Adenosine Triphosphate Treatment Research Articles

The dominant role of cooperation in fungal community drives the humification process of chicken manure composting under addition of regulatory factors

This study aimed to explore the action mechanism of fungal community on the enhancement of humification during chicken manure composting by regulating the core pathway of carbon metabolism - the tricarboxylic acid cycle. Regulators adenosine triphosphate (ATP) and malonic acid were added at the beginning of composting. The analysis of changes in humification parameters showed that the humification degree and stability of compost products were improved by adding regulators. Compared with CK, the humification parameters of adding regulators group increased by 10.98% on average. Meanwhile, adding regulators not only increased key nodes, but also strengthened the positive correlation between fungi, and network relationship was closer. Moreover, core fungi associated with humification parameters were identified by constructing OTU networks, and the division and cooperation mechanism of fungi were confirmed. Ultimately, the functional role of the fungal community acting on humification was confirmed by statistical means, that was, the fungal community promoting humification was the main group of composting process. And the contribution was more obvious in ATP treatment. This study was helpful to gain insight into the mechanism of regulators addition to advance the humification process, and provided new ideas for the safe, efficient and harmless disposal of organic solid waste.

Preharvest debagging alleviates external CO2 injury of ‘Fuji’ apple during storage by improving antioxidant capacity and energy status

‘Fuji’ apples that are commercially debagged before harvest are more resistant to external CO2 injury, a physiological disorder, than without debagging. In this study, debagged (bags removed 30 d before harvest) and bagged (bags not removed until harvest) apple fruit were stored in air and in controlled atmosphere storage regimes of 4 kPa O2/0 kPa CO2 and 4 kPa O2/5 kPa CO2 to explore the effects of preharvest debagging on the occurrence of external CO2 injury and the underlying mechanism of the injury. Debagging reduced disorder occurrence of the fruit from 8 d to at least 60 d. Preharvest debagging suppressed the accumulation of reactive oxygen species and malondialdehyde under CO2 stress by increasing the concentrations of antioxidant substances, including phenolics and ascorbic acid, and the activities of superoxide dismutase and catalase. In vitro free radicals scavenging activities further confirmed that the debagged fruit had higher antioxidant activity than the bagged fruit under elevated-CO2 storage conditions. Compared with the bagged fruit, the debagged fruit had higher contents of adenosine triphosphate (ATP) and adenosine diphosphate, level of energy charge, and activities of energy metabolism-related enzymes, including succinate dehydrogenase, cytochrome C oxidase, and Ca2+-ATPase under CO2 stress. ATP treatment inhibited the disorder occurrence ‘Fuji’ apple. These results reveal that the effect of preharvest debagging on inhibiting the occurrence of external CO2 injury of ‘Fuji’ apple was associated with the improvement of antioxidant capacity and energy status of apple peels under CO2 stress.

Adenosine Triphosphate Exerts Cardioprotective Effect on High-Dose Atorvastatin-Induced Heart Damage in Rats

Atorvastatin is a statin derivated hypolipidemic drug used in the treatment of hyperlipidemia. High-dose atorvastatin has been shown to significantly reduce adenosine triphosphate (ATP) levels in the heart tissue. Reduction of ATP by atorvastatin causes increased production of reactive oxygen species (ROS), decreased antioxidants, subsequent cell membrane and mitochondrial damage. The present study aimed to biochemically investigate the protective effect of ATP against possible cardiac damage caused by high dose atorvastatin in rats. Male Wistar rats were divided into atorvastatin (ATR), atorvastatin+ATP (AAT) and healthy control (HG) groups. ATP at a 25 mg/kg dose was injected intraperitoneally (ip) to the AAT (n-6) group. 0.9% NaCl as solvent was applied to the ATR (n-6) and HG (n-6) groups by the same route. Afterward, atorvastatin was administered orally at a dose of 20 mg/kg to the AAT and ATR groups. This procedure was repeated once daily for four weeks. At the end of this period, blood samples were taken into tubes to analyze troponin-I (TP-I) by cardiac puncture before animals were sacrificed with high-dose anesthesia. In addition, heart tissues were removed and malondialdehyde (MDA), total glutathione (tGSH), total oxidant (TOS) and total antioxidant (TAS) levels were measured. Biochemical test results showed that in the heart tissues of the ATR group, the oxidative parameters MDA and TOS significantly increased, while the antioxidant parameters tGSH and TAS significantly decreased compared to AAT and HG. Atorvastatin alone administration significantly increased blood TP-I levels, a marker of cardiac tissue damage. However, ATP administration to AAT group animals brought oxidative parameter levels closer to HG, despite high-dose atorvastatin treatment. In addition, the significant decrease in antioxidant levels was prevented by ATP application. High doses of atorvastatin can cause heart damage. ATP treatment was able to prevent atorvastatin-induced oxidative heart damage.

