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Adenosine 5′-triphosphate (ATP) is the energy currency in cells. It is involved in numerous cellular life activities and exhibits a close association with the development of certain diseases. Thus, the precise detection of ATP within cells holds immense significance in understanding cell biological events and related disease development. Fluorescent probes have obvious advantages in imaging ATP in cells and in vivo due to their high sensitivity, good selectivity, real-time imaging, and good biocompatibility. Thus far, an extensive array of fluorescent probes targeting ATP has been formulated to enable the visualization of ATP within cells and in vivo. This review summarizes the recent advances in ATP fluorescent probes according to different design strategies, mainly including those based on organic small molecules, metal complexes, and water-soluble conjugated polymers. In addition, the practical applications of ATP fluorescent probes in the imaging of target organelles, cell biological events, and disease markers are highlighted. Finally, the challenges and future trends of ATP detection based on fluorescent probes are discussed. [ABSTRACT FROM AUTHOR]

Adenosine 5′-triphosphate (ATP) is the energy currency in cells. It is involved in numerous cellular life activities and exhibits a close association with the development of certain diseases. Thus, the precise detection of ATP within cells holds immense significance in understanding cell biological events and related disease development. Fluorescent probes have obvious advantages in imaging ATP in cells and in vivo due to their high sensitivity, good selectivity, real-time imaging, and good biocompatibility. Thus far, an extensive array of fluorescent probes targeting ATP has been formulated to enable the visualization of ATP within cells and in vivo. This review summarizes the recent advances in ATP fluorescent probes according to different design strategies, mainly including those based on organic small molecules, metal complexes, and water-soluble conjugated polymers. In addition, the practical applications of ATP fluorescent probes in the imaging of target organelles, cell biological events, and disease markers are highlighted. Finally, the challenges and future trends of ATP detection based on fluorescent probes are discussed.

Whereas adenosine 5'-triphosphate (ATP) is the major energy source in cells, extracellular ATP (eATP) released from activated/damaged cells is widely thought to represent a potent damage-associated molecular pattern that promotes inflammatory responses. Here, we provide suggestive evidence that eATP is constitutively produced in the uninflamed lymph node (LN) paracortex by naïve T cells responding to C-C chemokine receptor type 7 (CCR7) ligand chemokines. Consistently, eATP was markedly reduced in naïve T cell-depleted LNs, including those of nude mice, CCR7-deficient mice, and mice subjected to the interruption of the afferent lymphatics in local LNs. Stimulation with a CCR7 ligand chemokine, CCL19, induced ATP release from LN cells, which inhibited CCR7-dependent lymphocyte migration in vitro by a mechanism dependent on the purinoreceptor P2X7 (P2X7R), and P2X7R inhibition enhanced T cell retention in LNs in vivo. These results collectively indicate that paracortical eATP is produced by naïve T cells in response to constitutively expressed chemokines, and that eATP negatively regulates CCR7-mediated lymphocyte migration within LNs via a specific subtype of ATP receptor, demonstrating its fine-tuning role in homeostatic cell migration within LNs. [ABSTRACT FROM AUTHOR]

Whereas adenosine 5’-triphosphate (ATP) is the major energy source in cells, extracellular ATP (eATP) released from activated/damaged cells is widely thought to represent a potent damage-associated molecular pattern that promotes inflammatory responses. Here, we provide suggestive evidence that eATP is constitutively produced in the uninflamed lymph node (LN) paracortex by naïve T cells responding to C-C chemokine receptor type 7 (CCR7) ligand chemokines. Consistently, eATP was markedly reduced in naïve T cell-depleted LNs, including those of nude mice, CCR7-deficient mice, and mice subjected to the interruption of the afferent lymphatics in local LNs. Stimulation with a CCR7 ligand chemokine, CCL19, induced ATP release from LN cells, which inhibited CCR7-dependent lymphocyte migration in vitro by a mechanism dependent on the purinoreceptor P2X7 (P2X7R), and P2X7R inhibition enhanced T cell retention in LNs in vivo. These results collectively indicate that paracortical eATP is produced by naïve T cells in response to constitutively expressed chemokines, and that eATP negatively regulates CCR7-mediated lymphocyte migration within LNs via a specific subtype of ATP receptor, demonstrating its fine-tuning role in homeostatic cell migration within LNs.

Jacopo M Fontana,1 Huijuan Yin,1 Yun Chen,2 Ricardo Florez,1 Hjalmar Brismar,1 Ying Fu1 1Section of Cellular Biophysics, Department of Applied Physics, Royal Institute of Technology, Science for Life Laboratory, Solna, 2Department of Molecular and Clinical Medicine/Clinical Physiology, The Sahlgrenska Academy and University Hospital, University of Gothenburg, Gothenburg, Sweden Abstract: Colloidal semiconductor quantum dots (QDs) have been extensively researched and developed for biomedical applications, including drug delivery and biosensing assays. Hence, it is pivotal to understand their behavior in terms of intracellular transport and toxicological effects. In this study, we focused on 3-mercaptopropionic acid-coated CdSe-CdS/ZnS core-multishell quantum dots (3MPA-QDs) converted from the as-grown octadecylamine-coated quantum dots (ODA-QDs) and their direct and dynamic interactions with human umbilical vein endothelial cells (HUVECs). Live cell imaging using confocal fluorescence microscopy showed that 3MPA-QDs first attached to and subsequently aggregated on HUVEC plasma membrane ~25 min after QD deposition. The aggregated QDs started being internalized at ~2 h and reached their highest internalization degree at ~24 h. They were released from HUVECs after ~48 h. During the 48 h period, the HUVECs responded normally to external stimulations, grew, proliferated and wound healed without any perceptible apoptosis. Furthermore, 1) 3MPA-QDs were internalized in newly formed LysoTracker-stained early endosomes; 2) adenosine 5'-triphosphate-induced [Ca2+]i modulation caused a transient decrease in the fluorescence of 3MPA-QDs that were attached to the plasma membrane but a transient increase in the internalized 3MPA-QDs; and 3) fluorescence signal modulations of co-stained LysoTracker and QDs induced by the lysosomotropic agent Gly-Phe-β-naphthylamide were spatially co-localized and temporally synchronized. Our findings suggest that 3MPA-QDs converted from ODA-QDs are a potential nontoxic fluorescent probe for future use in clinical applications. Moreover, the photophysical strategy and techniques reported in this work are easily applicable to study of direct interactions between other nanoparticles and live cells; contributing to awareness and implementation of the safe applications of nanoparticles. Keywords: colloidal semiconductor quantum dots, human umbilical vein endothelial cell, intracellular labeling, nanotoxicity, adenosine 5'-triphosphate, endosome

