scholarly journals The regulatory principles of glycolysis in erythrocytes in vivo and in vitro. A minimal comprehensive model describing steady states, quasi-steady states and time-dependent processes

1976 ◽  
Vol 154 (2) ◽  
pp. 449-469 ◽  
Author(s):  
T A. Rapoport ◽  
R Heinrich ◽  
S M. Rapoport
Keyword(s):  
Steady State ◽  
Atp Synthesis ◽  
Steady States ◽  
Time Dependent ◽  
Glycolytic Flux ◽  
Stable Steady State ◽  
Time Interval ◽  
Fructose 1,6 Bisphosphate

A simple mathematical model for glycolysis in erythrocytes is presented which takes into account ATP synthesis and consumption. The system is described by four ordinary differential equations. Conditions in vivo are described by a stable steady state. The model predicts correctly the metabolite concentrations found in vivo. The parameters involved are in agreement with data on the separate steps. The metabolite changes found in pyruvate kinase-deficient erythrocytes and the species variations among erythrocytes from different animals are described satisfactorily. The roles of the enzymes in the control of metabolites and glycolytic flux are expressed in the form of a control matrix and control strengths [R. Heinrich & T.A. Rapoport (1974) Eur. J. Biochem. 42, 89-95] respectively. Erythrocytes from various species are shown to be adapted to a maximal ATP-consumption rate. The calculated eigenvalues reveal the pronounced time-hierarchy of the glycolytic reactions. Owing to the slowness of the 2,3-bisphospho-glycerate phosphatase reaction, quasi-steady states occur during the time-interval of about 0.5-2h incubation, which are defined by perturbed 2,3-bisphosphoglycerate concentrations. The theoretical predictions agree with experimental data. In the quasi-steady state the flux control is exerted almost entirely by the hexokinase-phosphofructokinase system. The model describes satisfactorily the time-dependent changes after addition of glucose to starved erythrocytes. The theoretical consequences are discussed of the conditions in vitro with lactate accumulation and the existence of a time-independent conservation quantity for the oxidized metabolites. Even in this closed system quasi-steady states occur which are characterized by approximately constant concentrations of all glycolytic metabolites except for the accumulation of lactate, fructose 1,6-bisphosphate and triose phosphate.

Oxidative phosphorylation system during steady-state hypoxia in the dog brain

1990 ◽  
Vol 68 (6) ◽  
pp. 2527-2535 ◽  
Author(s):  
S. Nioka ◽  
D. S. Smith ◽  
B. Chance ◽  
H. V. Subramanian ◽  
S. Butler ◽  
...  
Keyword(s):  
Steady State ◽  
Oxidative Phosphorylation ◽  
Maximum Velocity ◽  
Atp Synthesis ◽  
Reciprocal Relationship ◽  
Phosphorylation Potential ◽  
Sagittal Sinus ◽  
Dog Brain

The relationship between biochemical and physiological responses and tissue O2 during hypoxia was investigated in vivo in the dog brain by 31P nuclear magnetic resonance (NMR) spectroscopy. Our findings demonstrate how ATP synthesis in the brain can be maintained during hypoxia because of compensatory changes in NADH, ADP, and Pi. Eleven beagle dogs were anesthetized and mechanically ventilated, and a steady-state graded hypoxia was induced by decreasing the fraction of inspired O2 (FIO2) stepwise at 20-min intervals. Biochemical metabolites were measured using 31P-NMR and fluorescence spectroscopy. When sagittal sinus O2 partial pressure (PVO2) had decreased to 15 Torr, NADH increased by 30%, Pi increased by 50%, and phosphocreatine (PCr) decreased by 20%. In contrast, ATP remained constant. There was a 10% increase in ADP in dogs that maintained a steady temperature, but ADP decreased by as much as 30% in dogs in which body temperature decreased with the falling PVO2. PCr/Pi was logarithmically related to the phosphorylation potential during steady-state hypoxia. Compensation for the O2 lack is attributed to increases in ADP, Pi, and NADH as a result of the reciprocal relationship of the Michaelis-Menten equation. If the Michaelis-Menten constants (Km) of ADP, Pi, and O2 are the same as determined in vitro in mitochondria, the minimum brain cytosolic O2 capable of maintaining a steady-state ATP is near its Km (0.1 Torr) at a PVO2 of 7.5 Torr. At this critical O2 level, PCr/Pi is 0.9, intracellular pH is 6.75, phosphorylation potential is 38.5 mM-1, and the calculated maximum velocity of ATP formation by oxidative phosphorylation is 55% of normal.

