Quantities Of ATP Research Articles

The compartment-specific manipulation of the NAD+/NADH ratio affects the metabolome and the function of glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive type of cancer in the brain and has an inferior prognosis because of the lack of suitable medicine, largely due to its tremendous invasion. GBM has selfish metabolic pathways to promote migration, invasion, and proliferation compared to normal cells. Among various metabolic pathways, NAD (nicotinamide adenine dinucleotide) is essential in generating ATP and is used as a resource for cancer cells. LbNOX (Lactobacillus brevis NADH oxidase) is an enzyme that can directly manipulate the NAD+/NADH ratio. In this study, we found that an increased NAD+/NADH ratio by LbNOX or mitoLbNOX reduced intracellular glutamate and calcium responses and reduced invasion capacity in GBM. However, the invasion was not affected in GBM by rotenone, an ETC (Electron Transport Chain) complex I inhibitor, or nicotinamide riboside, a NAD+ precursor, suggesting that the crucial factor is the NAD+/NADH ratio rather than the absolute quantity of ATP or NAD+ for the invasion of GBM. To develop a more accurate and effective GBM treatment, our findings highlight the importance of developing a new medicine that targets the regulation of the NAD+/NADH ratio, given the current lack of effective treatment options for this brain cancer.

Impaired Long-Chain Fatty Acid Metabolism in Carnitine Palmitoyltransferase II Knockout Mouse Leads to Chronic Pancreatitis

Background: To support high rates of protein synthesis, pancreatic acinar cells must generate large quantities of ATP. Mitochondrial dysfunction and impaired energy metabolism play a significant role in the development of pancreatitis. Patients with elevated plasma triglycerides are at increased risk for development of severe acute pancreatitis. Carnitine palmitoyltransferase II (CPT2) is required for importing and thus metabolizing long-chain fatty acids (LCFAs) in mitochondria. To inhibit LCFA metabolism, we utilized the CPT2acinarKO mouse to probe the consequences of impaired LCFA import on pancreatic function. Hypothesis: Loss of CPT2 causes an accumulation of long-chain acylcarnitines and disruption of the tissue acylcarnitine/free carnitine ratio which is thought to alter cellular function. Given acinar cells’ high metabolic requirement, impaired LCFA utilization would reduce energy production, secretory function, and alter pancreatic lipid composition. Methods: We generated mice lacking CPT2 specifically in acinar cells (CPT2acinarKO) by crossing tamoxifen-inducible, Elastase-Cre mice with CPT2 floxed mice. Mice were fed a high fat (60% kcal fat), low fat (10% kcal fat), or chow (14% kcal fat) diet for 12 weeks. Analysis of fat accumulation, edema, and fibrosis was performed with H&E and picrosirius staining. Mass Spec. lipid profiling was performed in pancreas and serum. Acinar cells were isolated from wildtype, CPT2 floxed, and CPT2acinarKO mice to assess secretory function, ATP levels, and necrosis via lactate dehydrogenase (LDH) release. Results: Histologic examination revealed CPT2acinarKO mice on all three diets displayed pancreatic fat accumulation. Strikingly, CPT2acinarKO mice fed a chow or low fat diet developed chronic pancreatitis with a 25% loss in pancreatic weight, immune cell infiltration, edema, and fibrosis. As anticipated, long-chain acylcarnitines were significantly elevated in CPT2acinarKO pancreas. Assessment of secretion in 2 weeks post tamoxifen cells revealed significantly reduced maximal (10 pM) and supramaximal (1 nM) cholecystokinin (CCK) stimulated amylase release as a percent of total cellular amylase. Acini isolated at 4 weeks post tamoxifen revealed significantly increased basal secretion and reduced maximal and supramaximal CCK stimulated amylase release. When normalized to cellular DNA, secretion was similar between CPT2acinarKO and CPT2 floxed acinar cells indicating CPT2acinarKO cells had reduced amylase levels. Wildtype acinar cells treated with unsaturated and saturated free fatty acid concentrations as low as 100 μM for 3 hours caused significant necrosis. CPT2acinarKO pancreas had significantly decreased ATP levels indicative of mitochondrial dysfunction. Conclusion: Loss of CPT2 in the pancreatic acinar cells impairs LCFA import, reduces ATP levels, disrupts the secretory pathway, promoting chronic pancreatitis. In contrast to other CPT2 knockout models, pancreatitis occurs independent of high fat feeding in CPT2acinarKO mice. Further investigation is needed to identify metabolic alterations leading to acinar damage and mitochondrial dysfunction following the loss of CPT2. UW-Madison Comprehensive Diabetes Center Pilot Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical [Formula: see text] of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle.