Hypophosphatasia (HPP) is a rare heritable metabolic bone disease caused by hypomorphic mutations in the ALPL (in human) or Akp2 (in mouse) gene, encoding the tissue-nonspecific alkaline phosphatase (TNAP) enzyme. In addition to skeletal and dental malformations, severe forms of HPP are also characterized by the presence of spontaneous seizures. Initially, these seizures were attributed to an impairment of GABAergic neurotransmission caused by altered vitamin B6 metabolism. However, recent work by our group using knockout mice null for TNAP (TNAP-/-), a well-described model of infantile HPP, has revealed a deregulation of purinergic signaling contributing to the seizure phenotype. In the present study, we report that adult heterozygous (TNAP+/-) transgenic mice with decreased TNAP activity in the brain are more susceptible to adenosine 5′-triphosphate (ATP)-induced seizures. Interestingly, when we analyzed the extracellular levels of ATP in the cerebrospinal fluid, we found that TNAP+/- mice present lower levels than control mice. To elucidate the underlying mechanism, we evaluated the expression levels of other ectonucleotidases, as well as different proteins involved in ATP release, such as pannexin, connexins, and vesicular nucleotide transporter. Among these, Pannexin-1 (Panx1) was the only one showing diminished levels in the brains of TNAP+/- mice. Altogether, these findings suggest that a physiological regulation of extracellular ATP levels and Panx1 changes may compensate for the reduced TNAP activity in this model of HPP.

The cerebral microcapillary endothelium, known as the blood–brain barrier (BBB), acts as a barrier between the blood and the interstitial fluid of the brain. The BBB therefore controls the passage of nutrients into the central nervous system (CNS). Microglia show a specific affinity for migration into the CNS, and this migration appears to occur independently of BBB integrity. To study the migration of microglia across the BBB, we developed an in vitro co-culture system of mouse brain endothelial cells (MBECs) and Ra2 microglia using Transwell inserts. We first investigated the influence of microglia or ATP, a microglial chemotactic factor, on MBEC barrier integrity. The addition of microglia or ATP led to the disruption of the MBEC monolayer and significantly decreased barrier function as measured by trans-endothelial electrical resistance (TEER) and electric cell–substrate impedance sensing (ECIS). Furthermore, ATP promoted the migration of microglia but not macrophages across the MBEC monolayer. An inhibitor of matrix metalloproteinases (MMPs) decreased the transmigration of microglia in our system, indicating that MMPs play a role in microglial chemotaxis. We specifically identify a role for microglia-derived MMP-2. In conclusion, we offer evidence that microglia migration across the brain endothelial cell monolayer is increased in the presence of ATP in a manner that involves MMP secretion. [ABSTRACT FROM AUTHOR]

The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ 1, γ2 or γ 3 isoform. When assayed at a physiological ATP concentration (5 mM), γ 1- and γ 2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ 3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr172 phosphorylation, when cells were incubatedwith mitochondrial inhibitors that increase cellular AMP. With γ 1 complexes, activation and Thr172 phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKKβ)] in cellfree assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ2 and γ 3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr172 dephosphorylation; with γ 2 complexes, ADP had similar potency to AMP, but with γ1 and γ 3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles. [ABSTRACT FROM AUTHOR]

Tribbles (TRIB) proteins are pseudokinase mediators of eukaryotic signalling that have evolved important roles in lipoprotein metabolism, immune function and cellular differentiation and proliferation. In addition, an evolutionary-conserved modulation of PI3K/AKT signalling pathways highlights them as novel and rather unusual pharmaceutical targets. The three human TRIB family members are uniquely defined by an acidic pseudokinase domain containing a 'broken' a C-helix and a MEK (MAPK/ERK)-binding site at the end of the putative C-lobe and a distinct C-terminal peptide motif that interacts directly with a small subset of cellular E3 ubiquitin ligases. This latter interaction drives proteasomal-dependent degradation of networks of transcription factors, whose rate of turnover determines the biological attributes of individual TRIB family members. Defining the function of individual Tribs has been made possible through evaluation of individual TRIB knockout mice, siRNA/overexpression approaches and genetic screening in flies, where the single TRIB gene was originally described 15 years ago. The rapidly maturing TRIB field is primed to exploit chemical biology approaches to evaluate endogenous TRIB signalling events in intact cells. This will help define how TRIB-driven protein-protein interactions and the atypical TRIB ATP-binding site, fit into cellular signalling modules in experimental scenarios where TRIB-signalling complexes remain unperturbed. In this minireview, we discuss how small molecules can reveal rate-limiting signalling outputs and functions of Tribs in cells and intact organisms, perhaps serving as guides for the development of new drugs. We predict that appropriate small molecule TRIB ligands will further accelerate the transition of TRIB pseudokinase analysis into the mainstream of cell signalling. [ABSTRACT FROM AUTHOR]

The ubiquitous pannexin 1 (Panx1) ion- and metabolitepermeable channelmediates the release ofATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1-EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp74). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease. [ABSTRACT FROM AUTHOR]

There are nineteen different receptor proteins for adenosine, adenine and uridine nucleotides, and nucleotide sugars, belonging to three families of G protein-coupled adenosine and P2Y receptors, and ionotropic P2X receptors. The majority are functionally expressed in blood vessels, as purinergic receptors in perivascular nerves, smooth muscle and endothelial cells, and roles in regulation of vascular contractility, immune function and growth have been identified. The endogenous ligands for purine receptors, ATP, ADP, UTP, UDP and adenosine, can be released from different cell types within the vasculature, as well as from circulating blood cells, including erythrocytes and platelets. Many purine receptors can be activated by two or more of the endogenous ligands. Further complexity arises because of interconversion between ligands, notably adenosine formation from the metabolism of ATP, leading to complex integrated responses through activation of different subtypes of purine receptors. The enzymes responsible for this conversion, ectonucleotidases, are present on the surface of smooth muscle and endothelial cells, and may be coreleased with neurotransmitters from nerves. What selectivity there is for the actions of purines/pyrimidines comes from differential expression of their receptors within the vasculature. P2X1 receptors mediate the vasocontractile actions of ATP released as a neurotransmitter with noradrenaline (NA) from sympathetic perivascular nerves, and are located on the vascular smooth muscle adjacent to the nerve varicosities, the sites of neurotransmitter release. The relative contribution of ATP and NA as functional cotransmitters varies with species, type and size of blood vessel, neuronal firing pattern, the tone/pressure of the blood vessel, and in ageing and disease. ATP is also a neurotransmitter in non-adrenergic non-cholinergic perivascular nerves and mediates vasorelaxation via smooth muscle P2Y-like receptors. ATP and adenosine can act as neuromodulators, with the most robust evidence being for prejunctional inhibition of neurotransmission via A 1 adenosine receptors, but also prejunctional excitation and inhibition of neurotransmission via P2X and P2Y receptors, respectively. P2Y 2 , P2Y 4 and P2Y 6 receptors expressed on the vascular smooth muscle are coupled to vasocontraction, and may have a role in pathophysiological conditions, when purines are released from damaged cells, or when there is damage to the protective barrier that is the endothelium. Adenosine is released during hypoxia to increase blood flow via vasodilator A 2A and A 2B receptors expressed on the endothelium and smooth muscle. ATP is released from endothelial cells during hypoxia and shear stress and can act at P2Y and P2X4 receptors expressed on the endothelium to increase local blood flow. Activation of endothelial purine receptors leads to the release of nitric oxide, hyperpolarising factors and prostacyclin, which inhibits platelet aggregation and thus ensures patent blood flow. Vascular purine receptors also regulate endothelial and smooth muscle growth, and inflammation, and thus are involved in the underlying processes of a number of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