Extracellular ATP promotes angiogenesis and adhesion of TNBC cells to endothelial cells via upregulation of CTGF.

Our previous works have indicated that extracellular ATP is an important prometastasis factor. However, the molecular mechanism involved needs to be further studied. We demonstrated that extracellular ATP treatment could upregulate the expression of connective tissue growth factor (CTGF) in both triple‐negative breast cancer (TNBC) cells and endothelial cells (ECs). Extracellular ATP stimulated the migration of TNBC cells and ECs, and angiogenesis of ECs via the P2Y2––YAP‐CTGF axis. Furthermore, we demonstrated that adenosine triphosphate (ATP) stimulated TNBC cell adhesion to ECs and transmigration through the EC layer via CTGF by upregulation of integrin β1 on TNBC cells and VCAM‐1 on ECs. Both apyrase (ATP‐diphosphohydrolase) and CTGF shRNA treatments could inhibit the metastasis of inoculated tumors to lung and liver in a mouse model, and these treated tumors had fewer blood vessels. Collectively, our data indicated that extracellular ATP promotes tumor angiogenesis and the interactions between TNBC cells and ECs through upregulation of CTGF, thereby stimulating TNBC metastasis. The pleiotropic effects of ATP in angiogenesis and cell adhesion suggest that extracellular ATP or CTGF could be an effective target for TNBC therapy.

Exogenous ATP attenuated fermentative metabolism in postharvest strawberry fruit under elevated CO2 atmosphere by maintaining energy status

Elevated CO2 shows adverse effects in horticultural crops including off-flavor formation and carbohydrate consumption. Here, 1 mM adenosine triphosphate (ATP) was applied to strawberry fruit under 20 % CO2 atmosphere to investigate its regulation on fermentative and carbohydrate metabolism. The results showed that ATP treatment increased endogenous ATP content by 27 %, and delayed the decrease of energy charge under elevated CO2 atmosphere. Exogenous ATP showed no adverse effects on fruit firmness and color but attenuated the accumulation of acetaldehyde and ethanol, which were 72 % and 75 % lower in ATP+CO2-treated fruit compared CO2-treared fruit, respectively. The inhibition of fermentative metabolism resulted from the repression of pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) activities, as well as FaADH expression. Meanwhile, ATP treatment also maintained carbohydrate levels under elevated CO2 atmosphere, with 6 %, 7 %, and 11 % more glucose, fructose and sucrose observed at the end of the storage period as compared with the CO2-treated group, respectively. The down-regulation of enzymes activities and gene expressions involved in sucrose catabolism and glycolysis may account for the inhibition of carbohydrate consumption. These results indicated that exogenous ATP might be a strategy to optimize elevated CO2 treatment to avoid its adverse effects.

Paxillin mediates ATP-induced activation of P2X7 receptor and NLRP3 inflammasome

BackgroundExtracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets. ATP directly activates the plasma membrane channel P2X7 receptor (P2X7R), leading to an intracellular influx of K+, a key trigger inducing NLRP3 inflammasome activation. However, the mechanism underlying P2X7R-mediated activation of NLRP3 inflammasome is poorly understood, and additional molecular mediators have not been identified. Here, we demonstrate that Paxillin is the molecule connecting the P2X7 receptor and NLRP3 inflammasome through protein interactions.ResultsWe show a distinct mechanism by which Paxillin promotes ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and then facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K+ efflux. Moreover, we demonstrated that USP13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from the cytosol to the plasma membrane and facilitates P2X7R-Paxillin interaction and PaxillinNLRP3 association, resulting in the formation of the P2X7R-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as well as in human PBMCs and THP-1-differentiated macrophages.ConclusionsWe have identified paxillin as a mediator of NLRP3 inflammasome activation. Paxillin plays key roles in ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7R-Paxillin-NLRP3 complex.

