DNA-based artificial molecular signaling system that mimics basic elements of reception and response

Availability of strategies for molecular biosensing over a finely adjustable dynamic range is essential for understanding and controlling vital biological processes. Herein we report design principles of highly responsive pH sensors based on a DNA i-motif where both response sensitivity and transition midpoint can be tuned with high precision over the physiologically relevant pH interval. The tuning is accomplished via rational manipulations of an i-motif structure as well as incorporation of allosteric control elements. This strategy delivers molecular sensing systems with a transition midpoint tunable with 0.1 pH units precision and with a total response range as narrow as 0.2 pH units which can be adjusted to a variety of outputs (e.g., fluorescent readout). The potential of the presented approach is not limited by pH sensing but may extend toward manipulation of other quadruplex based structures or the development of ultraresponsive elements for artificial molecular machines and signaling systems.

In the previous paper (S. Honda, T. Toraya, and S. Fukui, J. Bacteriol., 143, 1458-1465 (1980)), we reported that the glycerol-inactivated holoenzymes of adenosylcobalamin-dependent glycerol dehydratase and diol dehydratase are rapidly and continually reactivated in toluene-treated cells (in situ) by adenosine 5'-triphosphate (ATP) and divalent metal ions in the presence of free adenosylcobalamin. To elucidate the mechanism of this in situ reactivation, the nature of the binding of various irreversible cobalamin inhibitors to the dehydratases in situ was investigated. In the presence of ATP and Mn2+, enzyme-bound hydroxocobalamin, cyanocobalamin and methylcobalamin were rapidly displaced by added adenosylcobalamin. Without ATP and Mn2+, such displacement did not take place. In contrast, enzyme-bound adeninylbutylcobalamin and adenosylethylcobalamin were essentially not displaceable by the free coenzyme even in the presence of ATP and Mn2+. Inosylcobalamin was a very weak inhibitor irrespective of the presence of ATP and Mn2+. These results indicate that the relative affinity of the enzymes in situ for the cobalamins with simple Co beta ligands was markedly lowered in the presence of ATP and Mn2+, whereas that for the cobalamins with adenine-containing ligands was not. When the glycerol-inactivated holoenzymes in situ were dialyzed against a buffer containing ATP and Mg2+, the inactivated coenzyme moiety dissociated from the enzymes leaving apoproteins. Kinetic evidence was also obtained with the dehydratases in situ that continual displacement of the inactivated coenzyme moiety by adenosylcobalamin takes place during the glycerol dehydration reaction in the presence of ATP and Mn2+. Since the adenosyl group of the bound coenzyme is irreversibly removed from the cobalamin moiety during inactivation by glycerol, all of these data constitute clear evidence that the inactivated holo-dehydratases are reactivated in situ in the presence of ATP and Mn2+ by displacement of the modified coenzyme moiety by free intact adenosylcobalamin (i.e. selective B12-exchange mechanism).

Inhibition of ecto-ATPase activity by curcumin in hepatocellular carcinoma HepG2 cells.

Aptamer-based strip assay is an easy, highly efficient and low-cost detection method, which has been developed and easily applied to onsite detection. A new sensitive sandwich dipstick assay for adenosine triphosphate (ATP) detection was successfully developed based on specific recognition between split aptamer fragments and the target. In this method, the thiolated aptamer was first conjugated to the surface of gold nanoparticles (AuNPs), while the biotin-aptamer was immobilized on the surface of a nitrocellulose filter in the test line. In the presence of ATP, the thiol-aptamer/ATP/biotin-aptamer complexes were generated, which led to an obvious increase in optical signals at the test line. Under the optimal determination conditions, an excellent linear logarithmic response to the ATP concentration was obtained within the range of 0.5μM to 5mM. The limit of detection (LOD) of 0.5μM was reached at a signal-to-noise ratio of 3. The dipstick assay showed a good average recovery of 96-108% with the RSD of less than 20% in urine samples. The proposed method exhibited high specificity against other nucleotides such as the uridine triphosphate (UTP), cytidine triphosphate (CTP), and guanosine triphosphate (GTP). The results indicated that the dipstick strip may be considered as an inexpensive screening tool for onsite ATP determination. Graphical Abstract A simple split aptamer fragments based sandwich-type dipstick assay was developed for ATP detection.

Monitoring the fluctuation of adenosine 5'-triphosphate (ATP) at the subcellular level is important for the study of cell energy metabolism. Herein, we fabricated an electrochemical nanoaptasensor for continuously monitoring ATP fluctuation at the subcellular level. A gold nanoelectrode with a diameter of 120 nm was fabricated, and ferrocene (Fc)-labeled anti-ATP aptamer was self-assembled onto the nanoelectrode surface to form a nanoaptasensor. In the presence of ATP, the ferrocene-labeled anti-ATP aptamer bound with two ATP units to form an ATP-aptamer conjugation, resulting in the close proximity of Fc to the nanoelectrode surface and then an increase of oxidation current of Fc. ATP can be detected with a detection limit of 26 μM within 2 min. Cell viability assays indicated that the nanoaptasensor was biocompatible with negligible biological effects. By taking advantage of the good biocompatibility of the nanoaptasensor, ATP fluctuation at the subcellular level was monitored under glucose starvation and Ca2+ induction. This work demonstrates that the nanoaptasensor is a useful tool for investigating ATP-relevant biological processes via the electrochemical method.