Downregulating Mitochondrial DNA Polymerase γ in the Muscle Stimulated Autophagy, Apoptosis, and Muscle Aging-Related Phenotypes in Drosophila Adults.

Reactive oxygen species, generated as by-products of mitochondrial electron transport, can induce damage to mitochondrial DNA (mtDNA) and proteins. Here, we investigated whether the moderate accumulation of mtDNA damage in adult muscles resulted in accelerated aging-related phenotypes in Drosophila. DNA polymerase γ (Polγ) is the sole mitochondrial DNA polymerase. The muscle-specific silencing of the genes encoding the polymerase subunits resulted in the partial accumulation of mtDNA with oxidative damage and a reduction in the mtDNA copy number. This subsequently resulted in the production of abnormal mitochondria with reduced membrane potential and, consequently, a partially reduced ATP quantity in the adult muscle. Immunostaining indicated a moderate increase in autophagy and mitophagy in adults with RNA interference of Polγ (PolγRNAi) muscle cells with abnormal mitochondria. In adult muscles showing continuous silencing of Polγ, malformation of both myofibrils and mitochondria was frequently observed. This was associated with the partially enhanced activation of pro-apoptotic caspases in the muscle. Adults with muscle-specific PolγRNAi exhibited a shortened lifespan, accelerated age-dependent impairment of locomotor activity, and disturbed circadian rhythms. Our findings in this Drosophila model contribute to understanding how the accumulation of mtDNA damage results in impaired mitochondrial activity and how this contributes to muscle aging.

‘Reverse Warburg effect’ of cancer‑associated fibroblasts (Review)

Metabolic reprogramming is one of the main characteristics of malignant tumors. The metabolic reprogramming of tumors is not only related to the characteristics of cancer cells, but also closely related to the tumor microenvironment (TME). 'Aerobic glycolysis' is considered to be the classic metabolic mode of tumor cells. However, recent experiments have shown that the TME plays a key role in carcinogenesis and epithelial‑mesenchymal transition. Cancer‑associated fibroblasts (CAFs) dominate in the microenvironment and affect the homeostasis of the TME. The interaction between cancer cells and the surrounding CAFs markedly affects the growth, metabolism, metastasis, and progression of cancer. Based on this, a 'dual‑chamber' model, also known as the 'Reverse Warburg effect', is proposed. Specifically, cancer cells secrete hydrogen peroxide into the TME to induce oxidative stress in neighboring stromal cells. CAFs undergo aerobic glycolysis and produce high levels of energy‑rich 'fuels' (such as pyruvate, ketone bodies, fatty acids, and lactic acid). In turn, these energy‑rich 'fuels' then 'feed' cancer cells. The mitochondrial oxidative phosphorylation system produces a large quantity of ATP, such that tumor cells have a higher proliferation ability. The proposed 'Reverse Warburg effect' redefines the tumor cell microenvironment and tumor metabolic reprogramming. Therefore, understanding the 'Reverse Warburg effect' of CAFs and its related mechanisms will help us to understand the association between the microenvironment, the matrix, and cancer cells, and may lead to new treatment strategies and targets.

Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes.

To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method.