Current status of fertility preservation for women with collagen disease undergoing gonadotoxic therapy in Nagasaki University Hospital

Alterations in the number and protein/gene expression of Hofbauer cells (HBCs) may play a role in microbial-driven/cytokine-mediated placental inflammation, and in subsequent pregnancy complications such as villitis, histologic chorioamnionitis, and the fetal inflammatory response syndrome. Pyroptosis is an inflammatory form of cell death mediated by the inflammasome, a multi-protein complex which drives the processing and secretion of interleukin 1 beta (IL-1β). Pyroptosis can be triggered by bacterial lipopolysaccharide (LPS) and adenosine triphosphate (ATP) in non-placental macrophages through activation of the NLRP3 inflammasome. However, the role of inflammasome activation and pyroptosis in HBC pathophysiology remains unclear. HBCs isolated from human term placentas were treated with or without LPS or ATP, alone or in combination. Treatment of HBCs with both LPS and ATP induced the rapid secretion of high levels of IL-1β and at the same time, cell death associated with nuclear condensation and cellular swelling. HBC treatment with both LPS and ATP induced caspase-1 activation, gasdermin D (GSDMD) cleavage, which mediates pyroptosis, and IL-1β processing. Caspase-1 activation, GSDMD cleavage, IL-1β processing, and IL-1β secretion were all significantly reduced following NLRP3 knockdown; inhibition of caspase-1; and inhibition of P2X7, the receptor that mediates K+ efflux. Together, our data indicate that LPS and ATP treatment stimulated NLRP3 inflammasome activation and pyroptosis in HBCs leading to the rapid release of IL-1β. Since the localization of HBCs confers a unique ability to influence microbial-associated placental and fetal inflammation, these studies suggest a key role for the inflammasome and pyroptosis in mediating HBC driven inflammation.

NLRP3 inflammasome function and pyroptotic cell death in human placental Hofbauer cells

Alterations in the number and protein/gene expression of Hofbauer cells (HBCs) may play a role in microbial-driven/cytokine-mediated placental inflammation, and in subsequent pregnancy complications such as villitis, histologic chorioamnionitis, and the fetal inflammatory response syndrome. Pyroptosis is an inflammatory form of cell death mediated by the inflammasome, a multi-protein complex which drives the processing and secretion of interleukin 1 beta (IL-1β). Pyroptosis can be triggered by bacterial lipopolysaccharide (LPS) and adenosine triphosphate (ATP) in non-placental macrophages through activation of the NLRP3 inflammasome. However, the role of inflammasome activation and pyroptosis in HBC pathophysiology remains unclear. HBCs isolated from human term placentas were treated with or without LPS or ATP, alone or in combination. Treatment of HBCs with both LPS and ATP induced the rapid secretion of high levels of IL-1β and at the same time, cell death associated with nuclear condensation and cellular swelling. HBC treatment with both LPS and ATP induced caspase-1 activation, gasdermin D (GSDMD) cleavage, which mediates pyroptosis, and IL-1β processing. Caspase-1 activation, GSDMD cleavage, IL-1β processing, and IL-1β secretion were all significantly reduced following NLRP3 knockdown; inhibition of caspase-1; and inhibition of P2X7, the receptor that mediates K+ efflux. Together, our data indicate that LPS and ATP treatment stimulated NLRP3 inflammasome activation and pyroptosis in HBCs leading to the rapid release of IL-1β. Since the localization of HBCs confers a unique ability to influence microbial-associated placental and fetal inflammation, these studies suggest a key role for the inflammasome and pyroptosis in mediating HBC driven inflammation.

Intraspecific Variation in Growth and Alkaline Phosphatase Activity of Cylindrospermopsis raciborskii Strains in Response to Different Phosphorus Concentrations and Sources

