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There has been a remarkable growth of papers published about purinergic signalling via ATP since 1972. I am most grateful to the wonderful PhD students and postdoctoral fellows who have worked with me over the years to pursue the purinergic hypothesis despite early opposition and to the many outstanding scientists around the world who are currently extending the story. Recently, therapeutic approaches to pathological disorders include the development of selective P1 and P2 receptor subtype agonists and antagonists, as well as of inhibitors of extracellular ATP breakdown and of ATP transport enhancers and inhibitors. Medicinal chemists are starting to develop small molecule purinergic drugs that are orally bioavailable and stable in vivo., (© 2012 The Author. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Lists titles of research papers on biochemistry. "Redox control of catalysis in ATP-citrate lyase from rat liver"; "Construction and molecular dynamics of a soluble, enzymatically active form of the Escherichia coli penicillin-binding protein 5 and purification by dye chromatography"; "Studies on the variants of the protein toxins ricin and abrin".

Phosphatidic acid (PA) from swine and beef RBCs was isolated by chromatography on silicic acid columns. It comprised about 1 per cent of the total lipid phosphate in RBCs, but was eluted nearly pure from columns. An uncharacterized inositide accounted for 5 to 10 per cent of the phosphate in the PA-containing fraction. When cells were incubated with HP(32)O(4) (=), the fraction containing PA became more radioactive than any of the other fractions obtained. However, analysis of the labeled material by paper chromatography showed that most of the P(32) was in the inositide, not in PA. With the assumption of kinetic homogeneity for cellular PA, compartmental analysis of the kinetics of tracer incorporation showed that PA turnover is 3 to 4 orders of magnitude too slow to account for sodium extrusion by these cells.

Presents several research papers related to biochemistry. "Effect of Thiol Reagents on the Proton Conductivity of the H+ATPase of Mitochondria," by F. Guerrieri and S. Papa; "Purification of Penicillin-Binding Protein 3 from Streptococcus pneumoniae," by R. Hakenbeck and M. Kohiyama; "Reutilization of Preformed Pyrimidines for the Synthesis of DNA in Rat Liver and Kidney," by J. Seifert; "Human Progastriesin: Analysis of Intermediates during Activation in Gastriesin and Determination of the Amino Acid Sequence of the Propart," by B. Foltmann and A. L. Jensen; Others.

Background and Purpose: Amitriptyline is a tricyclic antidepressant that is also widely used to treat neuropathic pain in humans, but the mechanism of this anti-hyperalgesic effect is unknown. Microglia in the mouse spinal cord become activated in neuropathic pain, and expression of P2X4 receptors by these microglia is increased. Antisense RNA targeting P2X4 receptors suppresses the development of tactile allodynia in rats. This suggests that blockade of P2X4 receptors might be the mechanism by which amitriptyline relieves neuropathic pain.Experimental Approach: We expressed human, rat and mouse P2X receptors (P2X2, P2X4, P2X7) in human embryonic kidney cells and evoked inward currents by applying ATP. We compared the action of ATP on control cells and cells treated with amitriptyline.Key Results: Amitriptyline (10 microM), either applied acutely or by pre-incubation for 2-6 h, had no effect on inward currents evoked by ATP (0.3-100 microM) at human P2X4 receptors. At rat and mouse receptors, amitriptyline (10 microM) caused a modest reduction in the maximum responses to ATP, without changes in EC(50) values, but it had no effect at 1 microM. Amitriptyline also had no effects on currents evoked by ATP at rat P2X2 receptors, or at rat or human P2X7 receptors.Conclusion and Implications: The results do not support the view that amitriptyline owes its pain-relieving actions in man to the direct blockade of P2X4 receptors. [ABSTRACT FROM AUTHOR]

Background and Purpose: Extracellular nucleotides produce vasodilatation through endothelial P2 receptor activation. As these autacoids are actively metabolized by the ecto-nucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), we studied the effects of this cell surface enzyme on nucleotide-dependent vasodilatation.Experimental Approach: Vascular NTPDase expression and activity were evaluated by immunohistochemistry and histochemistry. The vascular effects of nucleotides were tested in vivo by monitoring mean arterial pressure, and in vitro comparing reactivity of aortic rings using wild-type and Entpd1(-/-) (lacking NTPDase1) mice.Key Results: The absence of NTPDase1 in Entpd1(-/-) mice led to a dramatic drop in endothelial nucleotidase activity. This deficit was associated with an exacerbated decrease in blood pressure after nucleotide injection. Following ATP injection, mean arterial pressure was decreased in Entpd1(+/+) and Entpd1(-/-) mice by 5.0 and 17%, respectively, and by 0.1 and 19% after UTP injection (10 nmole.kg(-1) both). In vitro, the concentration-response curves of relaxation to ADP and ATP were shifted to the left, revealing a facilitation of endothelial P2Y1 and P2Y2 receptor activation in Entpd1(-/-) mice. EC(50) values in Entpd1(+/+) versus Entpd1(-/-) aortic rings were 14 microM versus 0.35 microM for ADP, and 29 microM versus 1 microM for ATP. In Entpd1(-/-) aortas, P2Y1 receptors were more extensively desensitized than P2Y2 receptors. Relaxations to the non-hydrolysable analogues ADPbetaS (P2Y1) and ATPgammaS (P2Y2) were equivalent in both genotypes confirming the normal functionality of these P2Y receptors in mutant mice.Conclusions and Implications: NTPDase1 controls endothelial P2Y receptor-dependent relaxation, regulating both agonist level and P2 receptor reactivity. [ABSTRACT FROM AUTHOR]

Background and Purpose: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors.Experimental Approach: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp.Key Results: No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2'(3')-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors.Conclusion and Implications: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans. [ABSTRACT FROM AUTHOR]