Circ\u03b2-catenin promotes tumor growth and Warburg effect of gallbladder cancer by regulating STMN1 expression

Gallbladder cancer (GBC) is the most malignant cancer of the biliary tract cancer and presents poor prognosis. CircRNAs have been identified as critical regulators of multiple stages in tumor progression. In the study, we first demonstrated that circular RNA circβ-catenin expression was upregulated in GBC tissues when compared to adjacent normal tissues and associated with advanced clinical stage and poor prognosis in GBC patients. Silencing of circβ-catenin obviously suppressed GBC cell proliferation and cell cycle progression in vitro, but circβ-catenin overexpression had the opposite effects. In vivo, silencing of circβ-catenin inhibited tumor growth. Furthermore, we also found that circβ-catenin promoted GBC cell lactate production, pyruvate production, ATP quantity, and extracellular acidification rate (ECAR), which suggested that circβ-catenin regulated Warburg effect in GBC. Mechanistic analysis further highlighted that circβ-catenin promoted Stathmin 1 (STMN1) expression through sponging miR-223 in GBC progression. In addition, knockdown of STMN1 inhibited cell growth and Warburg effect in GBC. In summary, our findings indicated that circβ-catenin/miR-223/STMN1 axis could regulate cell growth and Warburg effect in GBC. Targeting circβ-catenin might be a potential therapeutic strategy for GBC.

Circular RNA circCUL3 Accelerates the Warburg Effect Progression of Gastric Cancer through Regulating the STAT3/HK2 Axis

The Warburg effect is a significant hallmark of gastric cancer (GC), and increasing evidence emphasizes the crucial role of circular RNAs (circRNAs) in GC tumorigenesis. However, the precise molecular mechanisms by which circRNAs drive the GC Warburg effect are still elusive. The present study was designed to unveil the roles of circRNAs and the corresponding potential mechanism. High-regulated expression of circCUL3 was observed in both GC tissues and cell lines. Clinically, the high expression of circCUL3 was closely correlated with advanced clinical stage and overall survival in GC patients. Functionally, cellular experimental investigations demonstrated that circCUL3 promoted the proliferation, glucose consumption, lactate production, ATP quantity, and extracellular acidification rate (ECAR) of GC cells. In vivo, circCUL3 knockdown repressed tumor growth. Mechanistic analysis demonstrated that circCUL3 promoted signal transducer and activator of transcription (STAT)3 expression through sponging miR-515-5p; moreover, transcription factor STAT3 accelerated the transcriptional level of hexokinase 2 (HK2). In summary, the present findings provide mechanistic insights into circCUL3/miR-515-5p/STAT3/HK2 axis regulation on the GC Warburg effect, providing a novel possibility for an understanding of GC pathogenesis.

Bimetallic Cu–Rh Nanoparticles on Diazonium-Modified Carbon Powders for the Electrocatalytic Reduction of Nitrates

4-Benzenethiol-functionalized high-surface-area graphite powder was prepared and decorated with bimetallic Cu100-xRhx nanoparticles (NPs) to serve as electrocatalysts for the reduction of nitrates. In the first step, the HSAG powder was grafted with in-situ-generated diazonium compounds from 4-aminothiophenol (ATP) in an acidic medium using NaNO2 for the diazotization process. The surface composition was tuned using different initial quantities of ATP. The surface XPS-determined S/C atomic ratio was found to increase stepwise with the initial quantity of amine. In a second step, the grafted and untreated HSAG powders were decorated with Cu100-xRhx NPs by a wet chemical method and the elemental composition of the end composites was assessed by EDS-SEM and ICP, whereas TEM and EDS-TEM served to characterize the NP morphology and their composition on the nanometer scale. In all cases, the NP size was invariably found to be ∼1.7 nm but with a size distribution becoming narrower under an increasing grafting rate and the global composition enriched in copper. Voltammetry was performed with a cavity microelectrode to evaluate the electrocatalytic performance of the composites for nitrate reduction. Increasing diazonium grafting led to a progressive reduction of the peak current intensity and a shift of the peak potentials toward cathodic values. The maximum intensity was obtained for 0.005 μmol of diazonium salt per mg of HSAG, with a gain of 40% in comparison to the best untreated sample. This improvement and a change in the voltammogram characteristics after grafting seem to result from modifications of the local composition at the level of NPs that differ from the global composition. This work conclusively shows that diazonium surface modification is important not only in attaching electrocatalytic NPs to carbon supports but also in providing a narrower size distribution of the electrocatalysts together with finely tuned catalytic properties.