scholarly journals A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux

2020 ◽  
Author(s):  
Alvaro D. Ortega ◽  
Vakil Takhaveev ◽  
Silke Bonsing-Vedelaar ◽  
Yi Long ◽  
Neus Mestre-Farràs ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Hammerhead Ribozyme ◽  
Single Cells ◽  
Glycolytic Flux ◽  
Metabolic Heterogeneity ◽  
Fructose 1,6 Bisphosphate ◽  
Glycolytic Rate ◽  
Selection Of

ABSTRACTMetabolic heterogeneity, the occurrence of different metabolic phenotypes among cells, represents a key challenge in health and biotechnology. To unravel its molecular basis, tools probing metabolism of single cells are needed. While RNA devices harbor huge potential for the development of such tools, until today, it is challenging to create in vivo-functional sensors for any given metabolite. Here, we developed from scratch an RNA-based sensor for fructose-1,6-bisphosphate (FBP), a doubly phosphorylated intermediate of glycolysis. Starting from in vitro selection of an RNA aptamer and its structural analyses, we developed libraries of RNA-based regulatory devices with this aptamer and the hammerhead ribozyme as an actuator. Through FACS-seq-based high-throughput screening in yeast, we identified in vivo-functional FBP-sensing devices that generate fluorescent readout dependent on intracellular FBP concentration. As FBP reports the flux through glycolysis, the developed RNA device can be used to sense the glycolytic rate in single cells, offering unprecedented possibilities to investigate the causes of metabolic heterogeneity.

scholarly journals Analysis of Synchronous and Asynchronous In Vitro Infections with Homologous Murine Norovirus Strains Reveals Time-Dependent Viral Interference Effects

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 823
Author(s):  
Louisa F. Ludwig-Begall ◽  
Elisabetta Di Felice ◽  
Barbara Toffoli ◽  
Chiara Ceci ◽  
Barbara Di Martino ◽  
...  
Keyword(s):  
Rare Event ◽  
Time Dependent ◽  
Time Interval ◽  
Murine Norovirus ◽  
Temporal Separation ◽  
Viral Recombination ◽  
Copy Numbers ◽  
Secondary Infections

Viral recombination is a key mechanism in the evolution and diversity of noroviruses. In vivo, synchronous single-cell coinfection by multiple viruses, the ultimate prerequisite to viral recombination, is likely to be a rare event and delayed secondary infections are a more probable occurrence. Here, we determine the effect of a temporal separation of in vitro infections with the two homologous murine norovirus strains MNV-1 WU20 and CW1 on the composition of nascent viral populations. WU20 and CW1 were either synchronously inoculated onto murine macrophage cell monolayers (coinfection) or asynchronously applied (superinfection with varying titres of CW1 at half-hour to 24-h delays). Then, 24 h after initial co-or superinfection, quantification of genomic copy numbers and discriminative screening of plaque picked infectious progeny viruses demonstrated a time-dependent predominance of primary infecting WU20 in the majority of viral progenies. Our results indicate that a time interval from one to two hours onwards between two consecutive norovirus infections allows for the establishment of a barrier that reduces or prevents superinfection.

scholarly journals Activating Signal Cointegrator 2 Belongs to a Novel Steady-State Complex That Contains a Subset of Trithorax Group Proteins

2003 ◽  
Vol 23 (1) ◽  
pp. 140-149 ◽  
Author(s):  
Young-Hwa Goo ◽  
Young Chang Sohn ◽  
Dae-Hwan Kim ◽  
Seung-Whan Kim ◽  
Min-Jung Kang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Steady State ◽  
Nuclear Receptors ◽  
Transcriptional Activation ◽  
Retinoic Acid Receptor ◽  
Dependent Manner ◽  
Trithorax Group ◽  
Trithorax Group Proteins

ABSTRACT Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, α/β-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.