1. Myosin-ATPase prepared from dog heart and rabbit skeletal muscle catalyzed the hydrolysis of adenosine tetraphosphate into adenosine diphosphate and orthophosphate. 2. Maximum activities were measured in the presence of Ca++ ions and represented 15 to 50% (according to pH and to origin) of the maximum activities measured in the presence of adenosine triphosphate (ATP). Low activities were measured in the presence of Co++ > Ni++ > Mg++ ions. 3. Actomyosin-ATPase prepared from rabbit and from carp muscles hardly catalyzed the hydrolysis of adenosine tetraphosphate (2.5% of the rate of ATP hydrolysis) in the presence of Mg++ ions at low ionic strength. 4. Ultracentrifugation analysis showed that carp muscle actomyosin is split into actin and myosin by low concentrations of adenosine tetraphosphate whereas the contaminating ATP or the ATP formed from adenosine tetraphosphate during the centrifugation does not markedly influence the centrifugal pattern.

Background and Aims Kidney is the principal contributor to circulating αKlotho (αKL), and simultaneously, constitutes the major organ where circulating αKlotho is taken up from the circulation. Klotho is mainly expressed in proximal renal tubule and in distal tubular epithelium. In the kidney soluble αKlotho acts from the urinary luminal side as an autocrine or paracrine enzyme to regulate transporters and ion channels. Recently, it has been demonstrated that the glomerulus is another source of αKlotho production. NPP1 is the best characterized nucleotide pyrophosphatases/phosphodiesterases (NPP) family member, encoded by the ENPP1 gene, and is a type II transmembrane glycoprotein that forms disulfide-bonded homodimers in the plasma membrane and in the mineral-depositing matrix vesicles. The major function of NPP1 is generation of PPi from adenosine triphosphate (ATP). Recently, we demonstrated that extracellular nucleotides stimulate an increase in glomerular albumin permeability, in part due to glomerular NO production and actin reorganization in podocytes . Furthermore, both αKlotho and NPP1 regulate insulin-dependent signaling. Thus, a key aim of this study is to fully examine the role of αKlotho on NPP1 function and its role in the protection of glomerular filtration barrier and podocyte function in diabetes. Method We measured glomerular permeability to albumin (Palb) in a single isolated rat glomerulus based on the video-microscopy method and permeability to albumin across the podocyte monolayer. Expression of αKlotho and NPP1 was confirmed in podocytes using Western blotting and immunofluorescence. Ecto-nucleotide pyrophosphatase/phosphodiesterase activity in podocytes was assessed using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. The ATP concentration was measured using luciferin-luciferase reaction. Results Since NPP1 and Klotho regulate insulin-dependent signaling we investigated the role of Klotho on NPP1 dimerization and NPPs activity. Podocyte treatment withαKlotho (0.5 nM, 30 min) in non-reducing conditions (maleimide, 100 mM) increased the disulfide bond formation of NPP1 (Fig. 1A). The percentage of dimerized (260 kDa) NPP1 increased approximately by 45%. This effect was abolished in the presence of ATP (100 µM, 30 min). As a positive control, we used hydrogen peroxide (100 μM, 5 min) which induces oxidative disulfide bond formation (Fig. 1A). We also demonstrated time-dependent decrease of NPPs activity in the presence of αKL (Fig. 1B). Klotho also induces an increase of extracellular ATP concentration by 76% (from 3.19±0.01 to 5.62±0.43, n=6, P<0.05). Moreover, we confirmed the role of soluble αKlotho in ATP-evoked increases of glomerular and podocyte albumin permeability. Conclusion Summarizing, we have clearly demonstrated an interaction between αKL and NPP1 in podocytes. Moreover, we observed the protective role of αKlotho in podocyte permeability to albumin.

Experimental proof of a compartment of “energy-rich”-P in a subcellular system from Pseudomonas