Adenine nucleotide-mediated regulation of hepatic PTP1B activity in mouse models of type 2 diabetes.

Plasma 5'-AMP (pAMP) is elevated in mouse models of type 2 diabetes. However, the metabolic regulatory role of adenine nucleotides in type 2 diabetes remains unclear. Adenine nucleotides and their metabolites in plasma and liver were examined by HPLC. 1H NMR-based metabolomics analysis was performed to explore the changes of metabolites in mouse models of type 2 diabetes. Na+/K+ ATPase and Na+/H+ exchanger activity were measured in response to adenine nucleotide metabolites. Human recombinant protein tyrosine phosphatase 1B (PTP1B) was used for enzyme kinetic assays. Protein binding assays were performed with microscale thermophoresis. The intracellular pH of hepatocyte AML12 cell lines was measured using the BCECF-AM method. We also analysed pAMP levels in participants with type 2 diabetes. Elevation of pAMP was a universal phenomenon in all mouse models of type 2 diabetes including db/db vs lean mice (13.9 ± 2.3μmol/l vs 3.7 ± 0.9μmol/l; p < 0.01), ob/ob vs lean mice (9.1 ± 2.0μmol/l vs 3.9 ± 1.2μmol/l; p < 0.01) and high-fat diet/streptozotocin-induced vs wild-type mice (6.6 ± 1.5μmol/l vs 4.1 ± 0.9μmol/l; p < 0.05); this elevation was required for the occurrence of hyperglycaemia in obese mice. 1H NMR-based metabolomics study following HPLC analysis revealed that the metabolite profile in wild-type mice treated with 5'-AMP was similar to that in db/db diabetic mice, especially the accumulation of a large quantity of ATP and its metabolites. The glucose-lowering drug metformin reduced the severity of hyperglycaemia both in 5'-AMP-induced wild-type mice and db/db mice. Metformin decreased the accumulation of liver ATP but not its metabolites in these hyperglycaemic mice. ATP and metformin reciprocally change cellular pH homeostasis in liver, causing opposite shifts in liver activity of PTP1B, a key negative regulator of insulin signalling. Furthermore, pAMP levels were also elevated in individuals with type 2 diabetes (45.2 ± 22.7nmol/l vs 3.1 ± 1.9nmol/l; p < 0.01). These results reveal an emerging role for adenine nucleotide in the regulation of hyperglycaemia and provide a potential therapeutic target in obesity and type 2 diabetes.

Human Bone Marrow Derived Mesenchymal Stromal Cells Enhance the Number and Function of Umbilical Cord Blood Peripheral Tregs during IL-2 Driven Ex Vivo Expansion