Studies with GIP/Ins cells indicate secretion by gut K cells is KATPchannel independent

2003 ◽  
Vol 284 (5) ◽  
pp. E988-E1000 ◽  
Author(s):  
Song Yan Wang ◽  
Maggie M.-Y. Chi ◽  
Lin Li ◽  
Kelle H. Moley ◽  
Burton M. Wice
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Calcium Mobilization ◽  
Enteroendocrine Cells ◽  
Glycolytic Flux ◽  
K Cells ◽  
Antibody Staining ◽  
Methyl Pyruvate ◽  
Fructose 1,6 Bisphosphate ◽  
Insulinoma Cells

K cells are a subpopulation of enteroendocrine cells that secrete glucose-dependent insulinotropic polypeptide (GIP), a hormone that promotes glucose homeostasis and obesity. Therefore, it is important to understand how GIP secretion is regulated. GIP-producing (GIP/Ins) cell lines secreted hormones in response to many GIP secretagogues except glucose. In contrast, glyceraldehyde and methyl pyruvate stimulated hormone release. Measurements of intracellular glucose 6-phosphate, fructose 1,6-bisphosphate, and pyruvate levels, as well as glycolytic flux, in glucose-stimulated GIP/Ins cells indicated that glycolysis was not impaired. Analogous results were obtained using glucose-responsive MIN6 insulinoma cells. Citrate levels increased similarly in glucose-treated MIN6 and GIP/Ins cells. Thus pyruvate entered the tricarboxylic acid cycle. Glucose and methyl pyruvate stimulated 1.4- and 1.6-fold increases, respectively, in the ATP-to-ADP ratio in GIP/Ins cells. Glyceraldehyde profoundly reduced, rather than increased, ATP/ADP. Thus nutrient-regulated secretion is independent of the ATP-dependent potassium (KATP) channel. Antibody staining of mouse intestine demonstrated that enteroendocrine cells producing GIP, glucagon-like peptide-1, CCK, or somatostatin do not express detectable levels of inwardly rectifying potassium (Kir) 6.1 or Kir 6.2, indicating that release of these hormones in vivo may also be KATPchannel independent. Conversely, nearly all cells expressing chromogranin A or substance P and ∼50% of the cells expressing secretin or serotonin exhibited Kir 6.2 staining. Compounds that activate calcium mobilization were potent secretagogues for GIP/Ins cells. Secretion was only partially inhibited by verapamil, suggesting that calcium mobilization from intracellular and extracellular sources, independent from KATPchannels, regulates secretion from some, but not all, subpopulations of enteroendocrine cells.

The anterior and posterior ‘stomach’ regions of larval Aedes aegypti midgut: regional specialization of ion transport and stimulation by 5-hydroxytryptamine

1999 ◽  
Vol 202 (3) ◽  
pp. 247-252 ◽  
Author(s):  
T.M. Clark ◽  
A. Koch ◽  
D.F. Moffett
Keyword(s):  
Steady State ◽  
Aedes Aegypti ◽  
Ion Transport ◽  
Malpighian Tubules ◽  
Transport Mechanisms ◽  
Regional Specialization ◽  
Negative Deflection ◽  
Electrical Polarity

The ‘stomach’ region of the larval mosquito midgut is divided into histologically distinct anterior and posterior regions. Anterior stomach perfused symmetrically with saline in vitro had an initial transepithelial potential (TEP) of −66 mV (lumen negative) that decayed within 10–15 min to a steady-state TEP near −10 mV that was maintained for at least 1 h. Lumen-positive TEPs were never observed in the anterior stomach. The initial TEP of the perfused posterior stomach was opposite in polarity, but similar in magnitude, to that of the anterior stomach, measuring +75 mV (lumen positive). This initial TEP of the posterior stomach decayed rapidly at first, then more slowly, eventually reversing the electrical polarity of the epithelium as lumen-negative TEPs were recorded in all preparations within 70 min. Nanomolar concentrations of the biogenic amine 5-hydroxytryptamine (5-HT, serotonin) stimulated both regions, causing a negative deflection of the TEP of the anterior stomach and a positive deflection of the TEP of the posterior stomach. Phorbol 12,13-diacetate also caused a negative deflection of the TEP of the anterior stomach, but had no effect on the TEP of the posterior stomach. These data demonstrate that 5-HT stimulates region-specific ion-transport mechanisms in the stomach of Aedes aegypti and suggest that 5-HT coordinates the actions of the Malpighian tubules and midgut in the maintenance of an appropriate hemolymph composition in vivo.