The cyanobacterial species C. raciborskii are ubiquitous in tropical regions, and its successful invasion into temperate zones has been partially attributed to its ability of survival in low P availability and the existence of multiple ecotypes. To explore the physiological response of different strains to phosphorus fluctuations, four strains of C. raciborskii isolated from the Zhenhai Reservoir were used to investigate their growth and alkaline phosphatase (ALP) activity at different inorganic phosphorus (Pi) concentrations (HP=7.13 mg ·L-1, MP=0.64 mg ·L-1, LP=0.03 mg ·L-1) and different phosphorus forms [dipotassium hydrogen phosphate (K2HPO4), sodium pyrophosphate (K4P2 O7), sodium polyphosphate (K5P3O10), D-glucose-6-phosphate (D-G-6-P), adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP)]. Four C. raciborskii strains showed a similar growth response to phosphate changes: their biomass increased with an increase in Pi concentrations, while the ALP activity showed the opposite trend. The ALP activity of C. raciborskii N8 was significantly lower than that of other three strains, regardless of inorganic phosphorus concentrations, suggesting that this strain had a higher adaptability to phosphorus fluctuations. When cultured with different phosphorus forms, the biomass of C. raciborskii N8 and N9 in three dissolved inorganic phosphorus (DIP) compounds were significantly higher than those in three dissolved organic phosphorus (DOP) compounds, with the maximum and minimum specific growth rate in K2HPO4and ATP treatments, respectively. C. raciborskii preferred DIP although they can also utilize DOP to sustain its growth. Under the DOP conditions, the ALP activity of C. raciborskii N8 in the ATP treatment was significantly higher than that in the other two organic phosphorus compounds, while we did not observe similar results in C. raciborskii N9, indicating that strain N8 was more sensitive to DIP deficiency. Our results showed an intraspecific variation within C. raciborskii strains from the same reservoir. Compared with the other strains, strain N8 represented better adaptability to phosphorus fluctuations and DIP deficiency. Variations within C. raciborskii strains may make this species more adaptable to environmental changes and enhance its competitive advantage.

The influence of ATP treatment on energy dissipation system in postharvest longan fruit during senescence

Abstract The harvested longan fruit are prone to senescence, leading to a decline in fruit quality like pericarp browning. There are two energy dissipative proteins involving in fruit senescence: alternative oxidase (AOX) and plant uncoupling mitochondrial protein (PUMP). The purpose of this work was to elucidate the influence of adenosine triphosphate (ATP) treatment on energy dissipation system in postharvest longan fruit during senescence. The results showed that the ATP content in postharvest longan fruit exhibited negative correlations with the expressions of DlAOX1, DlAOX2, DlPUMP3, and DlPUMP5. Moreover, compared to the control longan fruit, the treatment of exogenous ATP prevented the increases in the expressions of DlAOX1, DlAOX2, DlPUMP3, and DlPUMP5, helped maintain higher contents of ATP, ADP and higher level of energy charge, but lower respiration rate, lower cell membrane permeability, and lower pericarp browning index in postharvest longan fruit. These data indicated that the supply of exogenous ATP could reduce the induction of the expressions of DlAOX1, DlAOX2, DlPUMP3 and DlPUMP5, and consequently reduce the energy dissipation and retard the decline of energy level, which lead to the delayed senescence in postharvest longan fruit.

Manipulation of cellular energy reveals the relationship between ultraweak luminescence and cellular energy during senescence of strawberry (Fragaria × ananassa) fruits

The goal of this study was to investigate the ultraweak luminescence (UWL) of strawberry fruits in relation to mitochondrial functions and energy production during strawberry senescence. Fully ripe strawberry fruits and mitochondria isolated from those fruits were treated with either adenosine triphosphate (ATP) or the respiratory chain uncoupler 2,4-dinitrophenol (DNP). The activities of H+-ATPase and Ca2+-ATPase, the content of ATP, the free radical O2− as well as the UWL intensity were measured. Our results showed that activities of H+-ATPase and Ca2+-ATPase as well as the ATP content gradually decreased during fruit senescence in all three groups. Compared with the control, DNP treatment exacerbated, while ATP treatment reduced the decrease of H+-ATPase and Ca2+-ATPase activity, the energy charge and UWL intensity. UWL intensity was positively correlated with mitochondrial function and ATP content. Our results strongly suggest that mitochondria are a major source of UWL of strawberry fruits, and that the cellular energy ATP plays important roles in senescence of strawberry fruits, and in UWL formation. Our study provides convincing evidence of the interrelationship between cellular energy and UWL, which helps researchers to better understand the process of senescence in strawberry fruits.