Background and Purpose: Regulating macrophage-hepatocyte crosstalk through P2X7 receptors has led to new pharmacological strategies to reverse alcoholic hepatosteatosis. We investigated how tetrahydroxystilbene glucoside (2354glu), isolated from Polygonum multiflorum, modulates macrophage-hepatocyte crosstalk during alcoholic hepatosteatosis.Experimental Approach: A model of alcoholic hepatosteatosis was established by giving ethanol intragastrically to C57BL/6 mice. HepG2 cells were incubated in conditioned medium from LPS+ATP-activated THP-1 human macrophages with silenced or overexpressed P2X7 receptors. THP-1 macrophages or mouse peritoneal macrophages were pretreated with 2354glu for 1 hr prior to LPS+ATP stimulation. Western blots, RT-PCR and immunohistochemical analysis were used, along with over-expression and silencing of P2X7 receptors.Key Results: Knockdown or overexpression of P2X7 receptors in THP-1 macrophages affected release of mature IL-1β and, subsequently, modulated lipid metabolism in HepG2 cells via the LKB-AMPK pathway. 2354glu ameliorated alcoholic hepatosteatosis in mice by regulating LKB1-AMPK-SREBP1 pathway and its target genes. Suppression of P2X7 receptor activation by 2354glu inhibited IL-1β release and reduced macrophage and neutrophil infiltration. In macrophages stimulated with LPS+ATP, expression of P2X7 receptors, caspase-1 and NF-κB, release of IL-1β, calcium influx and PI uptake were reduced by 2354glu. SIRT1-LKB1-AMPK-SREBP1 axis-mediated lipid accumulation in HepG2 cells was reduced when they were cultured with conditioned media from LPS+ATP-activated THP-1 macrophages pretreated with 2354glu.Conclusion and Implications: Modulation of P2X7 receptors in macrophages regulated lipid accumulation in hepatocytes during alcoholic hepatosteatosis. 2354glu might be a promising candidate that targets P2X7 receptors in macrophages interacting with hepatocytes during alcoholic hepatosteatosis. [ABSTRACT FROM AUTHOR]

Study Question: Does lysine 2-hydroxyisobutyrylation, a newly identified protein posttranslational modification (PTM), occur in human sperm and affect human sperm function?Summary Answer: Lysine 2-hydroxyisobutyrylation mainly occurs in human sperm tail proteins, and excessive lysine 2-hydroxyisobutyrylation affects human sperm motility.What Is Known Already: PTM is regarded as an important pathway in regulating sperm function since mature sperm are almost transcriptionally silent. However, only phosphorylation was extensively studied in mature sperm to date. Lysine 2-hydroxyisobutyrylation, a newly characterised PTM, is broadly conserved in both eukaryotic and prokaryotic cells. Although histone lysine 2-hydroxyisobutyrylation has been shown to be associated with active gene expression in spermatogenic cells, the presence, regulatory elements and function of lysine 2-hydroxyisobutyrylation have not been characterised in mature sperm.Study Design, Size, Duration: Sperm samples were obtained from normozoospermic men and asthenozoospermic men who visited the reproductive medical centre at Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi, China, between May 2017 and November 2018. In total, 58 normozoospermic men and 65 asthenozoospermic men were recruited to participate in this study.Participants/materials, Setting, Methods: Lysine 2-hydroxyisobutyrylation was examined using immunoblotting and immunofluorescence assays using a previously qualified pan anti-lysine 2-hydroxyisobutyrylation antibody. The immunofluorescence assay was imaged using super-resolution structured illumination microscopy. Sperm viability was examined by using the eosin staining method, and sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm penetration ability was determined by evaluating the ability of the sperm to penetrate a 1% (w/v) methylcellulose solution. The level of intracellular adenosine triphosphate (ATP) was detected using a rapid bioluminescent ATP assay kit.Main Results and the Role Of Chance: Lysine 2-hydroxyisobutyrylation was present in several proteins (20-100 kDa) mainly located in the tail of human sperm. Sperm lysine 2-hydroxyisobutyrylation was derived from 2-hydroxyisobutyrate (2-Hib) and was regulated by acyltransferase P300 and nicotinamide adenine dinucleotide-dependent lysine deacylase sirtuins. Elevation of sperm lysine 2-hydroxyisobutyrylation by 2-Hib decreased total motility, progressive motility, penetration ability and ATP level of human sperm. Interestingly, the level of sperm lysine 2-hydroxyisobutyrylation was higher in asthenozoospermic men than that in normozoospermic men and was negatively correlated with the progressive motility of human sperm. Furthermore, high levels of lysine 2-hydroxyisobutyrylation in asthenozoospermic men accompanied decreased ATP levels.Limitations, Reasons For Caution: Although the present study indicated the involvement of sperm lysine 2-hydroxyisobutyrylation in regulating human sperm motility, the underlying mechanism needs to be further illustrated.Wider Implications Of the Findings: The findings of this study provide insight into the novel role of lysine 2-hydroxyisobutyrylation in human sperm and suggest that abnormality of sperm lysine 2-hydroxyisobutyrylation may be one of the causes for asthenozoospermia.Study Funding/competing Interest(s): National Natural Science Foundation of China (81771644 to T.L. and 81871207 to H.C.); Natural Science Foundation of Jiangxi province (20171ACB21006). The authors have no conflicts of interest to declare. [ABSTRACT FROM AUTHOR]

Background and Purpose: Capsaicin-mediated modulation of taste nerve responses is thought to be produced indirectly by the actions of neuropeptides, for example, CGRP and substance P (SP), on taste cells implying they play a role in taste sensitivity. During the processing of gustatory information in taste buds, CGRP shapes peripheral taste signals via serotonergic signalling. The underlying assumption has been that SP exerts its effects on taste transmitter secretion in taste buds of mice.Experimental Approach: To test this assumption, we investigated the net effect of SP on taste-evoked ATP secretion from mouse taste buds, using functional calcium imaging with CHO cells expressing high-affinity transmitter receptors as cellular biosensors.Key Results: Our results showed that SP elicited PLC activation-dependent intracellular Ca2+ transients in taste cells via neurokinin 1 receptors, most likely on glutamate-aspartate transporter-expressing Type I cells. Furthermore, SP caused Type I cells to secrete GABA.Conclusion and Implications: Combined with the recent findings that GABA depresses taste-evoked ATP secretion, the current results indicate that SP elicited secretion of GABA, which provided negative feedback onto Type II (receptor) cells to reduce taste-evoked ATP secretion. These findings are consistent with a role for SP as an inhibitory transmitter that shapes the peripheral taste signals, via GABAergic signalling, during the processing of gustatory information in taste buds. Notably, the results suggest that SP is intimately associated with GABA in mammalian taste signal processing and demonstrate an unanticipated route for sensory information flow within the taste bud. [ABSTRACT FROM AUTHOR]