The ATP:ADP molar ratio is an important physiological factor. However, in previous literatures, ATP and ADP could not be distinguished by Raman spectroscopy due to the high similarity of molecular structure. To challenge this problem, also considering that the γ phosphate group may interact with adenine group and cause a different variation of the Raman spectrum than that of ADP, a highly sensitive, low-cost, environment protecting, flexible and super-hydrophobic Au nanoparticles/cicada wing (Au/CW) substrate with three-dimension structure was fabricated and employed as an active surface-enhanced Raman scattering (SERS) substrate to detect the ATP:ADP molar ratios. The concentration as low as 10 −8 M for ATP and ADP was analyzed to determine the limit of detection. This SERS study on various ATP:ADP molar ratios demonstrates that ATP:ADP could be distinguished and the quantitative determination of ATP content was achieved. Moreover, a principle was speculated based on the molecular structures of ATP and ADP of the Raman peaks centered at ~685 and ~731 cm −1 to explain the linear relationship between the area ratio and the molar ratio. A new method has been developed for quantitative determination of ATP:ADP molar ratio based on Au/CW substrate by the SERS method. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is one of the major nutrients required for growth of phytoplankton. While dissolved inorganic phosphorus (DIP) is the preferred form of P, phytoplankton can utilize dissolved organic phosphorus (DOP) when DIP is limited. How DIP deficiency affects the progression of the cell division cycle and how well DOP can restore normal cell cycle in phytoplankton is unclear. Here we investigated the effects of DIP limitation and supply of adenosine 5′-triphosphate (ATP) as sole P-source on the growth and cell cycle in Prorocentrum donghaiense , one of the most common harmful algae bloom dinoflagellates in East China Sea. Part of the culture under DIP-replete condition progressed through a complete cell cycle (G1 → S → G2M) in 24 h with a typical diel rhythm of each phase, exhibiting a daily growth rate of 0.40 d −1 and average cell size 11.19 ± 0.06 μm in equivalent spherical diameter. Under phosphorus deficiency, population growth of P. donghaiense averaged at 0.09 d −1 and the cell cycle was blocked in G1 phase, while cell size averaged at 11.65 ± 0.05 μm. When DIP was replaced by ATP at equivalent molar concentration, the cultures displayed similar growth rate, cell size, and cell cycle oscillation as in the DIP-replete treatment. The results suggest that phosphorus deficiency blocks the cell cycle due to the inability to complete DNA duplication or check-point protein phosphorylation while still allowing photosynthesis and metabolisms, and that DOP such as ATP can fully complement DIP in P. donghaiense . [ABSTRACT FROM AUTHOR]

The human Tribbles (TRB)-related pseudokinases are CAMK (calcium/calmodulin-dependent protein kinase)-related family members that have evolved a series of highly unusual motifs in the 'pseudocatalytic' domain. In canonical kinases, conserved amino acids bind to divalent metal ions and align ATP prior to efficient phosphoryl-transfer to substrates. However, in pseudokinases, atypical residues give rise to diverse and often unstudied biochemical and structural features that are thought to be central to cellular functions. TRB proteins play a crucial role in multiple signalling networks and overexpression confers cancer phenotypes on human cells, marking TRB pseudokinases out as a novel class of drug target. In the present paper, we report that the human pseudokinase TRB2 retains the ability to both bind and hydrolyse ATP weakly in vitro. Kinase activity is metalindependent and involves a catalytic lysine residue, which is conserved in TRB proteins throughout evolution alongside several unique amino acids in the active site. A similar low level of autophosphorylation is also preserved in the closely related human TRB3. By employing chemical genetics, we establish that the nucleotide-binding site of an 'analogue-sensitive' (AS) TRB2 mutant can be targeted with specific bulky ligands of the pyrazolopyrimidine (PP) chemotype. Our analysis confirms that TRB2 retains low levels of ATP binding and/or catalysis that is targetable with small molecules. Given the significant clinical successes associated with targeting of cancer-associated kinases with small molecule inhibitors, it is likely that similar approaches will be useful for further evaluating the TRB pseudokinases, with the translation of this information likely to furnish new leads for drug discovery. [ABSTRACT FROM AUTHOR]

Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure-function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs. [ABSTRACT FROM AUTHOR]

Highlights: [•] Some cancer cells recover from severe damage that causes cell death in majority of cells. [•] Damage-Recovered (DR) cancer cells show reduced mitochondria, mDNA and mitochondrial enzymes. [•] DR cells show increased aerobic glycolysis, ATP, cell proliferation, and resistance to damage. [•] DR cells recovered from in vivo damage also show increased glycolysis and proliferation rate. [Copyright &y& Elsevier]

Abstract: Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine kinases, which function as key modulators of survival and metastasis signals in cancer cells. Previously, we reported that small molecule chlorpyramine hydrochloride (C4) specifically targets the interaction between FAK and VEGFR3 and exhibits anti-tumor efficacy. In this study, we designed and synthesized a series of 1 (C4) analogs on the basis of structure activity relationship and molecular modeling. The resulting new compounds were evaluated for their binding to the FAT domain of FAK and anti-cancer activity. Amongst all tested analogs, compound 29 augmented anti-proliferative activity in multiple cancer cell lines with stronger binding to the FAT domain of FAK and disrupted the FAK–VEGFR3 interaction. In conclusion, we hope that this work will contribute to further studies of more potent and selective FAK–VEGFR3 protein–protein interaction inhibitors. [Copyright &y& Elsevier]