Tumor growth factor β (TGF-β)-induced peripheral regulatory T cells (pTreg) are a promising therapeutic cell source that exhibit Foxp3 expression and suppressive functions similar to natural regulatory T cells. Nonetheless, their clinical potential is limited by the instability of Foxp3 expression and T cell exhaustion that occurs during ex vivo expansion. We postulated that mesenchymal stromal cells (MSCs) could enhance the number, function and Foxp3 expression stability of pTregs during IL-2 driven 21 day expansion due to their diverse immunomodulatory properties. In this study, we observed that use of a human bone marrow mesenchymal stromal cells (hBM-MSC) platform significantly enhanced the number of pTreg during IL-2 driven 21 day ex vivo expansion vs. standard suspension culture condition (MSC platform: 80.2 x 106 vs. IL2/media: 39.3 x 106, n=6; p<0.01). Also the number of pTreg expressing a naive phenotype (CD4+CD45RA+ and CD4+CD62L+ ) were significantly increased (CD45RA+; MSC platform: 74.4 ± 1.6 x 106 vs. IL2/media: 45.9 ± 2.9 x 106, n=6, p<0.001; CD62L+; MSC platform: 79.1 ± 1.3 x 106 vs. IL2/media: 54.5 ± 2.1 x 106, n=6, p<0.001), as well as stability of Foxp3 expression (IL-2/media: 88.2 ± 1.7% vs. MSC platform: 96.2 ± 1.1%, n=7; p<0.05). In addition, pTreg suppressive function was noted to be more potent during 21 day IL-2 driven ex vivo expansion compared to standard IL-2/media culture condition (MSC platform: 79% vs. media: 35% inhibition of T cell proliferation in 10:1 ratio, n=6; p<0.01). pTreg expanded over a hBM-MSC platform exhibited higher surface CD25, CTLA-4, and ICOS MFI expression (CD25; MSC platform: 1410 vs. Media: 774; p<0.001, CTLA-4; MSC platform: 1084 vs. Media: 318; p<0.001, ICOS; MSC platform: 4386 vs. Media: 2641, p<0.01, n=6). Notably, hBM-MSC enhancement of pTreg ex vivo expansion requires direct cell-cell contact, as Foxp3 expression in pTreg was not enhanced by hBM-MSC conditioned media (CM:73.4 ± 6.8% vs. MSC platform: 96.2 ± 1.0%, p<0.001; and IL2/media: 88.8 ± 1.6% vs. MSC platform: 96.2 ± 1.0%, p<0.01) nor in a trans-well culture experiments (Transwell: 83.4 ± 2.5% vs. IL2/media: 88.8 ± 1.6%; and Transwell: 83.4 ± 2.5% vs. MSC platform: 96.2 ± 1.0%, p<0.01). Importantly, optical sectioning microscopy and flow cytometry revealed that hBM-MSC supports Treg number and function via direct contact-dependent mitochondrial transfer (Figure 1A-B). Cytochalasin B treatment blocked mitochondrial transfer, suggesting that tunneling nanotubes (TNT) facilitate mitochondrial transfer from hBM-MSC to pTreg during IL-2 driven ex vivo expansion (Mock: 2208 ± 122.1 vs. Cyto B: 923.8 ± 89 MFI, n=6, p<0.0001). Moreover, the quantity of ATP (n=6; p<0.01) mitochondrial potential of pTreg (MSC platform: 9010 ± 224.5 vs. media: 7316 ± 122.7 MFI, n=6; p<0.01) were significantly enhanced in pTreg during IL-2 driven ex vivo expansion over a hBM-MSC platform. Taken together, hBM-MSC significantly improves the number, maturation, and function of pTreg during 21 day IL-2 driven ex vivo expansion. We have identified one key mechanism of action of hBM-MSC underlying these favorable effects on pTreg during ex vivo expansion to be mitochondrial transfer via TNT. Notably, these studies identify a novel role of hBM-MSC to overcome current limitations in IL-2/media suspension culture conditions including T cell senescence, and loss of Foxp3 expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Regulation of Insulin-Stimulated Cardiac Glucose Transport via Protein Acetylation.

Cellular catabolism is the cell capacity to generate energy from various substrates to sustain its function. To optimize this energy production, cells are able to switch between various metabolic pathways in accordance to substrate availability via a modulation of several regulatory enzymes. This metabolic flexibility is essential for the healthy heart, an organ requiring large quantities of ATP to sustain its contractile function. In type 2 diabetes, excess of non-glucidic nutrients such as fatty acids, branched-chain amino-acids, or ketones bodies, induces cardiac metabolic inflexibility. It is characterized by a preferential use of these alternative substrates to the detriment of glucose, this participating in cardiomyocytes dysfunction and development of diabetic cardiomyopathy. Identification of the molecular mechanisms leading to this metabolic inflexibility have been scrutinized during last decades. In 1963, Randle demonstrated that accumulation of some metabolites from fatty acid metabolism are able to allosterically inhibit regulatory steps of glucose metabolism leading to a preferential use of fatty acids by the heart. Nevertheless, this model does not fully recapitulate observations made in diabetic patients, calling for a more complex model. A new piece of the puzzle emerges from recent evidences gathered from different laboratories showing that metabolism of the non-glucidic substrates induces an increase in acetylation levels of proteins which is concomitant to the perturbation of glucose transport. The purpose of the present review is to gather, in a synthetic model, the different evidences that demonstrate the role of acetylation in the inhibition of the insulin-stimulated glucose uptake in cardiac muscle.

