Total Amount Of ATP Research Articles

Synergistic Tumor Inhibition via Energy Elimination by Repurposing Penfluridol and 2-Deoxy-D-Glucose in Lung Cancer.

Simple SummaryDrug repurposing has been effective for discovering novel treatments for cancer. The antipsychotic agent penfluridol was reported to suppress lung cancer growth via ATP energy deprivation. The aim of our study was to investigate how penfluridol influences energy metabolism in lung cancer cells. We observed that penfluridol inhibited mitochondrial oxidative phosphorylation (OXPHOS), but induced glycolysis to compensate for the loss of ATP caused by suppression of mitochondrial OXPHOS. We also confirmed that inhibition of glycolysis by 2-deoxy-D-glucose (2DG) significantly augmented the antitumor effects caused by penfluridol in vitro and in vivo. Our studies provide novel insights into repurposing penfluridol combined with 2-DG for lung cancer treatment.Energy metabolism is the basis for cell growth, and cancer cells in particular, are more energy-dependent cells because of rapid cell proliferation. Previously, we found that penfluridol, an antipsychotic drug, has the ability to trigger cell growth inhibition of lung cancer cells via inducing ATP energy deprivation. The toxic effect of penfluridol is related to energy metabolism, but the underlying mechanisms remain unclear. Herein, we discovered that treatment of A549 and HCC827 lung cancer cells with penfluridol caused a decrease in the total amount of ATP, especially in A549 cells. An Agilent Seahorse ATP real-time rate assay revealed that ATP production rates from mitochondrial respiration and glycolysis were, respectively, decreased and increased after penfluridol treatment. Moreover, the amount and membrane integrity of mitochondria decreased, but glycolysis-related proteins increased after penfluridol treatment. Furthermore, we observed that suppression of glycolysis by reducing glucose supplementation or using 2-deoxy-D-glucose (2DG) synergistically enhanced the inhibitory effect of penfluridol on cancer cell growth and the total amount of mitochondria. A mechanistic study showed that the penfluridol-mediated energy reduction was due to inhibition of critical regulators of mitochondrial biogenesis, the sirtuin 1 (SIRT1)/peroxisome-proliferator-activated receptor co-activator-1α (PGC-1α) axis. Upregulation of the SIRT1/PGC-1α axis reversed the inhibitory effect of penfluridol on mitochondrial biogenesis and cell viability. Clinical lung cancer samples revealed a positive correlation between PGC-1α (PPARGC1A) and SIRT1 expression. In an orthotopic lung cancer mouse model, the anticancer activities of penfluridol, including growth and metastasis inhibition, were also enhanced by combined treatment with 2DG. Our study results strongly support that a combination of repurposing penfluridol and a glycolysis inhibitor would be a good strategy for enhancing the anticancer activities of penfluridol in lung cancer.

Solution to the Olympic Games challenge

The solution to the Olympic Games challenge [1] is adenosine 5′-triphosphate (ATP; Formula 1). To be precise, the NMR spectra presented in the Olympic Games challenge [1] were measured from the more stable ATP disodium salt (C10H14N5Na2O13P3), but the spectra measured in D2O are indistinguishable. At the 2012 Olympic Games in London, Usain Bolt, the reigning Olympic champion in three Olympic events, ran the 100 m and 200 m with nearly the same average speed of 10.38 and 10.35 m/s. In contrast, Stephen Kiprotich—the 2012 gold medalist in the men’s marathon—required 7,681 s for the 42,195 m. So, his average speed was “only” 5.49 m/s. Since endurance athletes are no less motivated than short-distance runners, the following question arises: Why must a marathon runner reduce his or her speed and run at almost half the speed of sprinters? The answer lies in the solution of this challenge, or more exactly, in the rate of synthesis of ATP in human muscle cells. The coenzyme ATP is the only direct fuel for muscle cells, regardless of whether an athlete is trained as a sprinter or as a marathon runner. The concentration of this direct fuel for muscles inside the cell is typically 1–10 mmol/L. Surprisingly, the total amount of ATP in working muscles is constant. Even extremely hard work does not lower the ATP concentration by more than about 20%. Several differing energy sources are used by working muscles to maintain the ATP level. During the short sprint period, the major driving forces are stored high-energy phosphates and anaerobic breakdown of glucose. Consequently, the 100 m sprinter can perform almost without breathing just by using the energy stored as ATP and creatine phosphate in the active N N

Compositions of Extractive Nitrogenous Constituents and Their Monthly Variation for Fresh Capsosiphon fulvescens

To elucidate the composition of extractive nitrogenous components in the fresh Capsosiphons fulvescens cultured off the southern coast of Korea, and to determine the monthly variation of these nitrogenous components, extract samples collected monthly from December to March at Jangheung-gun, Jeonnam Province were analyzed for total nitrogen, free and combined amino acids, ATP and related compounds, betaines, trimethylamine oxide (TMAO) and trimethylamine (TMA). The content of extractive nitrogen was 1,090～1,233 ㎎/100 g on dry basis. The number of 21～25 ninhydrin-positive substances was detected in the analysis of free amino acids, and their total amount was 3,710～4,788 ㎎/100 g on dry basis. Among them, free proline, asparagine, glutamic acid, alanine, taurine and glutamine were found to be abundant. The combined amino acids amounted to 1,573～2,121 ㎎/100 g in total and the total amount of ATP and related compound was 33.8～84.0 ㎎/100 g (1.06～2.46 μ㏖/g) on dry basis. Betaine, glycinebetaine, β-alaninebetaine, γ-butyrobetaine, homarine and trigonelline were detected in most of samples. Levels of free and combined amino acids, ATP and related compounds fluctuated from sample to sample, with their contents higher in December and January and lower in March.

Analysis of Growth of Lactobacillus plantarum WCFS1 on a Complex Medium Using a Genome-scale Metabolic Model

A genome-scale metabolic model of the lactic acid bacterium Lactobacillus plantarum WCFS1 was constructed based on genomic content and experimental data. The complete model includes 721 genes, 643 reactions, and 531 metabolites. Different stoichiometric modeling techniques were used for interpretation of complex fermentation data, as L. plantarum is adapted to nutrient-rich environments and only grows in media supplemented with vitamins and amino acids. (i) Based on experimental input and output fluxes, maximal ATP production was estimated and related to growth rate. (ii) Optimization of ATP production further identified amino acid catabolic pathways that were not previously associated with free-energy metabolism. (iii) Genome-scale elementary flux mode analysis identified 28 potential futile cycles. (iv) Flux variability analysis supplemented the elementary mode analysis in identifying parallel pathways, e.g. pathways with identical end products but different co-factor usage. Strongly increased flexibility in the metabolic network was observed when strict coupling between catabolic ATP production and anabolic consumption was relaxed. These results illustrate how a genome-scale metabolic model and associated constraint-based modeling techniques can be used to analyze the physiology of growth on a complex medium rather than a minimal salts medium. However, optimization of biomass formation using the Flux Balance Analysis approach, reported to successfully predict growth rate and by product formation in Escherichia coli and Saccharomyces cerevisiae, predicted too high biomass yields that were incompatible with the observed lactate production. The reason is that this approach assumes optimal efficiency of substrate to biomass conversion, and can therefore not predict the metabolically inefficient lactate formation.

A pulsed DC electric field affects P2-purinergic receptor functions by altering the ATP levels in in vitro and in vivo systems

Recently it was shown that extracellular ATP, acting through purinergic receptors, has many physiological functions, including opening of Ca2+-ion channels, activation and mediation of signal tranduction mechanisms as well as activation of the pain sensation. Since electrical stimulation is also known to affect many signal transduction processes as well as the alleviation of pain, we hypothesized that electric stimulation may affect the extracellular release of ATP. We investigated the effects of a small DC electric field (101–102 V m−1 range and with frequencies below 150 Hz) on the release of ATP in vitro (HeLa cells), and on the levels of ATP in vivo (the plasma of healthy volunteers). In HeLa cells ATP release was increased 50 fold, while the total amount of ATP in the cells was increased by 163%. In the plasma a significant decrease (P<0.05) in ATP concentration was seen after electrical stimulation, in all the volunteers. The small DC electric field also affected the cAMP signal transduction system in vitro (HeLa cells and human lymphocytes) and in vivo (human plasma). Decreased levels of cAMP (P<0.05) were seen in HeLa cells and increased levels of cAMP (P<0.05) in isolated human lymphocytes. The cAMP levels in the plasma of the electrically treated volunteers were lower than control values. These results show that the frequency, waveform and signal strength of the applied electric field are suitable for effecting measurable changes on signal transduction in vitro and in vivo.

K value of cultured southern bluefin tuna (Thunnus maccoyii) imported in chilled state, and its difference among parts of fish body

Cultured tuna is becoming one of important fish sources in Japan, which is used to be imported by airplane without freezing from abroad. It gives a better economical profit and taste, and also promises a constant supply through out a year. However, there are few data available for the freshness of tuna undergoing such kind of transportation. This study was conducted to evaluate the initial freshness indicated as Kvalue of chilled-southern bluefin tuna (Thunnus maccoyii), imported by airplane from Australia, considering the difference of K value among parts of this fish body. The total amount of ATP and its related compounds was 11.82, 9. 74, 7.83 and 5.88, u moles/ g for dorsal (red), dorsal (white), tail and abdomen muscles, respecively. Tail muscle was showing the highest K value followed by dorsal (white), abdomen and dorsal (red) muscles, respectively. As tail muscle was more active among parts of fish body before fish was being caught, so that degradation of ATP was thought to occur faster in tail. It was confirmed that the quality of the sample fish imported by airplane was fairly good, which has K value ranged between 8. 8-14. 7%. The tail muscle could be regarded as a suitable part for analyzing K value.

Postmortem Biochemical Changes in Muscle of &lt;i&gt;Buccinum striatissimum&lt;/i&gt; during Storage

The changes in content of ATP and its related compounds, free amino acids, organic acids and opines were investigated in the muscle of B. striatissimum during storage at 0°C and 5°C. Immediately after shelling, the total amount of ATP and its related compounds were 2.8μmol/g. ATP, ADP and AMP accounted for 98% of the total ATP and its related compounds. ATP reached the highest level on the 1st day at 0°C, then decreased slowly. ATP degraded faster at 0°C than at 5°C. AMP was decomposed to inosine through both pathways, since IMP and adenosine were detected. Hypoxanthine, xanthine and uric acid increased with the decrease of ATP. Immediately after shelling, the total amount of free amino acids in the muscle were 1, 719 mg/100 g. Taurine, arginine, proline, alanine, glutamic acid and glycine were major free amino acids and accounted for 87% of the total amount of free amino acids. Acetic acid increased rapidly during storage, but octopine and alanopine increased slowly.

Platelet activation by healing EPTFE grafts

The purpose of this study was to examine the effect of the in vivo maturing ePTFE graft surface on platelet activation. Ten canines were randomized to receive either a carotid to infrarenal aorta ePTFE graft or sham operation. Animals were sampled at specific time points up to 3 months postoperatively. Whole blood platelet aggregometry (arachidonic acid, ADP, and collagen agonists) and ATP secretion (in response to arachidonic acid, ADP, collagen, and thrombin) were measured. Additionally, complete hematologic analysis and histology were performed. With time, graft animals showed significantly more decrease in platelet aggregation in response to ADP compared to sham animals (P = .023). The total amount of ATP per platelet was not different, as demonstrated by equivalent ATP release per platelet in response to thrombin. Over the first week, grafted dogs developed a decrease in systemic platelet count of 50% (P < .001) that persisted over the 3-month follow-up period. With time, overall regression model slopes of graft and sham platelet count data were not statistically different (P = .29). Histologically, the grafts demonstrated limited cellular ingrowth at both anastomoses, with fibrin matrix along the remainder of the blood-biomaterial interface. These data suggest that, similar to Dacron, exposure to an ePTFE surface results in significant changes in platelet biology, and these platelet-ePTFE interactions persist even after the graft has formed a mature pseudointima. The pseudointima appears to be the primary determinant of the blood-biomaterial interaction.

Seasonal Variations of Extractive Components in the Muscle of Disk Abalone.

Seasonal variations of extractive components were examined in the muscle of disk abalone Haliotis discus collected from the coast of Amatsukominato, Chiba Prefecture at intervals of two or three months from July 1990 to July 1991. The moisture content was highest in December after spawning season, thereafter decreased and became lowest in May. The total amount of ATP and its related compounds was highest in September. AMP was detected in high amounts from July to September. The level of glycogen was high in summer (over 6%), decreased remarkably in the spawning season and low in winter. Succinate was high in amounts from May to September. The total amount of free amino acids was highest in September and decreased to half in other months. The contents of glutamic acid, glycine, and AMP which are taste-active components were highest (470mg/100g) in September and lowest (130mg/100g) in February. Present study can demonstrate that disk abalones are palatable in summer, and watery and unpalatable in winter, which is generally told in Amatsu-kominato district.

Depletion of Dense Granule Nucleotides during Storage of Human Platelets

The energy metabolism of human platelets was studied during storage of platelet concentrates. The platelets were prepared from buffy coats in PVC/DEHP bags and stored for 7 days at room temperature at a concentration of 1.0 x 10(9)/ml with horizontal agitation. The total amount of ATP and ADP decreased with 40% during this storage. This decrease correlated with the disc-to-sphere transformation associated with the loss of platelet viability. During storage, the ability to incorporate 3H-adenosine into metabolic ATP and ADP (45 min at 37 degrees C) decreased with 50%. Via measurement of the specific activity of actin-bound ADP and the amount of incorporated radioactivity into total ATP and ADP, we calculated the content of the metabolic and storage pools of ATP and ADP. The results indicate that the decrease in adenine nucleotide levels during storage was mainly caused by a depletion of ATP and ADP from the storage pool, whereas the metabolic pool remained nearly intact. After 7 days, the ATP:ADP ratio of the storage pool had decreased from 1.0 to 0.3, indicating hydrolysis of ATP. Diadenosine-triphosphate and diadenosine-tetraphosphate (present in the storage pool) decreased with only 30%, and the serotonin content remained nearly constant. Therefore, it is unlikely that the storage pool was completely secreted. Probably, the storage pool of nucleotides serves as an internal supply for maintaining the contents of the metabolic pool of ATP and ADP during storage of platelets.

Microencapsulated Chromatophores for the Production of ATP

Chromatophores, organelles for photophosphorylation in non-sulfur purple photosynthetic bacteria, were microencapsulated and utilized in ATP production. The microcapsules were formed by photocrosslinking with trimethylolpropane triacrylate (TMPTA). In batch experiments chromatophores microencapsulated in TMPTA capsules were repeatedly used in ATP production for more than 5 times. Continuous ATP production was then undertaken. ATP was produced at a production rate of 14 μmol h−1 L−1 over 200 hrs. The yield (from ADP to ATP) was 35%. The total amount of ATP produced was 0.7 mM (μM Bchl)−1. Therefore, this microencapsulation method was found to be suitable for the continuous ATP production using chromatophores.

The Status of Adenine Nucleotides and Malate in Chloroplasts, Mitochondria, and Supernatant of Guard Cell Protoplasts from Vicia faba

Summary A technique for rapid fractionation of guard cell protoplasts from Vicia faba L. (Weiskernige Hangdown) is described. This description includes: (1) selection of suitable markers (plastids: light-dependent oxygen evolution; mitochondria: fumarase; cytoplasm: phosphoenolpyruvate carboxylase; vacuoles: acid phosphatase), (2) the rapid quenching of metabolism, and (3) methods for determining metabolite concentrations. Using these techniques, we report the distribution of energy pool sizes and malate in plastids, mitochondria, and the supernatant. The total amount of ATP (2.2fmol protoplast-1 = 92pmol µg protein-1) is distributed between the supernatant (65%), the mitochondria (31%), and the plastids (3%) being similar to that of ADP (total amount 0.56 fmol protoplast- 1 = 24 pmol µg protein-1). Malate (272 fmol protoplast-1 = 12 nmol µg protein-1) is accumulated in the supernatant (60%) and in the mitochondria (35%). The technique is a prerequisite for future investigations of alterations in adenylate pool sizes and malate during changes in volume of guard cell protoplasts.

Hydrogen oxidation and nitrogen fixation in rhizobia, with special attention focused on strain ORS 571.

In this survey we describe the influence of hydrogen oxidation on the physiology of Rhizobium ORS 571. The presence of hydrogen is required for the synthesis of hydrogenase. Carbon substrates do not repress the synthesis of hydrogenase. The respiratory system contains cytrochromes of the b- and c-type. Cytochrome alpha 600 is present after growth at high oxygen tensions. The nature of the terminal oxidases functioning at low oxygen tensions has not been established yet----H+/O values with endogenous substrates are between 6 and 7. The results show the presence of two phosphorylation sites: site 1 (ATP/2e = 1.0) and site 2(ATP/2e = 1.33). By measuring molar growth yields it has been demonstrated that carbon-limited, nitrogen-fixing cultures obtain additional ATP from hydrogen oxidation, and that site 2 of oxidative phosphorylation is passed during hydrogen oxidation. A method is described to calculate ATP/N2 values (the total amount of ATP used by nitrogenase during the fixation of 1 mol N2) and H2/N2 ratios (mol hydrogen formed per mol N2 fixed) in aerobic organisms. For Rhizobium ORS 571 the ATP/N2 value is about 40 and the H2/N2 ratio is between 5 and 7.5. Cells obtained from oxygen-limited nitrogen-fixing cultures contain 30-40% poly-beta-hydroxybutyrate, which explains the high molar growth yields found. Hydrogen has not been detected in the effluent gas of these cultures, which may point to reoxidation of the hydrogen formed at nitrogen fixation. Calculations show that the effect of hydrogen reoxidation on the efficiency of nitrogen fixation (g N fixed X mol-1 substrate converted) is not very large and that the actual H2/N2 ratio is of much more importance. After addition of hydrogen to succinate-limited, ammonia-assimilating cultures, an initial increase of the Ysuccinate value (g dry wt X mol-1 succinate) is followed by a gradual decrease. This is accompanied by a large decrease of the YO2 value, and an increased permeability of the cytoplasmic membrane to protons. The results may be explained by a transition of the culture from an energy-limited state to a carbon-limited state.

Changes of RNA precursor pools (ATP, UTP) during embryogenesis ofDrosophila melanogaster (Diptera) andBruchidius obtectus (coleoptera)

The total amount of ATP and UTP was measured in embryos ofDrosophila melanogaster andBruchidius obtectus at different development stages. The pool-sizes were measured by their ability to enhance poly(AU) synthesis in an in vitro test, using RNA polymerase ofE. coli, poly-d(AT), ATP, and3H-UTP. In embryos ofD. melanogaster, the amount of UTP decreased from 4.5 pmoles/ embryo during cleavage and early blastoderm to 2 pmoles at later stages. The ATP pool decreased from 14 pmoles/ embryo to 6 pmoles at the same developmental stages. In embryos ofB. obtectus, the UTP pool expanded from about 1 pmole/embryo during cleavage and early blastoderm to about 3 pmoles during gastrulation and organogenesis. Much more ATP was found inB. obtectus embryos: during fertilization 23 pmoles/embryo were found. At late syncytial blastoderm stages the amount of ATP rose to about 50 pmoles/embryo. At cellular blastoderm stages and during gastrulation only 26 pmoles/embryo of ATP were found. During organogenesis the ATP pool decreased to about 14 pmoles/embryo.

Liver adenosine triphosphate content and bile flow rate in the rat.

The effects of a number of hepatotoxic and other agents on the ATP content of rat liver are described. Changes in the distribution of ATP between the cell sap and the large-particle fraction were determined at intervals after rats had been dosed with various substances. Ethionine produced a rapid decrease in total liver ATP but no alteration in its intracellular distribution. Carbon tetrachloride, sodium salicylate, dimethylnitrosamine, 2,4-dinitrophenol, icterogenin, sodium succinate, sodium malonate and sodium taurocholate did not significantly alter the total ATP content of liver in the periods studied but changes in intracellular distribution were found. Carbon tetrachloride, malonate and taurocholate decreased, and salicylate treatment increased, the proportion of ATP in the cell sap. Treatment with sodium phenobarbitone increased the total liver ATP and the total amount of ATP in the cell sap. The changes in ATP concentration and in the intracellular distribution of ATP are correlated with changes previously reported in bile flow (Delaney & Slater, 1969). No general correlation was found between changes in total ATP and changes in bile flow rate, but there was a relationship between changes in bile flow and in ATP content in the case of ethionine. With the exception of taurocholate and icterogenin, which possibly act on a membrane site, an approximate correlation was found between changes in bile flow and changes in the amount of ATP in the cell sap. The findings are discussed in terms of possible mechanisms for biliary secretion.