Background and Purpose: Dexpramipexole, a drug recently tested in patients with amyotrophic lateral sclerosis (ALS,) is able to bind F1Fo ATP synthase and increase mitochondrial ATP production. Here, we have investigated its effects on experimental ischaemic brain injury.Experimental Approach: The effects of dexpramipexole on bioenergetics, Ca2+ fluxes, electrophysiological functions and death were evaluated in primary neural cultures and hippocampal slices exposed to oxygen-glucose deprivation (OGD). Effects on infarct volumes and neurological functions were also evaluated in mice following proximal or distal middle cerebral artery occlusion (MCAo). Distribution of dexpramipexole within the ischaemic brain was evaluated by means of mass spectrometry imaging.Key Results: Dexpramipexole increased mitochondrial ATP production in cultured neurons or glia and reduces energy failure, prevents intracellular Ca2+ overload and affords cytoprotection when cultures are exposed to OGD. This compound also counteracted ATP depletion, mitochondrial swelling, anoxic depolarization, loss of synaptic activity and neuronal death in hippocampal slices subjected to OGD. Post-ischaemic treatment with dexpramipexole, at doses consistent with those already used in ALS patients, reduced brain infarct size and ameliorated neuroscore in mice subjected to transient or permanent MCAo. Notably, the concentrations of dexpramipexole reached within the ischaemic penumbra equalled those found neuroprotective in vitro.Conclusion and Implications: Dexpramipexole, a compound able to increase mitochondrial F1Fo ATP-synthase activity reduced ischaemic brain injury. These findings, together with the excellent brain penetration and favourable safety profile in humans, make dexpramipexole a drug with realistic translational potential for the treatment of stroke.Linked Articles: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc. [ABSTRACT FROM AUTHOR]

Interaction of GluN2B subunit of N-methyl-D-aspartate receptor with calcium/calmodulin dependent protein kinase II (CaMKII) is critical for the induction of long term potentiation at hippocampal CA3-CA1 synapses. We have previously reported that CaMKII binding to GluN2B increases its affinity but abolishes the cooperativity for ATP. In the present study, we demonstrate that the reduction in S0.5 for ATP of an individual CaMKII subunit seems to be directly induced by the binding of GluN2B to the same subunit, while any GluN2B induced effects on the cooperativity and maximal velocity would additionally require the CaMKII holoenzyme structure. We measured the apparent kinetic parameters for ATP using an association domain truncated monomeric CaMKII and a heteromultimeric CaMKII (having subunits that are either GluN2B binding defective or ATP binding defective), in the presence of GluN2A or GluN2B substrates. The S0.5 value for ATP of monomeric CaMKII is reduced ~ 3 fold by the presence of GluN2B suggesting that the induced change in affinity for ATP is independent of the holoenzyme structure. The heteromultimeric mutant of CaMKII, did not exhibit cooperativity of ATP binding probably because of the interspersing of ATP binding defective subunits in the holoenzyme. In contrast to the wild type holoenzyme, presence of GluN2B increased the Vmax of monomeric CaMKII which resulted in an approximately 4.0 fold increase in the apparent catalytic constant (Vmax/S0.5) as compared to GluN2A. The kinetic parameter values of the heteromultimeric CaMKII for ATP, on the other hand, did not show any significant difference between the phosphorylation of GluN2B and GluN2A suggesting that modulation requires binding of GluN2B to the same subunit. Overall, our present study provides insights into the role of multimeric structure of CaMKII in GluN2B-mediated regulation. [ABSTRACT FROM AUTHOR]

The article offers information on the Review Focus Issue for October 2010. It centers on mitochondria in cardiac disease, particularly on the new concepts about the role of mitochondria that are beyond its functions as the adenosine triphosphate (ATP)-generating powerhouse of the cell and as arbitrators of cell death. Preliminary topics of invited review articles for the issue include mitochondrial genetics and cardiac disease, mitochondrial aldehyde dehydrogenase and cardiac disease, and mitochondrial kinase signalling pathways.

Background: Mammalian oocyte activation occurs via a series of intracellular calcium (Ca(2+)) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLCζ). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular analysis of the PLCζ gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLCζ residue 398 (PLCζ(H398P)), leading to abnormal Ca(2+) release profiles and reduced oocyte activation efficiency.Methods and Results: In the present study, we used HEK293T cells to produce recombinant human wild-type PLCζ (PLCζ(WT)) protein which, upon microinjection into mouse oocytes, induced Ca(2+) oscillations characteristic of oocyte activation. Injection of recombinant PLCζ(H398P) was unable to elicit Ca(2+) oscillations in mouse oocytes. Loss of activity mutations, such as PLCζ(H398P) and an artificially induced frameshift mutation (PLCζ(ΔYC2)) did not affect Ca(2+) release when over-expressed in HEK293T cells, whereas PLCζ(WT) inhibited adenosine triphosphate-activated Ca(2+) release. Confocal imaging of fluorescently tagged PLCζ isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLCζ(WT) > PLCζ(H398P) > PLCζ(ΔYC2), indicating that loss of activity mutations may lead to protein instability. This was further indicated by the low proportion of sperm and the lower levels of total PLCζ immunofluorescence from the patient exhibiting PLCζ(H398P) compared with fertile controls.Conclusions: We demonstrate, for the first time, the production of active recombinant human PLCζ protein which retained the ability to elicit characteristic Ca(2+) oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLCζ, and provide a critical step forward in obtaining purified PLCζ protein as a potential therapeutic agent for oocyte activation deficiency. [ABSTRACT FROM AUTHOR]