ATP-synthesis capacity of pulsed light-exposed bacteria

The ability of four different bacteria to synthesize new ATP upon exposure to different doses of pulsed-light (PL) irradiation was investigated. The bacterial cells were PL treated on a gel surface, resuspended in phosphate buffered saline (PBS) and subsequently incubated in Tryptic Soy Broth (TSB) at 37°C. Cellular ATP levels were monitored during a 2h incubation period and compared to the respective colony count data. Although PL affected ATP production in a dose dependent manner, the results showed that bacteria, which had rendered unculturable after PL exposure, are still capable of generating significant quantities of ATP. Escherichia coli and Listeria innocua proved to be more resistant to PL than Salmonella enterica and Staphylococcus aureus, which was supported by the colony count data and the ATP synthesis capacity. These findings underline that bacteria undetectable by culture-based methods may still show cellular activity and synthesize new ATP.

ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

AbstractA new strategy is reported for the production of luminescence signals from DNA synthesis through the use of chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. ATP‐releasing nucleotides (ARNs) were synthesized as derivatives of the four canonical nucleotides. All four derivatives are good substrates for DNA polymerase, with Km values averaging 13‐fold higher than those of natural dNTPs, and kcat values within 1.5‐fold of those of native nucleotides. Importantly, ARNs were found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. When using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, thereby allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.

ATP-Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling.

A new strategy is reported for the production of luminescence signals from DNA synthesis through the use of chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. ATP-releasing nucleotides (ARNs) were synthesized as derivatives of the four canonical nucleotides. All four derivatives are good substrates for DNA polymerase, with Km values averaging 13-fold higher than those of natural dNTPs, and kcat values within 1.5-fold of those of native nucleotides. Importantly, ARNs were found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. When using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, thereby allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.

Impact of different temperatures on survival and energy metabolism in the Asian citrus psyllid, Diaphorina citri Kuwayama

Temperature influences the life history and metabolic parameters of insects. Asian citrus psyllid (ACP), Diaphorina citri is a tropical and subtropical pest. ACP invaded new regions around the world and threatened the citrus industry as a vector for Huanglongbing (HLB) disease. ACP is widely distributed and can survive high (up to 45°C) and low temperatures (as low as −6°C). The precise mechanism of temperature tolerance in ACP is poorly understood. We investigated adult survival, cellular energy balance, gene expression, and nucleotide and sugar-nucleotide changes under the effect of different temperature regimes (0°C to 45°C with 5°C intervals). The optimum temperatures for survival were 20 and 25°C. Low temperatures of 0°C and 5°C caused 50% mortality after 2 and 4days respectively, while one day at high temperature (40°C and 45°C) caused more than 95% mortality. The lowest quantity of ATP (3.69±1.6ng/insect) and the maximum ATPase enzyme activities (57.43±7.6μU/insect) were observed at 25°C. Correlation between ATP quantities and ATPase activity was negative. Gene expression of hsp 70, V-type proton ATPase catalytic subunit A and ATP synthase α subunit matched these results. Twenty-four nucleotides and sugar-nucleotides were quantified using HPLC in ACP adults maintained at low, high, and optimum temperatures. The nucleotide profiles were different among treatments. The ratios between AMP:ATP and ADP:ATP were significantly decreased and positively correlated to adults survival, whereas the adenylate energy charge was increased in response to low and high temperatures. Exploring energy metabolic regulation in relation with adult survival might help in understanding the physiological basis of how ACP tolerates newly invaded regions.

Ex vivo effects of ibogaine on the activity of antioxidative enzymes in human erythrocytes