scholarly journals Metformin Inhibited Growth, Invasion and Metastasis of Esophageal Squamous Cell Carcinoma in Vitro and in Vivo

2018 ◽  
Vol 51 (3) ◽  
pp. 1276-1286 ◽  
Author(s):  
Feng Liang ◽  
Yu-Gang Wang ◽  
Changcheng Wang
Keyword(s):  
Squamous Cell Carcinoma ◽  
Plasma Glucose Level ◽  
Caspase 3 ◽  
Time Dependent ◽  
Dependent Manner ◽  
Metformin Treatment ◽  
Invasion And Migration ◽  
And Migration

Background/Aims: This study aimed at investigating the effects of metformin on the growth and metastasis of esophageal squamous cell carcinoma (ESCC) in vitro and in vivo. Methods: Two human ESCC cell lines EC9706 and Eca109 were selected and challenged with metformin in this study. Western blot assay was performed to detect th level of Bcl-2, Bax and Caspase-3. Scratch wound assay, transwell assay and Millicell invasion assay were used to assay the invasion and migration of EC9706 and Eca109 cells. Nude mice tumor models were used to assay the growth and lung metastasis of ESCC cells after metformin treatment. The plasma glucose level was also assayed. Results: We found that metformin significantly inhibited proliferation and induced apoptosis of both ESCC cell lines in a dose- and time-dependent manner, and the expression of Bcl-2 was down-regulated and Bax and Caspase-3 were up-regulated. Metformin significantly inhibited the invasion and migration of EC9706 and Eca109 cells (p < 0.05). mRNA and protein levels of MMP-2 and MMP-9 decreased significantly upon treatment with metformin of 10mM for 12, 24 and 48h in a time-dependent manner (p < 0.05). In line with in vitro results, in vivo experiments demonstrated that metformin inhibited tumorigenicity, inhibited lung metastasis and down-regulated the expression of MMP-2 and MMP-9. Moreover, we showed that metformin treatment did not cause significant alteration in liver and renal functions and plasma glucose level. Conclusion: Our study for the first time demonstrated the anti-invasive and anti-metastatic effects of metformin on human ESCC cells both in vitro and in vivo, which might be associated with the down-regulation of MMP-2 and MMP-9. As a whole, our results indicate the potential of metformin to be developed as a chemotherapeutic agent for patients with ESCC and might stimulate future studies on this area.

Control of glycolysis in vertebrate skeletal muscle during exercise

1996 ◽  
Vol 270 (4) ◽  
pp. R821-R829 ◽  
Author(s):  
U. Krause ◽  
G. Wegener
Keyword(s):  
Gastrocnemius Muscle ◽  
Rana Temporaria ◽  
High Capacity ◽  
Repeated Exercise ◽  
Frog Rana Temporaria ◽  
Time Courses ◽  
Fructose 1,6 Bisphosphate ◽  
Assay Conditions

The gastrocnemius muscle of the frog (Rana temporaria) has a high capacity for anaerobic glycolysis from glycogen. Glycolytic metabolites and effectors of phosphofructokinase, particularly the hexose bisphosphates, were followed in muscle during exercise (swimming between 5 s and 5 min), recovery (rest for up to 2 h after 5 min of swimming), and repeated exercise (swimming for up to 60 s after 2 h of recovery). Glycogen phosphorylase and phosphofructokinase were swiftly activated with exercise. The hexose bisphosphates followed markedly different time courses. Fructose 1,6-bisphosphate was transiently increased in both exercise and repeated exercise. This appears to be an effect rather than a cause of phosphofructokinase activation. Glucose 1,6-biphosphate was accumulated only while phosphofructokinase was active and was unchanged at other times. Fructose 2,6-biphosphate showed a 10-fold transient increase on exercise in rested frogs, almost disappeared from the muscle during recovery, and did not change during repeated exercise. Fructose 2,6-biphosphate is a potent activator of phosphofructokinase in vitro under near physiological assay conditions, and it may serve this function also in vivo during exercise. Glucose 1,6-biphosphate could be an activator of phosphofructokinase in repeated exercise when fructose 2,6-biphosphate is not available.

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