Background and Purpose: Ionotropic purinoreceptors (P2X) in renal vascular smooth muscle cells (RVSMCs) are involved in mediating the sympathetic control and paracrine regulation of renal blood flow (RBF). Activation of P2X receptors elevates [Ca(2+)](i) in RVSMCs triggering their contraction, leading to renal vasoconstriction and decrease of RBF. The goal of the present work was to characterize the P2X receptor-mediated ionic current (I(P2X)) and to identify the types of P2X receptors expressed in myocytes isolated from interlobar and arcuate arteries of rat kidney.Experimental Approach: The expression of P2X receptors in isolated RVSMCs was analysed by reverse transcription (RT)-PCR. I(P2X) and membrane potential were recorded using the amphotericin B-perforated patch method.Key Results: RT-PCR analysis on single RVSMCs showed the presence of genes encoding P2X1 and P2X4 receptors. Under voltage clamp conditions, the selective P2X receptor agonist alphabeta-methylene ATP (alphabeta-meATP) evoked I(P2X) similar to that induced by ATP. Under current clamp conditions, both ATP and alphabeta-meATP evoked a spike-like membrane depolarization followed by a sustained depolarization, linking P2X receptors in RVSMCs to sympathetic control of renal vascular tone. A selective antagonist of P2X1 receptors, NF279, reduced I(P2X) amplitude by approximately 65% concentration-dependently manner within the nanomolar to sub-micromolar range. The residual current was resistant to micromolar concentrations of NF279, but was inhibited by sub-millimolar to millimolar concentrations of NF279.Conclusions and Implications: Two types of functional P2X receptors, monomeric P2X1 and heteromeric P2X1/4 receptors, are expressed in RVSMCs. Our study has identified important targets for possible pharmacological intervention in the sympathetic control of renal circulation. [ABSTRACT FROM AUTHOR]

Background and Purpose: ATP, released from urothelial cells, modulates afferent nerve firing from the urinary bladder. Here, we have characterized ATP release from the rat bladder mucosa in response to acid, capsaicin, electrical field stimulation (EFS) and stretch, using agonists and antagonists at transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs).Experimental Approach: Rat mucosal strips (containing urothelium and lamina propria) in Perspex microbaths were superfused with Krebs solution. ATP was measured after exposure of matched strips to acid (pH 6.6-5.0), capsaicin (0.1-10 microM), EFS or stretch (150% of original length).Key Results: Median basal ATP release was 3.46 nmol g(-1). The mucosal strips responded to stimuli with potency order (median, IQR): acid (pH 5.6-6.0) 286 (103-555) > 10 microM capsaicin 188 (117-431) > 10 Hz EFS 63.0 (13.3-96.4) > stretch 24.4 (6.73-55.1) nmol ATP g(-1). ATP release in response to acid was pH dependent (P < 0.05). Responses to capsaicin did not desensitize nor were they concentration dependent. TRPV1 antagonist, capsazepine (10 microM) abolished capsaicin-evoked ATP release, and reduced acid-evoked (pH 6.5) release to 30% (P < 0.001). The ASIC channel antagonists gadolinium (0.1 mM) and amiloride (0.3 microM) reduced (P < 0.05) the acid-evoked (pH 6.5) release to 40 and 6.5% respectively. ASIC (ASIC1, ASIC2a, ASIC2b, ASIC3) and two TRPV1 gene products were detected in mucosal and detrusor extracts.Conclusions and Implications: Capsaicin (at TRPV1) and acid (at both TRPV1 and ASIC) induce ATP release from the rat bladder mucosa. This ATP appears to be principally of urothelial origin. This study highlights the importance of ATP and acid as signalling molecules in modulating bladder function. [ABSTRACT FROM AUTHOR]

Background and Purpose: The expression and function of P2X(7) receptors in osteoclasts is well established, but less is known about their role in osteoblast-like cells. A study in P2X(7) receptor knockout mice suggested the involvement of these receptors in bone formation. We have investigated the expression and pharmacology of several P2X receptors in two human osteosarcoma cell lines to see if they could be involved in bone turnover in man.Experimental Approach: Reverse transcriptase-polymerase chain reaction and Western blotting were used to study P2X(2), P2X(4) and P2X(7) receptor expression at mRNA and protein levels, respectively, in human osteoblast-like cells. P2X(7) receptor pharmacology was studied by measuring pore formation in the presence of different agonists and antagonists using the YO-PRO 1 uptake method.Key Results: P2X(4) and P2X(7) receptor mRNA and protein were found to be expressed by these cell lines. No evidence was found for P2X(4)/P2X(7) receptor heteropolymerization. 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (DBzATP) was equipotent to ATP and the antagonists used were either ineffective or weakly blocked pore formation.Conclusions and Implications: This study demonstrates that P2X(4) and P2X(7) receptors are expressed by human osteoblast-like cells. The affinities of the different agonists suggest that the P2X(7) receptor is mainly responsible for pore formation although P2X(4) receptors may also be involved. The low affinity of DBzATP and the weak action of the antagonists support the previously described atypical pharmacology of the P2X(7) receptor in osteoblasts. Targeting the P2X(7) receptor in osteoblasts could represent a promising new treatment for bone diseases such as osteoporosis and rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Background and Purpose: The purine compounds, adenosine 5'-triphosphate (ATP) and adenosine, are known to accumulate in the extracellular space and to elicit various cellular responses during hypoxia/ischemia, whereas the roles of purines during hypercapnia are poorly understood. In this study, we examined the effects of various drugs affecting purine turnover on the responses to hypercapnia in the spinal cord.Experimental Approach: Electrically evoked reflex potentials were measured in an in vitro preparation of the isolated spinal cord of the neonatal rat by extracellular recording. Extracellular adenosine concentrations were assayed by high performance liquid chromatography (HPLC) methods.Key Results: Hypercapnia (20% CO2) depressed the reflex potentials, which were partially reversed by an adenosine A1 receptor antagonist, 8-cyclopentyl theophylline, but not by a P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. Exogenous adenosine and ATP also depressed the reflex potentials via adenosine A1 receptors. The hypercapnia-evoked depression was not reversed by inhibitors of gap junction hemichannels, anion channels, P2X7 receptors or equilibrative nucleoside transporters, all of which might be involved in purine efflux pathways. The adenosine accumulation evoked by hypercapnia was not inhibited by tetrodotoxin, ethylene glycol-bis(beta-amino ethyl ether) tetraacetic acid (EGTA) or an ecto-ATPase inhibitor, ARL 67156. Homocysteine thiolactone, used to trap intracellular adenosine, significantly reduced extracellular adenosine accumulation during hypercapnia.Conclusions and Implications: These results suggest that hypercapnia released adenosine itself from intracellular sources, using pathways different from the conventional exocytotic mechanism, and that this adenosine depressed spinal synaptic transmission via adenosine A1 receptors. [ABSTRACT FROM AUTHOR]