Clinical observations or animal studies implicate enteric glial cells in motility disorders, irritable bowel syndrome, inflammatory bowel disease, gastrointestinal (GI) infections, postoperative ileus, and slow transit constipation. Mechanisms underlying glial responses to inflammation in human GI tract are not understood. Our goal was to identify the "reactive human enteric glial cell (rhEGC) phenotype" induced by inflammation, and probe its functional relevance. Human enteric glial cells in culture from 15 GI-surgical specimens were used to study gene expression, Ca, and purinergic signaling by Ca/fluo-4 imaging and mechanosensitivity. A nanostring panel of 107 genes was designed as a read out of inflammation, transcription, purinergic signaling, vesicular transport protein, channel, antioxidant, and other pathways. A 24-hour treatment with lipopolysaccharide (200 μg/mL) and interferon-γ (10 μg/mL) was used to induce inflammation and study molecular signaling, flow-dependent Ca responses from 3 mL/min to 10 mL/min, adenosine triphosphate (ATP) release, and ATP responses. Treatment induced a "rhEGC phenotype" and caused up-regulation in messenger RNA transcripts of 58% of 107 genes analyzed. Regulated genes included inflammatory genes (54%/IP10; IFN-γ; CxCl2; CCL3; CCL2; C3; s100B; IL-1β; IL-2R; TNF-α; IL-4; IL-6; IL-8; IL-10; IL-12A; IL-17A; IL-22; and IL-33), purine-genes (52%/AdoR2A; AdoR2B; P2RY1; P2RY2; P2RY6; P2RX3; P2RX7; AMPD3; ENTPD2; ENTPD3; and NADSYN1), channels (40%/Panx1; CHRNA7; TRPV1; and TRPA1), vesicular transporters (SYT1, SYT2, SNAP25, and SYP), transcription factors (relA/relB, SOCS3, STAT3, GATA_3, and FOXP3), growth factors (IGFBP5 and GMCSF), antioxidant genes (SOD2 and HMOX1), and enzymes (NOS2; TPH2; and CASP3) (P < 0.0001). Treatment disrupted Ca signaling, ATP, and mechanical/flow-dependent Ca responses in human enteric glial cells. ATP release increased 5-fold and s100B decreased 33%. The "rhEGC phenotype" is identified by a complex cascade of pro-inflammatory pathways leading to alterations of important molecular and functional signaling pathways (Ca, purinergic, and mechanosensory) that could disrupt GI motility. Inflammation induced a "purinergic switch" from ATP to adenosine diphosphate/adenosine/uridine triphosphate signaling. Findings have implications for GI infection, inflammatory bowel disease, postoperative ileus, motility, and GI disorders.

The cationic water-soluble poly(diethynylfluorene-co-diethynylcarbazole-co-diethynylbenzothiadiazole) (P1–P3) were synthesized and used for adenosine triphosphate (ATP) detection. The incorporation of diethynylbenzothiadiazole (DBT) into the polymer backbone offers dual emissive polyelectrolytes with both blue and red emission bands in the dilute solution resulting from incomplete intramolecular energy transfer. In the solution of P2, addition of ATP can quench the emission of fluorene-co-carbazole units of P2 more efficiently than that of DBT segments, which causes the fluorescent color change from blue to red. The asymmetric quenching of the blue and red emission bands in the presence of ATP might become a possible method for a real-time ATP detection. In addition, when ATP is cleaved into fragments by enzyme alkaline phosphatase (ALP), the blue emission of P2 can be recovered. Thus, it is possible to assay the enzyme activity by triggering the change in color and intensity of the emission of the solution of P2.

Aptamer-conjugated silver nanoparticles for electrochemical detection of adenosine triphosphate

The binding mode between oxyhemoglobin (oxyHb) and adenosine triphosphate (ATP) has been studied using absorption and fluorescence spectroscopy. OxyHb forms a ground state complex with ATP supported by five isosbestic points which appear in absorption spectra of oxyHb in buffer solution on addition of ATP. Moreover, the changes in absorption spectra suggest an oxidative interaction between the particular interacting systems. The binding constant has been determined from the quenching of fluorescence of oxyHb in the presence of a varied concentration of ATP, and that is 3.8 × 10(3) M(-1) at 25 °C. The negative changes in entropy and enthalpy indicate that the binding is enthalpy driven and the hydrogen bond and van der Waals (stacking) interactions play a major role. The oxygen affinity of oxyHb decreases with simultaneous formation of metHb in the presence of ATP. ATP-induced structural changes have been affirmed using both circular dichroism spectroscopy and synchronous fluorescence. A theoretical docking study gives the molecular details about the binding site of ATP in oxyHb.

Myosin‐ATPase prepared from dog heart and rabbit skeletal muscle catalyzed the hydrolysis of adenosine tetraphosphate into adenosine diphosphate and orthophosphate. Maximum activities were measured in the presence of Ca++ ions and represented 15 to 50% (according to pH and to origin) of the maximum activites measured in the presence of adenosine triphosphate (ATP). Low activities were measured in the presence of Co++ &gt; Ni++ &gt; Mg++ ions. Actomyosin‐ATPase prepared from rabbit and from carp muscles hardly catalyzed the hydrolysis of adenosine tetraphosphate (2.5% of the rate of ATP hydrolysis) in the presence of Mg++ ions at low ionic strength. Ultracentrifugation analysis showed that carp muscle actomyosin is split into actin and myosin by low concentrations of adenosine tetraphosphate whereas the contaminating ATP or the ATP formed from adenosine tetraphosphate during the centrifugation does not markedly influence the centrifugal pattern.

A new electrochemical aptasensor based on electrocatalytic property of graphene toward ascorbic acid oxidation

Decision letter: Improved ANAP incorporation and VCF analysis reveal details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain