  
Real Data ◽  
Elimination Rate Constant ◽  
Arterial Blood

A semiparametric model for analysis of pharmacokinetic (PK) and pharmacodynamic (PD) data arising from non-steady-state experiments is presented. The model describes time lag between drug concentration in a sampling compartment, e.g., venous blood (Cv), and drug effect (E). If drug concentration at the effect site (Ce) equilibrates with arterial blood concentration (Ca) slower than with Cv, a non-steady-state experiment yields E vs. Cv data describing a counterclockwise hysteresis loop. If Ce equilibrates with Ca faster than with Cv, clockwise hysteresis is observed. To model hysteresis, a parametric model is proposed linking (unobserved) Ca to Cv with elimination rate constant kappa ov and also linking Ca to Ce with elimination rate constant kappa oe. When kappa oe is greater than (or less than) kappa ov clockwise (or counterclockwise) hysteresis occurs. Given kappa oe and kappa ov, numerical (constrained) deconvolution is used to obtain the disposition function of the arterial compartment (Ha), and convolution is used to calculate Ce given Ha. The values of kappa oe and kappa ov are chosen to collapse the hysteresis loops to single curves representing the Ce-E (steady-state) concentration-response curve. Simulations, and an application to real data, are reported.

scholarly journals Inferring differential subcellular localisation in comparative spatial proteomics using BANDLE

2021 ◽  
Author(s):  
Oliver M. Crook ◽  
Colin T. R. Davies ◽  
Laurent Gatto ◽  
Paul D.W. Kirk ◽  
Kathryn S. Lilley
Keyword(s):  
Steady State ◽  
High Throughput ◽  
Statistical Power ◽  
Subcellular Localisation ◽  
Type I ◽  
Disease States ◽  
Proteomic Data ◽  
Rational Drug ◽  
Context Specific ◽  
Type Ii Errors

AbstractThe steady-state localisation of proteins provides vital insight into their function. These localisations are context specific with proteins translocating between different sub-cellular niches upon perturbation of the subcellular environment. Differential localisation provides a step towards mechanistic insight of subcellular protein dynamics. Aberrant localisation has been implicated in a number of pathologies, thus differential localisation may help characterise disease states and facilitate rational drug discovery by suggesting novel targets. High-accuracy high-throughput mass spectrometry-based methods now exist to map the steady-state localisation and re-localisation of proteins. Here, we propose a principled Bayesian approach, BANDLE, that uses these data to compute the probability that a protein differentially localises upon cellular perturbation, as well quantifying the uncertainty in these estimates. Furthermore, BANDLE allows information to be shared across spatial proteomics datasets to improve statistical power. Extensive simulation studies demonstrate that BANDLE reduces the number of both type I and type II errors compared to existing approaches. Application of BANDLE to datasets studying EGF stimulation and AP-4 dependent localisation recovers well studied translocations, using only two-thirds of the provided data. Moreover, we implicate TMEM199 with AP-4 dependent localisation. In an application to cytomegalovirus infection, we obtain novel insights into the rewiring of the host proteome. Integration of high-throughput transcriptomic and proteomic data, along with degradation assays, acetylation experiments and a cytomegalovirus interactome allows us to provide the functional context of these data.

Nucleation rates and the kinetics of particle growth II. The birth and death process

1964 ◽  
Vol 277 (1369) ◽  
pp. 167-182 ◽  
Keyword(s):  
Steady State ◽  
Homogeneous Nucleation ◽  
Time Lag ◽  
Particle Growth ◽  
Statistical Problem ◽  
Death Process ◽  
Birth And Death Process ◽  
Growth Mechanisms ◽  
Nucleation Kinetics ◽  
Kinetics Of

The model considered in part I is generalized to include growth mechanisms in which the chemical reaction which proceeds at the particle-atm osphere interface is reversible, so that molecules may evaporate from a particle as well as condense upon it. The Becker-Döring-Zeldovich-Frenkel theory of homogeneous nucleation kinetics is then reviewed in the light of the known statistical problem of the birth -and -death process, and an improved approximation is introduced which significantly alters the calculated results. Both steady-state nucleation kinetics and the time lag problem are discussed.

scholarly journals Improved Fluorescence Methods for High-Throughput Protein Formulation Screening

2018 ◽  
Vol 23 (6) ◽  
pp. 516-528 ◽  
Author(s):  
Yangjie Wei ◽  
Nicholas R. Larson ◽  
Siva K. Angalakurthi ◽  
C. Russell Middaugh
Keyword(s):  
Steady State ◽  
High Throughput ◽  
Protein Formulation ◽  
Formulation Development ◽  
High Concentration ◽  
Direct Optimization ◽  
Biophysical Characterization ◽  
Formulation Optimization ◽  
Long Term Storage ◽  
High Protein Concentration

The goal of protein formulation development is to identify optimal conditions for long-term storage. Certain commercial conditions (e.g., high protein concentration or turbid adjuvanted samples) impart additional challenges to biophysical characterization. Formulation screening studies for such conditions are usually performed using a simplified format in which the target protein is studied at a low concentration in a clear solution. The failure of study conditions to model the actual formulation environment may cause a loss of ability to identify the optimal condition for target proteins in their final commercial formulations. In this study, we utilized a steady-state/lifetime fluorescence-based, high-throughput platform to develop a general workflow for direct formulation optimization under analytically challenging but commercially relevant conditions. A high-concentration monoclonal antibody (mAb) and an Alhydrogel-adjuvanted antigen were investigated. A large discrepancy in screening results was observed for both proteins under these two different conditions (simplified and commercially relevant). This study demonstrates the feasibility of using a steady-state/lifetime fluorescence plate reader for direct optimization of challenging formulation conditions and highlights the importance of performing formulation optimization under commercially relevant conditions.

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