Background and purpose:ZP120 (Ac-RYYRWKKKKKKK-NH2), is a new partial nociceptin/orphanin FQ (NOP) receptor agonist with sodium-potassium sparing aquaretic effects. The mechanisms of vasodilatation of ZP120 were examined in rat mesenteric resistance arteries.Experimental approach:Arterial segments (internal diameters 206±4 μm, n=224) were mounted in microvascular myographs for isometric tension recordings and electrical field stimulation (EFS).Key results:ZP120 and the endogenous NOP receptor ligand, N/OFQ, did not relax arteries contracted with noradrenaline or adenosine-triphosphate. EFS-evoked contractions were inhibited by a purinoceptor antagonist, suramin, and the α1-adrenoceptor antagonist prazosin. N/OFQ inhibited, concentration-dependently, EFS-evoked contractions with a maximal effect of 52±3% (n=8) at 1 μM. The maximal effect of 1 μM ZP120 was lower (27±5%, P<0.05, n=9) than for N/OFQ. Endothelial removal or pretreatment with capsaicin did not influence the vasodilator effects of ZP120 and N/OFQ. ZP120 and N/OFQ responses were preserved in the presence of suramin. The α2-adrenoceptor antagonist, rauwolscine, antagonized the effect of clonidine and brimonidine, but ZP120 and N/OFQ inhibition of EFS-evoked contraction was unaltered. The competitive NOP receptor antagonist, UFP-101 (10 μM), prevented the inhibitory effect of N/OFQ, but not ZP120 suggesting that N/OFQ and ZP120 have distinct modes of interaction with the NOP receptor.Conclusions and implications:Our findings suggest that the vasodilator effect of ZP120 and N/OFQ in rat mesenteric resistance arteries is mediated by prejunctional inhibition of adrenergic neurotransmission. These properties, that promote diuresis and attenuate the cardiovascular consequences of increased sympathetic nerve activity, make ZP120 a promising drug candidate.British Journal of Pharmacology (2008) 153, 1185–1194; doi:10.1038/sj.bjp.0707688; published online 14 January 2008 [ABSTRACT FROM AUTHOR]

Background and Purpose: The human, rat, and mouse P2X(7) receptors have been previously characterized, and in this study we report the cloning and pharmacological properties of the guinea pig orthologue.Experimental Approach: A cDNA encoding for the guinea pig P2X(7) receptor was isolated from a guinea pig brain library. The receptor was expressed in U-2 OS cells using the BacMam viral expression system. A monoclonal antibody was used to confirm high levels of cell surface expression and the functional properties were determined in ethidium bromide accumulation studies.Key Results: The predicted guinea pig protein is one amino acid shorter than the human and rat orthologues and over 70% identical to the rat and human receptors. In contrast to human and rat P2X(7) receptors, 2'-&3'-O-(4benzoylbenzoyl)ATP (BzATP) was a partial agonist of the guinea pig P2X(7) receptor when compared to ATP and acted as an antagonist in some assays. However, as at other species orthologues, BzATP was more potent than ATP. The guinea pig P2X(7) receptor possessed higher affinity for 1-[N,O-bis(5-isoquinoline sulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62), suramin and Coomassie Brilliant Blue than human or rat P2X(7) receptors suggesting that it is pharmacologically different to other rodent or human P2X(7) receptors.Conclusions and Implications: The guinea pig recombinant P2X(7) receptor displays a number of unique properties that differentiate it from the human, rat and mouse orthologues and this structural and functional information should aid in our understanding of the interaction of agonists and antagonist with the P2X(7) receptor. [ABSTRACT FROM AUTHOR]

Cancers are a heterogeneous mix of cells, some of which exhibit cancer stem cell-like characteristics including ATP-dependent drug efflux and elevated tumorigenic potential. To determine whether aerodigestive squamous cell carcinomas (SCCs) contain a subpopulation of cancer stem cell-like cells, we performed Hoechst dye efflux assays using four independent cell lines. Results revealed the presence of a rare, drug effluxing stem cell-like side population (SP) of cells within all cell lines tested (SCC-SP cells). These cells resembled previously characterised epithelial stem cells, and SCC-SP cell abundance was positively correlated with overall cellular density and individual cell quiescence. Serial SCC-SP fractionation and passaging increased their relative abundance within the total cell population. Purified SCC-SP cells also exhibited increased clonogenic potential in secondary cultures and enhanced tumorigenicity in vivo. Despite this, SCC-SP cells remained chemotherapeutically sensitive upon ATP-dependent transporter inhibition. Overall, these findings suggest that the existence of ATP transporter-dependent cancer stem-like cells may be relatively common, particularly within established tumours. Future chemotherapeutic strategies should therefore consider coupling identification and targeting of this potential stem cell-like population with standard treatment methodologies. [ABSTRACT FROM AUTHOR]