Ethnopharmacological relevanceIbogaine is a naturally occurring alkaloid with psychotropic and metabotropic effects, derived from the bark of the root of the West African Tabernanthe iboga plant. The tribes of Kongo basin have been using iboga as a stimulant, for medicinal purposes, and in rite of passage ceremonies, for centuries. Besides, it has been found that this drug has anti-addictive effects. Aim of the studyPrevious studies have demonstrated that ibogaine changed the quantity of ATP and energy related enzymes as well as the activity of antioxidant enzymes in cells thus altering redox equilibrium in a time manner. In this work, the mechanism of its action was further studied by measuring the effects of ibogaine in human erythrocytes in vitro on ATP liberation, membrane fluidity and antioxidant enzymes activity. Materials and methodsHeparinized human blood samples were incubated with ibogaine (10 and 20μM) at 37°C for 1h. Blood plasma was separated by centrifugation and the levels of ATP and uric acid were measured 10min after the addition of ibogaine using standard kits. The activity of copper–zinc superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were measured in erythrocytes after incubation period. The stability of SOD1 activity was further tested through in vitro incubation with H2O2 and scanning of its electrophoretic profiles. Membrane fluidity was determined using an electron paramagnetic resonance spin-labelling method. ResultsResults showed that ibogaine treatment of erythrocytes in vitro increased ATP concentration in the blood plasma without changes in neither erythrocytes membrane fluidity nor uric acid concentration. Ibogaine also increased SOD1 activity in erythrocytes at both doses applied here. Treatment with 20μM also elevated GR activity after in vitro incubation at 37°C. Electrophoretic profiles revealed that incubation with ibogaine mitigates H2O2 mediated suppression of SOD1 activity. ConclusionSome of the effects of ibogaine seem to be mediated through its influence on energy metabolism, redox active processes and the effects of discrete fluctuations of individual reactive oxygen species on different levels of enzyme activities. Overall, ibogaine acts as a pro-antioxidant by increasing activity of antioxidative enzymes and as an adaptagene in oxidative distress.

Factors affecting the interactions between beta-lactoglobulin and fatty acids as revealed in molecular dynamics simulations.

Beta-lactoglobulin (BLG), a bovine dairy protein, is a promiscuously interacting protein that can bind multiple hydrophobic ligands. Fatty acids (FAs), common hydrophobic molecules bound to BLG, are important sources of fuel for life because they yield large quantities of ATP when metabolized. The binding affinity increases with the length of the ligands, indicating the importance of the van der Waals (vdW) interactions between the hydrocarbon tail and the hydrophobic calyx of BLG. An exception to this rule is caprylic acid (OCA) which is two-carbon shorter but has a stronger binding affinity than capric acid. Theoretical calculations in the current literature are not accurate enough to shed light on the underlying physics of this exception. The computed affinity values are greater for longer fatty acids without respect for the caprylic exception and those values are generally several orders of magnitude away from the experimental data. In this work, we used hybrid steered molecular dynamics to accurately compute the binding free energies between BLG and the five saturated FAs of 8 to 16 carbon atoms. The computed binding free energies agree well with experimental data not only in rank but also in absolute values. We gained insights into the exceptional behavior of caprylic acid in the computed values of entropy and electrostatic interactions. We found that the electrostatic interaction between the carboxyl group of caprylic acid and the two amino groups of K60/69 in BLG is much stronger than the vdW force between the OCA's hydrophobic tail and the BLG calyx. This pulls OCA to the top of the beta barrel where it is easier to fluctuate, giving rise to greater entropy of OCA at the binding site.

Study of baicalin on sympathoexcitation induced by myocardial ischemia via P2X3 receptor in superior cervical ganglia.

After the myocardial ischemia, injured myocardial tissues released large quantity of ATP, which activated P2X3 receptor in superior cervical ganglia and made the SCG postganglionic neurons excited. Excitatory of sympathetic postganglionic efferent neurons increased the blood pressure and heart rates, which aggravated the myocardial ischemic injury. Baicalin has anti-inflammatory and anti-oxidant properties. Our study showed that baicalin reduced the incremental concentration of serum CK-MB, cTn-T, epinephrine and ATP, decreased the up-regulated expression levels of P2X3 mRNA and protein in SCG after MI, and then inhibited the sympathetic excitatory activity triggered by MI injury. These results indicated that baicalin acted on P2X3 receptor was involved in the transmission of sympathetic excitation after the myocardial ischemic injury. Baicalin might decrease sympathetic activity via inhibiting P2X3 receptor in rat SCG to protect the myocardium.

Commentary: micromanaging oxidative stress: how gametes and embryos survive in their cozy niches!

Commentary: micromanaging oxidative stress: how gametes and embryos survive in their cozy niches!