Abstract: The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNAPyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNAPyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs. [Copyright &y& Elsevier]

Abstract: Several regulatory and signaling molecules governing angiogenesis are targets of interest for the development of drugs in the cancer, including growth factors such as Vascular Endothelial Growth Factor (VEGF) and Platelet-Derived Growth Factor (PDGF). A series of 4-aryloxy-6,7-dimethoxyquinazolines, previously synthesized in our laboratory, has shown a nanomolar inhibition of kinase enzymatic activity of VEGFR, PDGFR and c-Kit. We have therefore studied the impact of the variation in the 7-position substitution of the quinazoline core. Substitution by aminoalkoxy chains led to new highly potent ATP-competitive inhibitors of VEGFR, PDGFR and c-Kit enzyme with IC50 values in the nanomolar range and this substitution has increased greatly antiproliferative activity on cancer cell lines (PC3, MCF7, HT29) and HUVEC (human umbilical vein endothelial cells). One of the most promising compounds (36) was assessed for its ability to limit the induction of web like network of capillary tubes by the human umbilical vascular endothelial cells (HUVEC) and for its ability to inhibit invasion. [Copyright &y& Elsevier]

Abstract: Sevoflurane is one of inhalation anesthetics and has been commonly used in obstetric and pediatric anesthesia. The widespread use of sevoflurane in newborns and infants has made its safety a health issue of concern. Voltage-gated Ca2+ channels (VGCCs) play an important role in neuronal excitability and are essential for normal brain development. However, the role of sevoflurane on regulating Ca2+ channels during the period of rapid brain development is still not well understood. The aim of this study is to explore the effects of sevoflurane on voltage-gated Ca2+ channels for hippocampal CA1 pyramidal neurons during the period of rapid brain development. 1-week-old Sprague-Dawley rats were randomly divided into 3 groups: control group, 2.1% sevoflurane group (exposed to 2.1% sevoflurane for 6h) and 3% sevoflurane group (exposed to 3% sevoflurane for 6h). Whole-cell patch clamp technique was used. I–V curve, steady-state activation and inactivation curves of Ca2+ channels were studied in rats of the both 3 treated groups at 5 different ages (1 week, 2 weeks, 3 weeks, 4 and 5 weeks old). After anesthesia with sevoflurane at 1-week-old rats, Ca2+ channels current density was significantly decreased at week 1 and week 2 (p<0.01). And 3% sevoflurane exposure resulted in a rightward shift in steady-state activation curve at week 1 and week 2, as well as the inactivation curve from week 1 to week 3. However, the 2.1% sevoflurane-induced rightward shift was only found in steady-state inactivation curve of Ca2+ channels at week 1 and week 2. Both the slope factor (k) of Ca2+ channels activation and inactivation curves increased by 3% sevoflurane at week 1 (p<0.05). Therefore, early exposure to sevoflurane persistently inhibits Ca2+ channels activity in hippocampal CA1 pyramidal neurons of developing rats but the development of Ca2+ channels recovers to normal level at juvenile age. Moreover, the inhibition of 3% sevoflurane on VGCCs is greater than that of 2.1% sevoflurane. [Copyright &y& Elsevier]

Highlights: [•] ARS administration produces nephrotoxicity. [•] ARS causes ultra structural alterations in the kidney. [•] The renal proximal tubule, particularly its BBM seems to be the major ARS target. [•] ARS appeared to shift energy dependence from aerobic to anaerobic glycolysis by causing damage to mitochondria. [•] ARS caused nephrotoxic and other deleterious effects by increasing ROS generation. [ABSTRACT FROM AUTHOR]

Abstract: Binding of extracellular adenosine 5′-triphosphate (ATP) or lipopolysaccharide (LPS) to the damage-associated molecular pattern receptor P2X7 or the pathogen-associated molecular pattern receptor Toll-like receptor (TLR)4, respectively, can induce the release of the pleiotropic cytokine interleukin (IL)-1β in humans and mice. However, the release of IL-1β in dogs remains poorly defined. Using a canine IL-1β enzyme-linked immunosorbent assay, this study investigated whether ATP or LPS could induce IL-1β release in a canine blood-based assay. Short-term incubations (30min) with ATP induced IL-1β release in LPS-primed canine blood, and this process could be near-completely impaired by the P2X7 antagonist, A438079. In contrast, ATP failed to induce IL-1β release from blood not primed with LPS. ATP-induced IL-1β release was observed with LPS-primed blood from eight different pedigrees or cross breeds. Long-term incubations (24h) with LPS induced IL-1β release in canine blood in a concentration-dependent manner. This process was not altered by co-incubation with A438079. LPS-induced IL-1β release was observed with blood from 10 different pedigrees or cross breeds. These results demonstrate that both extracellular ATP and LPS can induce IL-1β release in dogs, and that ATP- but not LPS-induced IL-1β release in blood is dependent on P2X7 activation. These findings support the role of both P2X7 and TLR4 in IL-1β release in dogs. [Copyright &y& Elsevier]

Abstract: ClpAP is an ATP-dependent protease that assembles through the association of hexameric rings of ClpA with the cylindrically-shaped protease ClpP. ClpA contains two nucleotide binding domains, termed Domain 1 (D1) or 2 (D2). We have proposed that D1 or D2 limits the rate of ClpA catalyzed polypeptide translocation when ClpP is either absent or present, respectively. Here we show that the rate of ClpA catalyzed polypeptide translocation depends on [ATPγS] in the absence of ClpP, but not in the presence of ClpP. We observe that ATPγS non-cooperatively binds to ClpA during polypeptide translocation with an apparent affinity of ~6μM, but that introduction of ClpP shifts this affinity such that translocation is not affected. Interpreting these data with our proposed model for translocation catalyzed by ClpA vs. ClpAP suggests that ATPγS competes for binding at D1 but not at D2. [Copyright &y& Elsevier]

The function of F1-ATPase relies critically on the intrinsic ability of its catalytic and noncatalytic subunits to interact with nucleotides. Therefore, the study of isolated subunits represents an opportunity to dissect elementary energetic contributions that drive the enzyme's rotary mechanism. In this study we have calorimetrically characterized the association of adenosine nucleotides to the isolated noncatalytic α-subunit. The resulting recognition behavior was compared with that previously reported for the isolated catalytic β-subunit (N.O. Pulido, G. Salcedo, G. Pérez-Hernández, C. José-Núñez, A. Velázquez-Campoy, E. García-Hernández, Energetic effects of magnesium in the recognition of adenosine nucleotides by the F1-ATPase β subunit, Biochemistry 49 (2010) 5258–5268). The two subunits exhibit nucleotide-binding thermodynamic signatures similar to each other, characterized by enthalpically-driven affinities in the μM range. Nevertheless, contrary to the catalytic subunit that recognizes MgATP and MgADP with comparable strength, the noncatalytic subunit much prefers the triphosphate nucleotide. Besides, the α-subunit depends more on Mg(II) for stabilizing the interaction with ATP, while both subunits are rather metal-independent for ADP recognition. These binding behaviors are discussed in terms of the properties that the two subunits exhibit in the whole enzyme. [ABSTRACT FROM AUTHOR]

Highlights: [•] Basal and ATP-activated phenotypes of microglia vary non-linearly with age. [•] Neurotoxicity and production of microglial effectors are highest in neonatal microglia. [•] 13–15-mo-old microglia show more effector activity than microglia from younger adults. [Copyright &y& Elsevier]

Highlights: [•] Rottlerin induces cytoskeletal changes consistent with astrocyte activation and stellation. [•] Rottlerin inhibits glutamate uptake via a decreased rate of transport, not a loss in cell surface glutamate transporters. [•] Loss of glutamate uptake was reversible and enhanced by co-treatment with monensin. [•] Na+/K+-ATPase activity and ATP levels are also reduced with exposure to rottlerin. [•] Recovery of Na+/K+-ATPase activity preceded Glu uptake rescue supporting partial uncoupling of these processes. [Copyright &y& Elsevier]

Highlights: [•] Chemiluminescence (CL) detection with magnetic microparticles and nanoparticles. [•] Principles of CL detection assisted by magnetic microparticles and nanoparticles. [•] Improved features of CL detection with magnetic microparticles and nanoparticles. [•] Improved selectivity of CL detection with magnetic microparticles and nanoparticles. [ABSTRACT FROM AUTHOR]

Highlights: [•] 8-Nitro-cGMP is a unique cGMP derivative formed in response to NO and ROS production. [•] 8-Nitro-cGMP mediates canonical cGMP signaling via PKG activation. [•] 8-Nitro-cGMP also mediates redox signaling via protein S-guanylation. [•] H2S involves 8-nitro-cGMP metabolism via sulfhydration to form 8-SH-cGMP. [Copyright &y& Elsevier]

A chip was developed with a 32×32 array of extended-gate field-effect transistor (FET)-based redox-potential sensors, each with a gold electrode modified with 11-ferrocenylundecane-1-thiol (11-FUT). The potential of the sensors was stable to within 0.5mV/h. Overall, for the 32×32 sensor cells, 80% showed potentials that were within ±5mV of the median and 92% were within ±1mV of the median. The sensor array detected the redox reaction of hexacyanoferrate(II) and hexacyanoferrate(III) as a change in the electric potential of the 11-FUT-modified electrode with a Nernstian response, at a slope of −58.0mV/decade at room temperature, and a dynamic range of more than five orders of magnitude. Two-dimensional and real-time visualization were made possible by imaging of the sensor array. With an enzyme-catalyzed redox reaction, the FET-based sensor array showed a slope of −59.5mV/decade for logarithmic concentrations of glucose in the range 0.1–2mM, and it successfully detected glucose levels from 22.5 to 360mg/dL. The limit of detection of glucose was 50μM. Finally, the FET-based enzyme sensor array successfully detected glucose levels in samples of human serum from 100.1 to 264.3mg/dL. [ABSTRACT FROM AUTHOR]

Highlights: [•] Uridine (Urd) affects the activity of some cortical and thalamic neurons. [•] Urd (i.p.) decreased absence epileptic activity in both Long Evans and WAG/Rij rats. [•] Higher dose of Urd decreased the number of SWDs even 24h after injection. [•] Uridine-sensitive neurons may be involved in the antiepileptic action of Urd. [ABSTRACT FROM AUTHOR]

Abstract: The effect of bicarbonate ion (HCO3 −) on the mobilization of iron (Fe) reserves from cotyledons to roots during early growth of citrus seedlings and its influence on the components of the iron acquisition system were studied. Monoembryonic seeds of Citrus limon (L.) were germinated “in vitro” on two iron-deprived media, supplemented or not with 10mM HCO3 − (−Fe+Bic and −Fe, respectively). After 21d of culture, Fe concentration in seedling organs was measured, as well as gene expression and enzymatic activities. Finally, the effect of Fe resupply on the above responses was tested in the presence and absence of HCO3 − (+Fe+Bic or +Fe, respectively). −Fe+Bic seedlings exhibited lower Fe concentration in shoots and roots than −Fe ones but higher in cotyledons, associated to a significative inhibition of NRAMP3 expression. HCO3 − upregulated Strategy I related genes (FRO1, FRO2, HA1 and IRT1) and FC-R and H+-ATPase activities in roots of Fe-starved seedlings. PEPC1 expression and PEPCase activity were also increased. When −Fe+Bic pre-treated seedlings were transferred to Fe-containing media for 15d, Fe content in shoots and roots increased, although to a lower extent in the +Fe+Bic medium. Consequently, the above-described root responses became markedly repressed, however, this effect was less pronounced in +Fe+Bic seedlings. In conclusion, it appears that HCO3 − prevents Fe translocation from cotyledons to shoot and root, therefore reducing their Fe levels. This triggers Fe-stress responses in the root, enhancing the expression of genes related with Fe uptake and the corresponding enzymatic activities. [Copyright &y& Elsevier]

Highlights: [•] Oxidative stress impairs 3-MST-derived H2S production in isolated enzyme and in isolated mitochondria. [•] This impairs the stimulatory bioenergetic effects of H2S in hepatocytes. [•] This has implications for the pathophysiology of diseases with oxidative stress. [ABSTRACT FROM AUTHOR]

Highlights: [•] We report a novel magnetic bead for phosphate-affinity separation. [•] The target molecules are phosphomonoester-type nucleotides and phosphopeptides. [•] The separation can be conducted by using aqueous solutions at neutral pH. [•] The operation time from sample addition to elution is less than 12min. [•] The phosphate-affinity beads are stable for multiple use and long-term storage. [Copyright &y& Elsevier]

The nucleotide adenosine 5′-triphosphate (ATP) has classically been considered the cell's primary energy currency. Importantly, a novel role for ATP as an extracellular autocrine and/or paracrine signalling molecule has evolved over the past century and extensive work has been conducted to characterize the ATP-sensitive purinergic receptors expressed on almost all cell types in the body. Extracellular ATP elicits potent effects on vascular cells to regulate blood vessel tone but can also be involved in vascular pathologies such as atherosclerosis. While the effects of purinergic signalling in the vasculature have been well documented, the mechanism(s) mediating the regulated release of ATP from cells in the blood vessel wall and circulation are now a key target of investigation. The aim of this review is to examine the current proposed mechanisms of ATP release from vascular cells, with a special emphasis on the transporters and channels involved in ATP release from vascular smooth muscle cells, endothelial cells, circulating red blood cells, and perivascular sympathetic nerves, including vesicular exocytosis, plasma membrane F1/F0-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channels. [ABSTRACT FROM AUTHOR]

Abstract: Purinergic receptors are present in the lateral parabrachial nucleus (LPBN), a pontine structure involved in the control of sodium intake. In the present study, we investigated the effects of α,β-methyleneadenosine 5′-triphosphate (α,β-methylene ATP, selective P2X purinergic agonist) alone or combined with pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, P2X purinergic antagonist) or suramin (non-selective P2 purinergic antagonist) injected into the LPBN on sodium depletion-induced 1.8% NaCl intake. Male Holtzman rats with stainless steel cannulas implanted into the LPBN were used. Sodium depletion was induced by treating rats with the diuretic furosemide (20mg/kg of body weight) followed by 24h of sodium-deficient diet. Bilateral injections of α,β-methylene ATP (2.0 and 4.0nmol/0.2μl) into the LPBN increased sodium depletion-induced 1.8% NaCl intake (25.3±0.8 and 26.5±0.9ml/120min, respectively, vs. saline: 15.2±1.3ml/120min). PPADS (4nmol/0.2μl) alone into the LPBN did not change 1.8% NaCl intake, however, pretreatment with PPADS into the LPBN abolished the effects of α,β-methylene ATP on 1.8% NaCl intake (16.9±0.9ml/120min). Suramin (2.0nmol/0.2μl) alone into the LPBN reduced sodium depletion-induced 1.8% NaCl intake (5.7±1.9ml/120min, vs. saline: 15.5±1.1ml/120min), without changing 2% sucrose intake or 24h water deprivation-induced water intake. The combination of suramin and α,β-methylene ATP into the LPBN produced no change of 1.8% NaCl intake (15.2±1.2ml/120min). The results suggest that purinergic P2 receptor activation in the LPBN facilitates NaCl intake, probably by restraining LPBN mechanisms that inhibit sodium intake. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine differences of susceptibility to oxidative stress of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) established from Meniere's disease (MD) patients and to examine the effect of ATP treatment in the prognosis and treatment MD. LCLs were established from 10 patients with MD and 10 healthy donors by EBV. Cell viabilities were calculated after treatment of H2O2 with or without ATP. The relationship between the sensitivity of H2O2-treated LCLs to ATP and the staging scale of MD was examined. The nuclear morphological changes of Hoechst 33258-stained LCLs after H2O2-treatment were observed under a fluorescence microscope. LCLs from MD were significantly more sensitive (p < 0.001) to H2O2 than LCLs from healthy donors after 3 h of H2O2 treatment. All of the ATP-sensitive LCLs were categorized as Stage 1 or 2, while others categorized as Stage 3 or 4 were not sensitive to ATP. There were significant differences (p < 0.01) of cell viabilities after addition of ATP between H2O2-treated LCLs classified as Stage 1 or 2 and as Stage 3 or 4 in MD. Both chromatin condensation and swelling of the cell body were observed in H2O2-treated LCLs. Our findings indicate that LCLs established from MD patients might be used as a unique model to detect susceptibility to oxidative stress and ATP treatment in MD patients. Also, the difference of the sensitivity of H2O2-treated LCLs to ATP might relate to prognosis and treatment of MD. This system may form the basis of tailor-made therapy for MD. [ABSTRACT FROM AUTHOR]

Abstract: Class II chitinase CHT11 is one of the 12 chitinases identified in rice (Oryza sativa L.). In order to elucidate its enzymatic properties, the Cht11 gene was cloned and overexpressed in Escherichia coli BL21 cells as a glutathione-S-transferase (GST) fusion protein. The optimal culture temperature and the optimal time after induction were found to be 16°C and 12h, respectively. In the process of purification, a non-specific 70-kDa protein bound to the fusion protein was successfully removed from recombinant CHT11 by adding 3mM ATP-Mg in wash buffer. In the culture conditions and purification procedures presented in this study, the final yield of recombinant CHT11 from 800ml of the culture supernatant was 4.1mg with 11.5-fold purification. The optimal temperature and pH of recombinant CHT11 were determined to be 35–40°C and 5.5–6.5, respectively. It inhibited the hyphal growth of both avirulent and virulent strains of Trichoderma viride at 100 and 300μg in medium, but the growth inhibition zone was subtle even when 300μg of protein was applied. This indicates that the antifungal activity of recombinant CHT11 is considerably lower than other chitinases. [Copyright &y& Elsevier]

Abstract: Mitochondrial dysfunction may play an important role in the pathogenesis of ageing and age-neurodegenerative diseases such as Alzheimer’s disease (AD). Platelet mitochondrial membrane potential (reflected by measurement of JC-1 fluorescence ratio) and adenosine 5′-triphosphate (ATP) contents of 24 moderate probable AD patients, 20 age-matched control subjects and 20 young control subjects were measured. Also, a beta-amyloid peptide (Aβ)-induced damage model of platelets was established. After the addition of Aβ, platelet JC-1 fluorescence ratio and ATP content of platelets were measured in 16 AD patients, 20 aged and 20 young control subjects. Young control subjects had higher JC-1 fluorescence ratio than both AD patients and aged control subjects. No significant differences in platelet ATP contents were found among AD patients, aged and young control subjects. After the addition of Aβ, platelet JC-1 fluorescence ratio and ATP content of aged and young control subjects lowered markedly, but no obvious decrease of platelet JC-1 fluorescence ratio of AD patients was found compared with those of aged and young control subjects. Decrease of platelet JC-1 fluorescence ratio of aged control subjects was lower than that of young control subjects following the addition of Aβ. These results indicated that mitochondrial dysfunction may occur during ageing and platelet mitochondria of AD patients and aged subjects showed a tolerance to Aβ-induced damage. Therefore, blood platelets might serve as a biomarker for detection of mitochondrial function and age-related disease. [Copyright &y& Elsevier]