Background and purpose:Activation of P2X receptors on macrophages is an important stimulus for cytokine release. This study seeks evidence for functional expression of P2X receptors in macrophages that had been only minimally activated.Experimental approach:Whole-cell recordings were made from macrophages isolated 2–6 h before by lavage from mouse peritoneum, without further experimental activation. ATP (1–1000 μM) elicited inward currents in all cells (holding potential −60 mV). The properties of this current were compared among cells from wild type, P2X1 −/− and P2X4 −/− mice.Key results:Immunoreactivity for P2X1 and P2X4 receptors was observed in wild type macrophages but was absent from the respective knock-out mice. In cells from wild type mice, ATP and αβmethyleneATP (αβmeATP) evoked inward currents rising in 10–30 ms and declining in 100–300 ms: these were blocked by pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 10 μM). ATP also elicited a second, smaller (∼10% peak amplitude), more slowly decaying (1–3 s) at concentrations 10 μM: this was resistant to PPADS and prolonged by ivermectin. Macrophages from P2X1 −/− mice responded to ATP (>100 μM) but not αβmeATP: these small currents were prolonged by ivermectin. Macrophages from P2X4 −/− mice responded to ATP and αβmeATP as cells from wild type mice, except that ATP did not evoke the small, slowly decaying component: these currents were blocked by PPADS.Conclusion:Mouse peritoneal macrophages that are minimally activated demonstrate membrane currents in response to ATP and αβmeATP that have the predominate features of P2X1 receptors.British Journal of Pharmacology (2007) 152, 1283–1290; doi:10.1038/sj.bjp.0707504; published online 15 October 2007 [ABSTRACT FROM AUTHOR]

Background and purpose:Noradrenaline and ATP are sympathetic co-transmitters. In the rat perfused mesenteric bed cannabinoids have been shown to modify the overall response to sympathetic nerve stimulation. This study has assessed whether cannabinoid receptor activation modulates differentially the noradrenergic and purinergic components of sympathetic vasoconstriction.Experimental approach:Rat mesenteric beds were perfused with physiological salt solution and the effects of cannabinoids on responses to nerve stimulation, or exogenous noradrenaline or α,β-methylene ATP (α,β-meATP; P2X receptor agonist) were determined after raising tone with U46619. The effects of cannabinoids on the noradrenaline and ATP components of sympathetic neurotransmission were assessed using the α1-adrenoceptor antagonist, prazosin, or after P2X receptor desensitization with α,β-meATP.Key results:Anandamide, WIN 55,212-2 and CP55,940 attenuated sympathetic neurogenic vasoconstrictor responses. The inhibitory actions of anandamide and WIN 55,212-2 were blocked by LY320135, a CB1 receptor antagonist, but not by SR144528, a CB2 receptor antagonist. The inhibitory actions of CP55,940 were unaffected by LY320135 and SR144528. WIN 55,212-3, the inactive S(−) enantiomer of WIN 55,212-2, had no effect on sympathetic neurogenic responses. None of the cannabinoids affected contractile responses to exogenous noradrenaline or α,β-meATP. Anandamide and WIN 55,212-2 inhibited both the noradrenaline and ATP components of the sympathetic neurogenic contractile responses, with effects on the ATP component being most marked.Conclusions and implications:These results indicate that prejunctional CB1-like receptors mediate the sympathoinhibitory action of anandamide and WIN 55,212-2, but not CP55,940, in the rat mesenteric bed. Cannabinoids inhibit both the noradrenergic and purinergic components of sympathetic neurotransmission.British Journal of Pharmacology (2007) 152, 725–733; doi:10.1038/sj.bjp.0707397; published online 16 July 2007 [ABSTRACT FROM AUTHOR]

Background and Purpose: Red blood cells (RBCs) are reservoirs of vasodilatory, antiaggregatory, and antiinflammatory lipid mediators-epoxyeicosatrienoic acids (EETs). This study addresses the formation and release of erythrocyte-derived EETs in response to ATP receptor stimulation that may represent an important mechanism regarding circulatory regulation.Experimental Approach: Erythrocyte EET formation and release were investigated by incubating rat RBCs in physiological salt solution with agents that effected ATP release via P2 receptor stimulation of phospholipase A2 and epoxygenase-like activities with activation of the ATP secretory mechanism. EETs were analyzed by gas and liquid chromatography-mass spectrometry.Key Results: EETs were released from rat RBCs: 14,15-, 11,12-, 8,9- and 5,6-EETs in a ratio of 1.2:1.0:0.9:0.8. EETs were produced by epoxidation of arachidonic acid catalyzed by hemoglobin. Spontaneous release of EETs, 0.66+/-0.14 ng per 10(9) RBCs, was dose-dependently increased by an ATP analog, BzATP, and inhibited by P2X(7) receptor antagonists. 5 microM ATP increased release of EETs over 20% to 0.83+/-0.15 ng per 10(9) RBCs; 10 microM BzATP tripled the amount of EET release to 1.87+/-0.20 ng per 10(9) RBCs. EET release by ATP or BzATP was not associated with hemolysis. Carbenoxolone, a gap junction inhibitor that inhibits ATP release, and glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), which is required for ATP release, inhibited the spontaneous and stimulated EET release from RBCs.Conclusions and Implications: EETs are produced and released from RBCs via a mechanism that is mediated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, pannexin-1 and CFTR. [ABSTRACT FROM AUTHOR]

