Changes In Adenosine Triphosphate Levels Research Articles

Highly sensitive and stable fluorescent aptasensor based on an exonuclease III-assisted amplification strategy for ATP detection

Fluctuations in intracellular adenosine triphosphate (ATP) concentration are closely associated with some cancer diseases. Thus, it is a worthwhile undertaking to predict sickness by monitoring changes in ATP levels. However, the detection limits of current fluorescent aptamer sensors for ATP detection are in the range of nmol L−1 to μmol L−1. It has become crucial to employ amplification strategies to increase the sensitivity of fluorescent aptamer sensors. In the current paper, a duplex hybrid aptamer probe was developed based on exonuclease III (Exo III)-catalyzed target recycling amplification for ATP detection. The target ATP forced the duplex probe configuration to change into a molecular beacon that can be hydrolyzed with Exo III to achieve the target ATP cycling to amplify the fluorescence signal. Significantly, many researchers ignore that FAM is a pH-sensitive fluorophore, leading to the fluorescence instability of FAM-modified probes in different pH buffers. The negatively charged ions on the surface of AuNPs were replaced by new ligands bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP) to improve the drawback of FAM instability in alkaline solutions in this work. The aptamer probe was designed to eliminate the interference of other similar small molecules, showing specific selectivity and providing ultra-sensitive detection of ATP with detection limits (3σ) as low as 3.35 nM. Such detection limit exhibited about 4-500-fold better than that of the other amplification strategies for ATP detection. Thus, a relatively general high sensitivity detection system can be established according to the wide target adaptability of aptamers, which can form specific binding with different types of targets.

Genetically‐Encoded ATP Sensors for Fluorescence Lifetime Measurements

Fluorescent protein‐based genetically‐encoded sensors of adenosine triphosphate (ATP) have proven valuable for the detection and imaging of both intracellular and extracellular ATP. Current ATP sensors employ either a Förster‐type resonance energy transfer (FRET) fluorescent protein pair or circularly‐permuted fluorescent proteins to report changes in ATP levels. Steady‐state fluorescence spectroscopy and imaging has been the predominant mode of measurement, but both single‐channel intensity and two‐channel ratiometric signals are subject measurements artifacts from a variety of confounding factors such as variance in sensor expression levels and wavelength‐dependent scattering. Recently, we demonstrated that time‐resolved measurements of fluorescence lifetime offer a robust and quantitative alternative mode of detection that relies only on an intrinsic photophysical parameter. Here, we characterize the fluorescence lifetime responses of a panel of the currently available ATP sensors that span different protein architectures. We find that ATP sensors can provide large fluorescence lifetime dynamic ranges, with some sensors exhibiting nearly 1 ns changes in response to ATP. Thus, our study demonstrates that time‐resolved measurements expand the application space of genetically‐encoded fluorescent protein ATP sensors and will facilitate multicolor, multiplexed quantitation of live‐cell energy metabolism and purinergic signaling dynamics.

AG-348 Activation of Pyruvate Kinase in Vivo Enhances Red Cell Glycolysis in Mice

Pyruvate kinase deficiency (PKD) is an autosomal recessive enzymopathy that is the most common cause of hereditary nonspherocytic hemolytic anemia (HNSHA). PKD is a rare disease characterized by a life-long chronic hemolysis with severe co-morbidities. It is hypothesized that insufficient energy production to maintain red cell membrane homeostasis promotes the chronic hemolysis. Treatment is generally palliative, focusing on the resultant anemia, and there are no approved drugs that directly target mutated pyruvate kinase.AG-348 is an allosteric activator of the red cell isoform of pyruvate kinase (PKR) that has recently entered Phase I clinical trials in normal healthy volunteers. AG-348 increases the catalytic efficiency and enhances the protein stability of a spectrum of recombinantly expressed PKR mutant proteins that have been associated with PKD. PKD red cells are characterized by changes in metabolism associated with defective glycolysis, including a build-up of the upstream glycolytic intermediate 2,3-DPG and deficiency in the PKR product adenosine triphosphate (ATP). PKR flux, e.g. the rate of carbon flow through the PKR enzyme reaction, was examined in PKD patient or wild type (WT) donor blood samples by incubation of whole blood with a stable isotope tracer, [U-13C6]-glucose. At various time points after the addition of [U-13C6]-glucose, metabolism was quenched and metabolites were extracted. Metabolite pool sizes and 13C label incorporation into glycolytic intermediates were monitored by LC/MS. The rate of label incorporation was found to be significantly slower in PKD patient red cells, consistent with decreased glycolytic activity. Treatment of PKD red cells with AG-348 ex-vivo induces changes in metabolism consistent with increased glycolytic activity including reduced 2,3-DPG levels, increased ATP levels, and increased PKR enzyme activity levels.The effect of AG-348 on red cell metabolism in vivo was evaluated in mice. C57/BL6 mice were dosed by oral gavage either with a single dose, or with multiple doses (BID) of AG-348 for 7 days. Dose levels tested were 1 mpk, 10 mpk, 50 mpk, and 150 mpk. Following the last dose, mice were bled to evaluate drug exposure and pharmacodynamic markers including 2,3-DPG and ATP levels, and PKR activity. AG-348 was demonstrated to be a well-behaved compound, with dose-proportional increase in exposure, both in the single-dose and multiple dose studies. A single dose of AG-348 resulted in a dose-dependent increase in PKR activity levels, concomitant with reduction in 2,3-DPG levels. There were no significant changes in ATP levels after a single administration of AG-348. In the multiple-dose studies, similar changes in PKR activity and 2,3-DPG levels were observed. In contrast to the single-dose study, ATP levels were observed to be robustly increased in a dose-dependent manner.The effect of AG-348 on PKR flux was assessed in whole blood from mice treated with AG-348. C57BL/6 mice were dosed by oral gavage with AG-348 (150 mg/kg twice daily [BID]) for 3 days. Whole blood was incubated with [U-13C6]-glucose and the metabolite pool sizes and rate of 13C label incorporation into glycolytic intermediates were assessed. The data were subsequently analyzed using a mathematical model to quantify flux through the PKR reaction and it was determined that AG-348 treatment significantly increased flux through the PKR reaction.Collectively, these data demonstrate that AG-348 not only potently binds to and activates the PKR enzyme in vivo, but this enzyme activation induces enhanced glycolytic pathway activity in red cells that results in profound changes in cellular metabolism, as reflected in dramatically increased ATP levels and reduced 2,3-DPG levels. As AG-348 has similar potency against the WT PKR enzyme as against tested mutant PKR enzymes in vitro, these data support the hypothesis that AG-348 treatment may similarly enhance glycolytic activity in PKD patients and thus correct the underlying pathology of PKD. [Display omitted] DisclosuresKung:Agios Pharmaceuticals: Employment, Stockholder Other. Hill:Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Jha:Agios Pharmaceuticals: Employment, Stockholder Other. Kosinski:Agios Pharmaceuticals: Employment, Stockholder Other. Clasquin:Agios Pharmaceuticals: Employment, Stockholder Other. Si:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Agios Pharmaceuticals: Employment, Stockholder Other. Hixon:Agios Pharmaceuticals: Employment, Stockholder Other. Dang:A: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. Silverman:Agios Pharmaceuticals: Employment, Stockholder Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other.

The Immunomodulatory Effects of <i>Sutherlandia frutescens</i> Extracts in Human Normal Peripheral Blood Mononuclear Cells

Sutherlandia frutescens (SF) is one of the medicinal plants used as an immune booster in the treatment of chronic ailments such as HIV/AIDS and cancer. Limited data suggest that its efficacy is based on its regulatory effect on cytokines, the critical components of the immune response. In this study, we investigated the in vitro immunomodulatory effects of SF extracts on normal human peripheral blood mononuclear cells (PBMCs). An ELISA-based assay was used to assess the levels of expression of 12 cytokines in treated cells. An adenosine triphosphate (ATP) assay was used to assess cell viability in relation to cytokine secretion. SF ethanol extracts induced changes in cytokine secretion relative to the dose of the extract. Generally cytokine expression and secretion was low in concentration because were not stimulated with any endotoxin. The high SFE dose (2.5 mg/ml) significantly (p<0.001) decreased some cytokines including TNF-α and IL 1β. Low doses of this extract (0.5 mg/ml) did not change TNF-α and IL 1β secretion from the baseline (untreated cells). Changes in cytokine secretion of SFE treated cells tracked changes in ATP levels (cell viability). The SFW extract-induced changes in cytokine secretion were independent of cell viability. TNF-α was decreased (p<0.001) by the high dose of SFW extract while IL 1β and IFNγ were increased (p<0.01) by the same dose. High doses decreased cell viability which was reflected in cytokine secretion. It is evident, from these results, that SF extracts can modulate cytokine secretion in unstimulated normal PBMCs in vitro. Further studies in animal models are recommended to advance understanding of this immunomodulatory activity.

A matter of energy stress: p38β meets mTORC1

A matter of energy stress: p38β meets mTORC1

Association between VDAC1 mRNA expression and intracellular ATP levels of cultured L-02 hepatocytes during hexavalent chromium toxicity

One way in which xenobiotics induce apoptotic cell death is to alter the selective permeability of the intracellular voltage-dependent anion channel (VDAC1) in the mitochondrial membrane. In this study, we explored the association between VDAC1 mRNA expression and mitochondrial function during hexavalent chromium compounds [Cr(VI)] treatment. Cultured L-02 hepatocytes were treated with 2, 4, 8, 16, or 32 μmol/L Cr(VI) for 12, 24, or 36 h. Expression of VDAC1 mRNA was measured by qualitative reverse transcription polymerase chain reaction (RT-PCR), whereas intracellular adenosine triphosphate (ATP) levels were determined by an ATP-specific bioluminescence assay. Over this range of Cr(VI) concentrations, average VDAC1 mRNA expression decreased by 84% after 12 h treatment and by 40% after 24 h of treatment, but it increased 2.33-fold at 36 h when compared with the control cultures. Treatment with Cr(VI) disrupted cellular metabolism as evidenced by changes in ATP levels. Cr(VI) treatment caused ATP levels to increase at 12 h, decrease at 24 h, and increase again at 36 h, resulting in a slant V-shaped curve. Correlation analysis revealed a moderate negative relationship between VDAC1 mRNA expression and intracellular ATP levels during Cr(VI) treatment (r = -0.557, P < 0.05). The negative association seemed to be more obvious in 32 μmol/L Cr(VI) group. Based on these results, heavy metal toxicity may induce over-expression of VDAC1 mRNA, which causes a decrease in ATP levels. Key words : Heavy metal toxicity, VDAC1 mRNA expression, ATP, relationship.

Age‐related decreases in NAD(P)H and glutathione cause redox declines before ATP loss during glutamate treatment of hippocampal neurons

Age-related glutamate excitotoxicity depends in an unknown manner on active mitochondria, which are key determinants of the cellular redox potential. Compared with embryonic and middle-aged neurons, old-aged rat hippocampal neurons have a lower resting reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and a lower redox ratio (NAD(P)H/flavin adenine nucleotide). Glutamate treatment resulted in an initial increase in NAD(P)H concentrations in all ages, followed by a profound calcium-dependent, age-related decline in NAD(P)H concentration and redox ratio. With complex I of the electron transport chain inhibited by rotenone, treatment with glutamate or ionomycin only resulted in the increase in NAD(P)H fluorescence. High-performance liquid chromatography analysis of adenine nucleotides in brain extracts showed 50% less nicotinamide adenine dinucleotide (NADH) and almost twice as much oxidized nicotinamide adenine dinucleotide, demonstrating a more oxidized ratio in old than middle-aged brain. Resting glutathione content also declined with age and further decreased with glutamate treatment without accompanying changes in adenosine triphosphate levels. We conclude that age does not affect production of NADH by dehydrogenases but that old-aged neurons consume more NADH and glutathione, leading to a catastrophic decline in redox ratio.

Assessing the Risk of Infection and Rejection in Hispanic Renal Transplant Recipients by Means of an Adenosine Triphosphate Release Assay

Introduction Because of the necessary immunosuppression, transplant recipients have a high risk of infection. Conversely, underimmunosuppression carries with it the risk of rejection. It would be quite useful to have a test that could differentiate between infection and rejection in renal transplant patients and better still, to predict which patients are at risk of complications. A new assay, which measures adenosine triphosphate (ATP) synthesis by CD4 + T cells in response to stimulation by phytohemagglutinine (Immuknow assay, Cylex, Inc) is undergoing clinical evaluations. Preliminary investigations suggest that this test could be useful to assess and predict the immune status of patients with other conditions. Methods We examined the records of all patients who received a kidney transplant in our program between August 2004 and January 2005. Of 64 patients, 58 had pretransplant and posttransplant ATP level determinations. We searched for associations between ATP levels and immunosuppression type, doses, and levels; creatinine levels; white blood cell count; tissue typing; preformed antibodies; as well as ATP levels on infection and rejection, and changes in ATP levels with time. Chi-square, Fisher, t test, analysis of variance (ANOVA), and relative risks were used for analysis of data. Results There was no relation between ATP levels and immunosuppression type, doses, or levels; creatinine levels; white blood cell counts; HLA; and panel-reactive antibody ( P > 0.05). However, patients with moderate or high pretransplant ATP levels had more rejection episodes (8/10) while patients with ATP levels in the low immune response had more infections (6/11) ( P < .001; relative risk [RR] for rejection = 1.2; RR for infection = 4.4). The mean ATP levels for rejection was 423.3 ng/mL versus 268.45 ng/mL for infection and 277.15 ng/mL for no events (ANOVA, P = .0145). Although acute rejections occurred mostly above 300, this was not significant ( P = .059; RR = 0.9). Infections were more frequent with ATP under 300 (RR = 7.3) and severe infection (endocarditis, meningitis, peritoneal abscesses, pneumonia, etc) were more frequent under 200 ( P < .001). Comparing pretransplant with posttransplant values at the second week an increase correlated with rejection ( P < .001, RR = 15.3), while a decrease did not correlate with the infection ( P = .845, RR = 1.4). Patients who received antirejection treatment had a decrease in their ATP levels at 5 days ( P = .002). Conclusion This ATP release assays helpful in determining the risk of developing infection or rejection, as well as follow-up in the response to therapy.

Warm ischemia-reperfusion injury is decreased by tacrolimus in steatotic rat liver

Ischemia-reperfusion (I-R) injury is poorly tolerated by fatty livers, most probably secondary to reduced cellular adenosine triphosphate (ATP) levels. We investigated the effectiveness of tacrolimus pretreatment on fatty liver I-R injury in obese Zucker rats. Tacrolimus (0.3 mg/kg, intravenously) was injected 24 hours before a 75-minute ischemic period and rats were sacrificed 6 hours later. Tacrolimus modified the response to I-R observed in obese Zucker rats, when compared to nontreated obese rats: a significant reduction in hepatocyte necrosis was associated with a significant increase in hepatocyte apoptosis. In addition, cell necrosis and apoptosis were significantly and inversely correlated in lean nontreated and treated obese Zucker rats following I-R. Tacrolimus also significantly increased the hepatic ATP levels, reduced in nontreated obese rats, toward values found in lean Zucker rat livers. This protective effect of tacrolimus was further confirmed in vivo by a significantly improved survival following pretreatment with tacrolimus, 24 hours prior to ischemia. In conclusion, in obese Zucker rat livers, tacrolimus pretreatment reversed the I-R injury toward the one found in lean Zucker rats. The correlations between ATP levels and the opposite changes in necrosis and apoptotic pathways strongly suggest a cause-effect relationship between tacrolimus and changes in ATP levels.

Cardiac energy metabolism at several stages of adriamycin-induced heart failure in rats

To evaluate the changes in myocardial energy metabolism in the progressively failing myocardium, we measured myocardial level of adenosine triphosphate (ATP) using high-performance liquid chromatography (HPLC), and levels of lactate, alanine and free carnitine using 1H-nuclear magnetic resonance (NMR) spectroscopy in rats injected with adriamycin. The drug was injected intraperitoneally 2.5 mg/kg 6 times over a period of 2 weeks. Measurements were obtained 1 day (1 d), 3 weeks (3 w) and 6 weeks (6 w) after the last injection. No deaths were observed until the end of the 3rd week. The cumulative mortality rate 6 weeks after the last injection was 48%. ATP and free carnitine levels were not significantly changed at 1 d, while myocardial lactate was increased by 33% from the control values ( P < 0.05). Lactate levels were reduced significantly, but not progressively, at 3 w (31% of control values) and at 6 w (69% of control values). Similar changes were observed in alanine levels. Free carnitine levels were progressively decreased at 3 w (74% of control values) and at 6 w (57% of control values). Changes in ATP levels paralleled those of free carnitine. Data suggest that a decrease in the myocardial level of free carnitine may be involved in progression of the heart failure induced by adriamycin in rats.

Aminoácidos libres en liquido cefalorraquídeo de recién nacidos con encefalopatía hipóxico isquémica

Cerebrospinal fluid free aminoacid (AA| composition depends on blood - CSF barrier, nervous tissue and ependimacaptation. It has been previously reported that in mammalian cerebral cortex a decrease of adenosinetriphosphate (ATP)is associated with an increase in asparlic acid, a deciease in glutarnic acid, aminobulyric acid and glutamine contentsand that fuel deprivation causes changes in brain levels of ATP and those mentioned aminoacids. Using gascromatography, after isobuthanol sterification of the carboxilic group and heptafluorbulyrate acylation of the aminogroup, free aminoacid content of cerebrospinal fluid and plasma were studied in eighl normal canlrol newborn infantsand eight patients with neonatal hipoxic ischemic encephalopathy in order to detect changes in these late, but nodifferences of aminoacid concentration in cerebrospinal fluid were recorded among both groups. This result is incontradiction with recent reports of increased excitatory aminoacid (glutamic and aspartic acid) concentration in thecerebrospinal fluid of six asphyxiated newborn infants, and of a good ccxfelalion of these changes and neurologicprognosis of patients. More experience is needed in these respecl.(Key words; amino acids, cerebiospinal fluid cerebral ischemia, newborn.)

Structure-function activity of azasterols and nitrogen-containing steroids.

Thirty-nine nitrogen-containing steroids were tested against two gram-negative, five gram-positive, and two yeast organisms. Many of these steroids have been previously reported to inhibit various metabolic processes involving sterol metabolism. While low minimal inhibitory concentration (MIC) values were recorded for sterol producing yeast, growth of bacteria which contain no sterols was also inhibited. Structure-function studies provided no relationship between biological activity and hypocholesteremic effects of these azasteroids. A hypothesis put forward is that amino and azasteroids are effectors of membrane which, in the case of mitochondria, lead to changes in adenosine triphosphate levels and/or dehydrogenase activity. Their effects on sterol metabolism, therefore, may be of secondary consideration.