The universal stress protein UspF (YnaF) is a small cytoplasmic bacterial protein. The expression of stress proteins is enhanced when cells are exposed to heat shock, nutrition starvation and certain other stress-inducing agents. YnaF promotes cell survival during prolonged exposure to stress and may activate a general mechanism for stress endurance. This manuscript reports preliminary crystallographic studies on YnaF from Salmonella typhimurium. The gene coding for YnaF was cloned and overexpressed and the protein was purified by Ni–NTA affinity chromatography. Purified YnaF was crystallized using vapour-diffusion and microbatch methods. The crystals belong to space group P21, with unit-cell parameters a = 37.51, b = 77.18, c = 56.34 Å, β = 101.8°. A data set was collected to 2.5 Å resolution with 94.6% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. Attempts to determine the structure are in progress. [ABSTRACT FROM AUTHOR]

Concentrations of fungal metabolites were measured in 32 wheat grain samples from north-eastern Poland in 2003. The samples originated from fields cultivated conventionally (but varying in chemical protection level) or cultivated organically. Concentrations of Fusarium toxins (HT-2, DON, 3-AcDON, NIV), trichodiene, microbial biomass indicators (fungal ergosterol and general adenosine 5'-triphosphate (ATP)) and seed vigour were assessed. A large variation between samples was observed, depending on their origin. Seed from organic farms contained similar amounts of Fusarium toxins but more ergosterol and ATP than conventionally grown and chemically protected seed. The highest levels of toxins and ergosterol were detected in samples from conventional cultivation lacking chemical protection. Intensive agronomic practices (including complete chemical protection) significantly lowered the levels of ergosterol, ATP and trichodiene, as compared with other cultivation systems. [ABSTRACT FROM AUTHOR]

The changes in the contents of adenosine 5’-triphosphate (ATP) and its related breakdown compounds were investigated in the adductor muscle, mantle, gill and body trunk of oyster ( Crassostrea gigas) during frozen storage at −20°C and −30°C and compared with that of the fresh oyster. The investigation was performed using an HPLC system. Different extents of ATP decomposition were found in various tissues frozen at the two temperatures. The K, K’ and A.E.C values were calculated as the chemical freshness indices. Considering the results of sensory evaluation, the A.E.C. value in body trunk at −20°C and −30°C could be used as the best freshness index for frozen oyster. [ABSTRACT FROM AUTHOR]

Biomolecular motors such as the muscle protein myosin with its partner protein actin hold great promise for actuation in hybrid nanoscale biomicroelectromechanical systems devices (bio-MEMS), particularly for future biomedical applications that involve highly localized delivery of biomolecules over short distances (e.g., micrometers) to specific tissue or cellular locations. Two fundamental issues in the construction and packaging of actomyosin-based nanoactuators are the ability to electrically insulate microelectrical components while maintaining both biocompatibility and also compatibility with our functional assays for prototype development and identification of conditions for storage of assembled devices. Here, we show that sputter coating with SiO2 provides a straightforward method for electrical insulation that can be readily integrated into existing assays of myosin function in a bio-MEMS setting. We also report using in vitro motility analysis that both rabbit skeletal muscle heavy meromyosin (HMM) and fish (Fundulus heteroclitus) myosin remained functional for at least six days in a bio-MEMS setting when hydrating conditions were maintained. The speed of actin sliding was faster after six days when rabbit HMM was stored in 10% DMSO than in its absence, but there was no effect of DMSO during storage on fish myosin. The speed of actin translocation by fish myosin was significantly increased when adenosine 5'-triphosphate (ATP), the chemical energy source for myosin function, was replaced by the analog 2' deoxy-ATP, as has been previously reported for rabbit HMM. Taken together, these results provide new direction for modulation and control of actomyosin-based nanoactuators and also for long-term storage of assembled nanoactuators. [ABSTRACT FROM AUTHOR]

This review summarizes clinical application of adenosine and adenosine 5′-triphosphate (ATP) in pain conditions. Investigations have been performed in patients with acute perioperative pain or chronic neuropathic pain treated with intravenous adenosine or ATP, or intrathecal adenosine. Characteristic central adenosine A1 receptor-mediated pain-relieving effects have been observed after intravenous adenosine infusion in human inflammation/sensitization pain models and in patients with chronic neuropathic pain. Adenosine compounds, in low doses, can reduce allodynia/hyperalgesia more consistently than spontaneous pain, suggesting that these compounds affect neuronal pathophysiological mechanisms involved in central sensitization. Such pain-relieving effects, which are mostly mediated via central adenosine A1 receptor activation, have a slow onset and long duration of action, lasting usually for hours or days and occasionally for months. With acute perioperative pain, treatment with a low-dose infusion of adenosine compounds and the A1 receptor-mediated central antisensitization mechanisms may play only a minor part in the total perioperative pain experience. By administering sufficient doses of adenosine compounds during surgery, however, significant and long-lasting perioperative pain relief can be achieved via central A1 receptor-mediated antinociceptive/analgesic actions as well as via peripheral A2a or A3 receptor-mediated antiinflammatory actions. Thus, adenosine compounds have significant potential for alleviating various types of pain. [ABSTRACT FROM AUTHOR]

Abstract: Rapid, non-genomic effects of glucocorticoids on extracellular adenosine 5′-triphosphate (ATP)-induced intracellular Ca2+ concentration ([Ca2+]i) changes and nitric oxide (NO) production were investigated in type I spiral ganglion neurons (SGNs) of the guinea-pig cochlea using the Ca2+-sensitive dye fura-2 and the NO-sensitive dye 4,5-diaminofluorescein (DAF-2). Pretreatment of SGNs with 1 μM dexamethasone for 10 min, a synthetic glucocorticoid hormone, enhanced the ATP-induced [Ca2+]i increase in SGNs. RU 38486, a competitive glucocorticoid receptor antagonist eliminated the effects of dexamethasone on the ATP-induced [Ca2+]i increase in SGNs. These acute effects of dexamethasone were dependent on the presence of extracellular Ca2+, thereby suggesting that dexamethasone may rapidly enhance the Ca2+ influx through the activation of ionotropic P2X receptors which may interact with glucocorticoid-mediated membrane receptors. Extracellular ATP increased the intensity of DAF-2 fluorescence, indicating NO production in SGNs. The ATP-induced NO production was mainly due to the Ca2+ influx through the activation of P2 receptors. S-nitroso-N-acetylpenicillamine, a NO donor, enhanced the ATP-induced [Ca2+]i increase in SGNs while l-NG-nitroarginine methyl ester (l-NAME), a NO synthesis inhibitor, inhibited it. Dexamethasone enhanced the ATP-induced NO production in SGNs. The augmentation of dexamethasone on ATP-induced NO production was abolished in the presence of l-NAME. It is concluded that the ATP-induced [Ca2+]i increase induces NO production which enhances a [Ca2+]i increase in SGNs by a positive-feedback mechanism. Dexamethasone enhances the ATP-induced [Ca2+]i increase in SGNs which results in the augmentation of NO production. The present study suggests that NO may play an important role in auditory signal transduction. Our results also indicate that glucocorticoids may rapidly affect auditory neurotransmission due to a novel non-genomic mechanism. [Copyright &y& Elsevier]

Abstract: Conformational changes of the cAMP-dependent protein kinase (PKA) catalytic (C) subunit are critical for the catalysis of γ-phosphate transfer from adenosine 5′-triphosphate (ATP) to target proteins. Time-resolved fluorescence anisotropy (TRFA) was used to investigate the respective roles of Mg2+, ATP, MgATP, and the inhibitor peptide (IP20) in the conformational changes of a 5,6-carboxyfluorescein succinimidyl ester (CF) labeled C subunit (CFC). TRFA decays were fit to a biexponential equation incorporating the fast and slow rotational correlation times ϕF and ϕS. The CFC apoenzyme exhibited the rotational correlation times ϕF=1.8±0.3 ns and ϕS=20.1±0.6 ns which were reduced to ϕF=1.1±0.2 ns and ϕS=13.3±0.9 ns in the presence of MgATP. The reduction in rotational correlation times indicated that the CFC subunit adopted a more compact shape upon formation of a CFC·MgATP binary complex. Neither Mg2+ (1–3 mM) nor ATP (0.4 mM) alone induced changes in the CFC subunit conformation equivalent to those induced by MgATP. The effect of MgATP was removed in the presence of ethylenediaminetetraacetic acid (EDTA). The addition of IP20 and MgATP to form the CFC·MgATP·IP20 ternary complex produced rotational correlation times similar to those of the CFC·MgATP binary complex. However, IP20 alone did not elicit an equivalent reduction in rotational correlation times. The results indicate that binding of MgATP to the C subunit may induce conformation changes in the C subunit necessary for the proper stereochemical alignment of substrates in the subsequent phosphorylation. [Copyright &y& Elsevier]

Abstract: The influence of the thyroid hormones on the normal function of the mammalian central nervous system depends on the brain region and on the developmental stage. Adenine nucleotides and their products also affect the brain function; ATP is an excitatory neurotransmitter, and adenosine has inhibitory effects on neurotransmission. Thus, this study aimed to evaluate the effects of hypothyroidism on the hydrolysis of ATP to adenosine in hippocampal and cortical synaptosomes and blood serum of rats during different phases of development. Rats aged 60 and 420 days old were divided into three groups: control, sham-operated and hypothyroid. Hypothyroidism was induced in these rats by thyroidectomy and methimazole (0.05%) added to their drinking water for 14 days. Neonatal hypothyroidism was induced by adding 0.02% methimazole in the drinking water from day 9 of gestation, and continually until 14 days old. Hypothyroidism increased the AMP hydrolysis in both hippocampus and cerebral cortex synaptosomes of rats in all aged tested. In blood serum, thyroid hormones deficiency increased the AMP hydrolysis in 14-day-old rats and the hydrolysis of ATP, ADP and AMP in 60-day-old rats; however, no alteration was observed in 420-day-old rats. Thus, our results suggest the involvement of the 5′-nucleotidase in synaptic function control in hypothyroidism throughout brain development. [Copyright &y& Elsevier]

Abstract: To further characterize the nature of the regulatory metal-binding sites of the rat P2X4 receptor, several transition heavy metals were tested to examine their ability to mimic the facilitator action of zinc or the inhibitory action of copper. cDNA coding for the rat P2X4 receptor was injected into Xenopus laevis oocytes; the two-electrode voltage-clamp technique was used to measure and quantify the ATP-evoked currents in the absence or presence of the metals. Cadmium facilitated the ATP-gated currents in a reversible and voltage-independent manner; maximal potentiation occurred within less than 1 min. Cadmium displaced leftward, in a concentration-dependent manner, the ATP concentration–response curve. In contrast, mercury reduced the ATP-gated currents in a reversible, time, and concentration manner. Maximal inhibition occurred after about 5 min of metal application. Cobalt also augmented the ATP-evoked currents, but its action was long lasting and did not reverse even after 45 min of metal washout. Other metals such as lead, nickel, manganese, silver, or gallium did not significantly alter the ATP-gated currents. The co-application of cadmium plus zinc or mercury plus copper caused additive effects. Mutation of H140 by alanine (H140A) augmented both the cadmium-induced facilitation and the mercury-induced inhibition. In contrast, the H241A mutant showed characteristics indistinguishable from the wild type. The H286A mutant showed a normal cadmium-induced potentiation, but an increased mercury inhibition. Out of the metals examined, only cadmium mimicked closely the action of zinc, evidencing commonalities. While mercury mimicked the action of copper, both metals apparently interact at distinct metal-binding sites. The present findings allow us to infer that heavy metals modulate the P2X4 receptor by acting in at least three separate metal-binding sites. [Copyright &y& Elsevier]

The P2X7 nucleotide receptor is an adenosine 5′-triphosphate (ATP) -gated ion channel, which is widely expressed in cells of hematopoietic origin and functions as a non-selective cation channel permeable to Na+, Ca2+, etc upon stimulation. Here, we investigated P2X7 expression in 11 human hematopoietic cell lines, representing different lineages, as well as bone marrow mononuclear cells (BMMC) samples from 87 leukemia and 10 myelodysplastic syndrome (MDS) patients. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry results showed that both P2X7 mRNA and protein were detected in eight cell lines with a non-lineage-specific manner. Samples from 69 leukemia and 9 MDS patients were P2X7 positive at mRNA level. Moreover, both positive rates and relative expression levels were significantly higher in acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and MDS groups than that in normal donor group. The expression levels varied among AML subtypes with higher levels being observed in M4, M5, and M6 groups but not in M1 or M2 group. Furthermore, after one course of standard induction therapies, the remission rate in high P2X7 expression group was lower than that in either P2X7 negative group or low P2X7 expression group. Cytoplasmic free calcium increase was detected in five of eight P2X7+ cell lines as well as P2X7+ normal donor and patient samples tested, but not in three Epstein–Barr virus (EBV) positive cell lines (J6-1, Namalwa, and LCL-H) in Locke’s solution upon stimulation by extracellular ATP or the more potent and specific agonist, 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP). The possible mechanisms causing the loss of P2X7 function were discussed. [Copyright &y& Elsevier]