Background and Purpose: Hepatic stellate cells play an important role in liver fibrosis but little is known about liver myofibroblasts located around the central vein and in the portal area. In this study, intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured to assess the response to endothelin-1 (ET-1), platelet derived growth factor (PDGF) and ATP in rat liver myofibroblasts.Experimental Approach: Rat liver myofibroblasts were compared in 'quiescent' (cultured on Matrigel-coated dishes) and 'activated' (cultured on non-coated plastic dishes) conditions. [Ca(2+)](i) was measured with the fluorescent dye fura-2 and mRNA for ET-1, PDGF and their receptors by RT-PCR.Key Results: ET-1 increased [Ca(2+)](i) in quiescent cells but not in activated cells, whereas PDGF-BB increased [Ca(2+)](i) in activated cells but not in quiescent cells. However, there was no difference between responses to ATP in quiescent or activated cells. ET-1 (in quiescent cells), PDGF-BB (in activated cells) and ATP (in both cells) all induced transient increases in [Ca(2+)](i) in the absence of extracellular Ca(2+) (with EGTA), indicating the involvement of Ca(2+) release from intracellular Ca(2+) stores. The sustained increase in [Ca(2+)](i) in the presence of external Ca(2+) in activated cells (ATP and PDGF) was significantly reduced by nicardipine, a L-type Ca(2+) channel blocker, but not in quiescent cells (ATP and ET-1).Conclusions and Implications: The different pharmacological profiles of [Ca(2+)](i)-response in quiescent and activated myofibroblasts suggest that ET-1 and PDGF contribute differently to myofibroblast activation during the process of liver fibrosis. [ABSTRACT FROM AUTHOR]

Background and purpose:The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X3 receptor-channels.Experimental approach:Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X3 receptors (HEK293-hP2X3 cells) and rat dorsal root ganglion (DRG) neurons.Key results:In both cell types, the P2Y1,12,13 receptor agonist, ADP-β-S, inhibited P2X3 currents evoked by the selective agonist, α,β-methylene ATP (α,β-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-β-S, a procedure known to block all G protein heterotrimers. P2X3 currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X3 receptor agonist α,β-meATP. In the presence of intracellular GDP-β-S, ATP- and α,β-meATP-induced currents were identical. Recovery from P2X3 receptor desensitization induced by repetitive ATP application was slower than the recovery from α,β-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-β-S, the recovery from the ATP- and α,β-meATP-induced desensitization were of comparable speed.Conclusions and Implications:Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X3 receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X3 and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity.British Journal of Pharmacology (2007) 151, 226–236. doi:10.1038/sj.bjp.0707217; published online 12 March 2007 [ABSTRACT FROM AUTHOR]

Background and Purpose: The 2-propyl-1,4 benzoxazine (AM10) shows a peculiar behaviour in skeletal muscle, inhibiting or opening the ATP-sensitive K(+) (KATP) channel in the absence and presence of ATP, respectively. We focused on tissue selectivity and mechanism of action of AM10 by testing its effects on pancreatic KATP channels by means of both in vitro and in vivo investigations.Experimental Approach: In vitro, patch-clamp recordings were performed in native pancreatic beta cells and in tsA201 cells expressing the Kir6.2 Delta C36 channel. In vivo, an intraperitoneal glucose tolerance test was performed in normal mice.Key Results: In contrast with what observed in the skeletal muscle, AM10, in whole cell perforated mode, did not augment KATP current (I(KATP)) of native beta cells but it inhibited it in a concentration-dependent manner (IC(50): 11.5 nM; maximal block: 60%). Accordingly, in current clamp recordings, a concentration-dependent membrane depolarization was observed. On excised patches, AM10 reduced the open-time probability of KATP channels without altering their single channel conductance; the same effect was observed in the presence of trypsin in the bath solution. Moreover, AM10 inhibited, in an ATP-independent manner, the K(+) current resulting from expressed Kir6.2 Delta C36 (maximal block: 60% at 100 microM; IC(50): 12.7 nM) corroborating an interaction with Kir. In vivo, AM10 attenuated the glycemia increase following a glucose bolus in a dose-dependent manner, without, at the dose tested, inducing fasting hypoglycaemia.Conclusion and Implications: Altogether, these results help to gain insight into a new class of tissue specific KATP channel modulators. [ABSTRACT FROM AUTHOR]

Background and purpose:Our knowledge of the effects of P2-receptor activation on renal vascular tone comes mostly from in vitro models. We aimed to characterise the pharmacology of ATP in the renal circulation in vivo.Experimental approach:In pentobarbitone anaesthetized rabbits, we examined total renal and medullary vascular responses to ATP (0.2 and 0.8 mg kg-1), β,γ-methylene ATP (β,γ-mATP, 7 and 170 μg kg-1), α,β-mATP (0.2 and 2 μg kg-1) and adenosine (2 and 6 μg kg-1) using transit-time ultrasound and laser Doppler flowmetry, respectively. We also determined whether adenosine receptors, NO or prostanoids contribute to the actions of the purinoceptor agonists.Key results:Renal arterial boluses of ATP, β,γ-mATP, and adenosine produced biphasic changes; ischaemia followed by hyperaemia, in total renal and medullary blood flow. α,β-mATP induced only ischaemia. The adenosine receptor antagonist 8-(p-sulphophenyl)theophylline reduced the responses to adenosine and the hyperaemic responses to ATP and β,γ-mATP only. NO synthase inhibition (Nω-nitro-L-arginine) did not significantly alter responses to the P2 receptor agonists. Subsequent cyclooxygenase inhibition (ibuprofen) reduced the ATP- and β,γ-mATP-induced increases in renal blood flow. All other responses remained unchanged.Conclusions and implications:In the rabbit kidney in vivo, α,β-mATP sensitive receptors mediate vasoconstriction. β,γ-mATP and ATP induce vasodilation at least partly through adenosine receptors. ATP induced renal vasodilatation is independent of NO and partly dependent on prostanoids in the bulk of the kidney, but not in the vasculature controlling medullary blood flow.British Journal of Pharmacology (2006) 149, 523–531. doi:10.1038/sj.bjp.0706901; published online 18 September 2006 [ABSTRACT FROM AUTHOR]