Effect of NaHS on ATP-induced P2X receptor expression in rat microglia

To observed the effect of sodium hydrosulphide (NaHS), a donor of H2S on the cell viability,the membrane permeability and the expression of P2X7 receptor induced by adenosine triphosphate(ATP) in rat microglia. Rat microglia in logarithmic growth phase was randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treatment with ATP after cultured for 24 hours. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, the others were as the same as NaHS+ATP group. The cell viability was detected by MTT. Fluorescent dyes YO-PRO-1 was used to observe the membrane permeability. The expression of P2X7 receptor was examined by immunofluorescence staining. ① Compared with control group, the cell viability dropped after treatment with ATP (1、3、5、10 mmol/L) for 3 hours. When pre-incubation with NaHS(200 μmol/L), the cell viability was apparently higher than that of ATP alone group(P<0.01), while 400 μmol/L had no further beneficial.②The YO-PRO-1 fluorescence intensity was obviously elevated by ATP in rat microglia, but this effect was counteracted by NaHS pretreatment (P<0.01). ③ The expression of P2X7 receptor protein was significantly increased after ATP(3 mmol/L) for 3 h. While the expression upregulation of P2X7 receptor protein induced by ATP was significantly counteracted by pretreating with NaHS(200 μmol/L) (P<0.01). NaHS could reduce the expression of P2X7 receptor, decrease membrane permeability, and increase the cell viability in rat microglia injured by ATP. So the cytoprotection of hydrogen sulfide may be related to the expression and function of P2X7 receptor.

Sanitation indicators as a tool to evaluate a food safety and sanitation training program for farmstead cheese processors

This project evaluated the impact of a food safety and sanitation training program, developed for farmstead cheesemakers, by investigating its effects on the cleanliness of cheesemaking rooms of dairy farms in Pennsylvania. Participating farms (n = 16) were divided randomly into control (n = 6; no training) and two treatments, consisting of a food safety and sanitation training program without a video vignette (treatment 1; n = 5) and a training program supplemented with a video vignette (treatment 2; n = 5). Before the training and again 3–4 months after the training, environmental samplings were conducted on select surfaces in cheesemaking rooms. Surfaces were swabbed and evaluated for aerobic bacterial counts (AC), Enterobacteriaceae (EB), yeast and molds (YM), Listeria spp., and for levels of adenosine triphosphate (ATP). The results demonstrate that the training program, with or without the video vignette, significantly reduced (p < 0.05) populations (log10 CFU/100 cm2) of AC (treatment 2 = 1.23), EB (treatment 1 = 1.18; treatment 2 = 0.89), and ATP (treatment 1 = 0.41; treatment 2 = 0.61) in samples taken from floors and drains. The results from this study may serve as a reference for future evaluations of food safety-related training programs that look beyond changes in employee knowledge, attitudes, skills, or behavior, and address or correlate to other potential indicators of sanitation, such as microbial counts and ATP levels.

ATP-Induced Inflammasome Activation and Pyroptosis Is Regulated by AMP-Activated Protein Kinase in Macrophages

Adenosine triphosphate (ATP) is released by bacteria and host cells during bacterial infection as well as sterile tissue injury, acting as an inducer of inflammasome activation. Previous studies have shown that ATP treatment leads to AMP-activated protein kinase (AMPK) activation. However, it is unclear whether AMPK signaling has been involved in the regulation of ATP-induced inflammasome activation and subsequent pyroptosis. In this study, we aimed to investigate this issue in lipopolysaccharide-activated murine macrophages. Our results showed that AMPK signaling was activated in murine macrophages upon ATP treatment, which was accompanied by inflammasome activation and pyroptosis as evidenced by rapid cell membrane rupture as well as mature interleukin (IL)-1β and active caspase-1p10 release. The ATP-induced inflammasome activation and pyroptosis were markedly suppressed by an AMPK inhibitor compound C or small-interfering RNA-mediated knockdown of AMPKα, but could be greatly enhanced by metformin (a well-known AMPK agonist). Importantly, metformin administration increased the mortality of mice with bacterial sepsis, which was likely because metformin treatment enhanced the systemic inflammasome activation as indicated by elevated serum and hepatic IL-1β levels. Collectively, these data indicated that the AMPK signaling positively regulated ATP-induced inflammasome activation and pyroptosis in macrophages, highlighting the possibility of AMPK-targeting therapies for inflammatory diseases involving inflammasome activation.

Antihyperlipidemic activity of adenosine triphosphate in rabbits fed a high-fat diet and hyperlipidemic patients

Context Recently, adenosine triphosphate (ATP) was occasionally found to decrease the triglyceride (TG) levels in several hyperlipidemic patients in our clinical practice.Objective The study investigates the anti-hyperlipidemic effects of ATP in a high-fat fed rabbit model and hyperlipidemic patients.Materials and methods Twenty-four rabbits were randomly divided into three groups of eight animals each as follows: normal diet, high-fat diet and high-fat diet + ATP group. ATP supplementation (40 mg/day) was started at the 20th day and lasted for 10 days. Serum concentrations of total cholesterol (TC), TG, LDL-C, HDL-C were measured on the 20th day and 30th day. Heart, liver and aorta were subjected histopathological examination. Twenty outpatients diagnosed primary hyperlipidemia took ATP at a dose of 60 mg twice a day for 1 week.Results Feeding rabbits with a high-fat diet resulted in a significant elevation of lipid parameters including TC, TG, LDL-C, VLDL-C compared to the normal diet group (p < 0.01). ATP treatment significantly decreased serum TG level (p < 0.01), whilst other parameters remained statistically unaltered. Meanwhile, ATP significantly reduced the thickness of fat layer in cardiac epicardium (p < 0.05) and pathological gradation of ballooning degeneration in hepatocytes (p < 0.05). After taking ATP for 1 week, hyperlipidemia patients exhibited a significant decrease of TG (p < 0.01), but other lipid parameters had no significant change.Discussion and conclusion The study indicates that ATP selectively decreases serum TG levels in high-fat diet rabbits and hyperlipidemic patients. Therefore, ATP supplementation may provide an effective approach to control TG level.

Establishment of a flow cytometry-based test for the detection of pyroptosis of BMDM

Objective To establish a flow cytometry-based test for the detection of pyroptosis of murine bone marrow-derived macrophages (BMDM). Methods Bone marrow cells were isolated from wild type (WT) C57BL/6 mice and/or caspase-1-/- C57BL/6 mice and then stimulated with macrophage colony-stimulating factor (M-CSF) to differentiate into murine BMDM. PBS, LPS and LPS+ adenosine triphosphate (ATP) were respectively used to stimulate the BMDM. Western blot assay was performed to detect the cleavage of IL-1β and caspase-1. The levels of IL-1β in the supernatants of cell culture were measured by ELISA. Lactate dehydrogenase (LDH) released in the culture media was detected by using LDH kit. The pyroptosis of murine BMDM was detected by using flow cytometry analysis after double-staining with TMR red+ caspase-1, AnnexinⅤ+ caspase-1 and propidium iodide (PI)+ caspase-1. Results IL-1β was detected in the culture medium of BMDM treated with LPS+ ATP and the cleavage of IL-1β and caspase-1 was confirmed by Western blot assay, which indicated that the NLRP3 inflammasome was activated by LPS+ ATP treatment. Compared with the caspase-1-/- mice group, higher levels of LDH were detected in the culture medium of BMDM isolated form the WT mice. Results of the flow cytometry analysis after staining BMDM with caspase-1 plus AnnexinⅤ or PI showed that more cells undergoing pyroptosis were detected in the LPS+ ATP treatment group than that in LPS or PBS treatment group, which were consistent with the results of the reported flow cytometry with caspase-1+ TMR red staining. Conclusion The flow cytometry-based test with double-staining of caspase-1 plus AnnexinⅤ or PI could be used for the detection of pyroptosis of murine BMDM. Key words: NLRP3 inflammasome; Pyroptosis; Flow cytometry

ATP Induces IL-1β Secretion in Neisseria gonorrhoeae-Infected Human Macrophages by a Mechanism Not Related to the NLRP3/ASC/Caspase-1 Axis.

Neisseria gonorrhoeae (Ngo) has developed multiple immune evasion mechanisms involving the innate and adaptive immune responses. Recent findings have reported that Ngo reduces the IL-1β secretion of infected human monocyte-derived macrophages (MDM). Here, we investigate the role of adenosine triphosphate (ATP) in production and release of IL-1β in Ngo-infected MDM. We found that the exposure of Ngo-infected MDM to ATP increases IL-1β levels about ten times compared with unexposed Ngo-infected MDM (P < 0.01). However, we did not observe any changes in inflammasome transcriptional activation of speck-like protein containing a caspase recruitment domain (CARD) (ASC, P > 0.05) and caspase-1 (CASP1, P > 0.05). In addition, ATP was not able to modify caspase-1 activity in Ngo-infected MDM but was able to increase pyroptosis (P > 0.01). Notably ATP treatment defined an increase of positive staining for IL-1β with a distinctive intracellular pattern of distribution. Collectively, these data demonstrate that ATP induces IL-1β secretion by a mechanism not related to the NLRP3/ASC/caspase-1 axis and likely is acting at the level of vesicle trafficking or pore formation.