Background and purpose: The ATP-gated P2X7 receptor is an unusual ion channel that couples to multiple downstream signalling cascades. We noted differences in mouse cDNA sequences that may indicate polymorphisms; the aim of this study was to compare function and expression of these mouse P2X7 receptor mutations. Experimental approach: There are three differences in the sequences of P2X7 cDNA cloned from mouse NTW8 microglial cells or C57 BL/6 mice: [Phe11,Ala221,Met283]P2X7 in the former and [Leu11,Thr221,Thr283]P2X7 in the latter. We expressed these receptors and measured membrane currents, ethidium uptake, calcium influx and surface membrane expression. We also carried out these assays on the previously described polymorphism observed between C57 BL/6 and Balb/c mice ([Leu451]P2X7 vs [Pro451]P2X7). Key results: Maximum current densities at [Phe11,Ala221,Met283]P2X7 were <12% of those at [Leu11,Thr221,Thr283]P2X7 without change in the agonist concentration-response. Replacing methionine with threonine at residue 283 yielded a receptor whose properties were the same as [Leu11,Thr221,Thr283]P2X7. Replacing T283 in the rat P2X7 receptor with methionine yielded currents that were <10% of wildtype and no ethidium uptake was associated with its activation. Maximum current densities and agonist EC50 values were the same at mouse [Thr283,Leu451]P2X7 and [Thr283,Pro451]P2X7 but ethidium uptake and Fluo4 fluorescence were significantly reduced at the [Thr283,Leu451]P2X7 receptor. There was equivalent surface membrane expression of all P2X7 receptors. Conclusions: This study has revealed a residue (Thr283) in the ectodomain that is critical for P2X7 receptor function and suggests that the intracellular residue 451 alters downstream signalling independently of ion channel activity. [ABSTRACT FROM AUTHOR]

Background and Purpose: Absorptive epithelia express apical receptors that allow nucleotides to inhibit Na(+) transport but ATP unexpectedly stimulated this process in an absorptive cell line derived from human bronchiolar epithelium (H441 cells) whilst UTP consistently caused inhibition. We have therefore examined the pharmacological basis of this anomalous effect of ATP.Experimental Approach: H441 cells were grown on membranes and the short circuit current (I(SC)) measured in Ussing chambers. In some experiments, [Ca(2+)](i) was measured fluorimetrically using Fura -2. mRNAs for adenosine receptors were determined by the polymerase chain reaction (PCR).Key Results: Cross desensitization experiments showed that the inhibitory response to UTP was abolished by prior exposure to ATP whilst the stimulatory response to ATP persisted in UTP-pre-stimulated cells. Apical adenosine evoked an increase in I(SC) and this response resembled the stimulatory component of the response to ATP, and could be mimicked by adenosine receptor agonists. Pre-stimulation with adenosine abolished the stimulatory component of the response to ATP. mRNA encoding A(1), A(2A) and A(2B) receptor subtypes, but not the A(3) subtype, was detected in H441 cells and adenosine receptor antagonists could abolish the ATP-evoked stimulation of Na(+) absorption.Conclusions and Implications: The ATP-induced stimulation of Na(+) absorption seems to be mediated via A(2A/B) receptors activated by adenosine produced from the extracellular hydrolysis of ATP. The present data thus provide the first description of adenosine-evoked Na(+) transport in airway epithelial cells and reveal a previously undocumented aspect of the control of this physiologically important ion transport process. [ABSTRACT FROM AUTHOR]

The effects of combined and simultaneously applied localised 43 degrees C hyperthermia (HT) and an antivascular bacteriochlorophyll-serine-based photodynamic therapy (Bchl-ser-PDT) on tumour growth and several microenvironmental parameters were examined. Rats bearing DS-sarcomas were allocated to treatment groups: (i) sham-treatment (control), (ii) Bchl-ser-PDT (20 mg kg(-1) i.v.), (iii) localised HT, (iv) Bchl-ser-PDT+HT. The light source used was an infrared-A irradiator, which, by use of appropriate filters, delivered the different ranges of wavelengths required. Following treatment, tumour volume was monitored. The greatest tumour growth inhibition was seen with Bchl-ser-PDT+HT, and subsequent experiments identified the pathophysiological basis for this effect. Red blood cell flux in tumour microvessels declined rapidly upon Bchl-ser-PDT+HT, reaching approximately 10% of initial values by the end of treatment. Similarly, tumour oxygenation worsened, reaching almost anoxic levels by the end of the treatment period. Assessment of metabolic parameters showed a pronounced increase in lactate levels and a decrease in ATP concentrations after combined treatment. The results presented suggest that vascular collapse and flow stasis resulting in a deterioration of tumour oxygenation and a switch from oxidative to glycolytic glucose turnover are key elements in the tumour eradication seen with this novel approach in which an antivascular PDT and HT are combined and simultaneously applied. [ABSTRACT FROM AUTHOR]

Background: Cleaning and disinfection processes must be improved so that there is a reduction in environmental contamination of frequent-contact surfaces. The objective of this study was to evaluate cleaning and disinfection of surfaces at a specialized healthcare unit after an intervention program.Methods: Exploratory, longitudinal, and correlational study carried out in a medium-complexity clinic. Two hundred and forty samples from five surfaces were collected during three phases: diagnosis; implementation of an intervention program; and evaluation of immediate and long-term effects. In total, 720 evaluations were made, performed through three monitoring methods: visual inspection; adenosine triphosphate bioluminescence assay (ATP); and aerobic colony count (ACC). The Wilcoxon, Mann-Whitney, and Fisher's Exact tests were run to analyze data statistically.Results: Cleaning and disinfection of surfaces were not being performed properly in most cases. Failure rates of surfaces reached 37.5 and 100% when the ATP and ACC procedures were used, respectively. However, after an intervention program, an improvement occurred. Success rates increased by 43.96% (ATP) and 12.46% (ACC) in phase I, by 70.6% (ATP) and 82.3% (ACC) immediately after interventions, and by 76.52% (ATP) and 85.76% (ACC) two months after the changes, showing that the program was effective.Conclusion: The present study reveals that implementing intervention actions with a cleaning and healthcare team brings benefits to prevent the spread of pathogenic agents through frequently touched hospital surfaces. [ABSTRACT FROM AUTHOR]

The article reports on developments in research as of March 8, 2007. Rocky wedges of debris act as "sandbags" to protect the Antarctic ice from rising sea levels. Vitamin B1 or thiamine, which occurs naturally in complex with adenosine triphosphate, is the first vitamin to do so. An animal model to test cancer drugs that target the blood supply of a tumor has been developed.