Effects of Adenosine Triphosphate (ATP) Treatment on Postharvest Physiology, Quality and Storage Behavior of Longan Fruit

The effects of adenosine triphosphate (ATP) on postharvest physiology, quality, and storage behavior of harvested longan fruit were investigated. Fruit of “Fuyan” longan were dipped in 0.8 mM ATP solution or distilled water (control) for 20 min. The fruit were dried prior to packaging and then stored at 28 °C and 90 % relative humidity. The results showed that as compared with control fruit, application of ATP could significantly reduce fruit respiration rate; delay an increase of cell membrane permeability in pericarp; maintain higher contents of chlorophyll, carotenoid, anthocyanin, and flavonoid; and subsequently retard the change of apparent color on longans. In addition, lower polyphenol oxidase (PPO) activity and higher total phenolics content were also found in ATP-treated fruit, which contributed to lower browning index in longan pericarp. The application of ATP could effectively inhibit the decreases in contents of total soluble solids (TSS), total soluble sugars, and vitamin C with a limited increase of titratable acidity (TA) content in pulp, which maintain better quality and flavor in longan pulp. Furthermore, ATP treatment could keep a higher rate of commercially acceptable fruit, effectively prevent weight loss from longans, and retard the developments of aril breakdown and fruit disease in longan fruit. It could be concluded that 0.8 mM ATP treatment for 20 min could greatly delay longan fruit senescence, maintain higher fruit quality, and exhibit better storage behavior which might suggest a promising method for keeping quality and extending the shelf life of harvested “Fuyan” longan fruit.

ATP promotes extracellular matrix biosynthesis of intervertebral disc cells.

We have recently found a high accumulation of extracellular adenosine triphosphate (ATP) in the center of healthy porcine intervertebral discs (IVD). Since ATP is a powerful extracellular signaling molecule, extracellular ATP accumulation might regulate biological activities in the IVD. The objective of this study was therefore to investigate the effects of extracellular ATP on the extracellular matrix (ECM) biosynthesis of porcine IVD cells isolated from two distinct anatomical regions: the annulus fibrosus (AF) and nucleus pulposus (NP). ATP treatment significantly promotes ECM deposition and corresponding gene expression (aggrecan and type II collagen) by both cell types in three-dimensional agarose culture. A significant increase in ECM accumulation has been found in AF cells at a lower ATP treatment level (20 μM) compared with NP cells (100 μM), indicating that AF cells are more sensitive to extracellular ATP than NP cells. NP cells also exhibit higher ECM accumulation and intracellular ATP than AF cells under control and treatment conditions, suggesting that NP cells are intrinsically more metabolically active. Moreover, ATP treatment also augments the intracellular ATP level in NP and AF cells. Our findings suggest that extracellular ATP not only promotes ECM biosynthesis via a molecular pathway, but also increases energy supply to fuel that process.

Phenolic components, antioxidant enzyme activities and anatomic structure of longan fruit pericarp following treatment with adenylate triphosphate

Fruit senescence and pericarp browning are strongly related to the antioxidant properties and are affected by the cellular energy status. In the present study, longan fruits were treated with 1mmolL−1 adenosine triphosphate (ATP) before storage for six days at 25°C. The phenolic contents and antioxidant enzyme activities in relation to pericarp browning were determined, and the fruit pericarp structure was observed under a confocal laser scanning microscope (CLSM) and transmission electron microscope. Increases in the browning index, membrane permeability, peroxidase (POD) activity and gallic acid content were slowed during storage following exogenous ATP application, whereas the flavonoids accumulation was increased significantly by ATP treatment. In addition, decrease in catechin, corilagin, epicatechin and gallocatechin gallate were accelerated by exogenous ATP treatment. The cuticle in the pericarp of the untreated longan fruit was incomplete, the organelle was swollen or disintegrated, the grana lamellae was destroyed and osmiophilic particles accumulated in the chloroplast, the cytoplasm was granulated and the fruiting body of a possible pathogenic organism was observed after storage. However, the pericarp structure maintained its relative integrity without any visible pathogenic organisms, as determined with CLSM in the ATP-treated fruit. It is suggested that ATP application protects longan fruit from pathogen attack, maintains the pericarp structure and delays senescence, which are closely related to the fruit's higher total phenolic levels and lower POD activity.