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Following the injection of 99mTechnetium-polyphosphate for bone scanning, a diffuse accumulation of radioactivity in post-pneumonectomy fibrothorax was demonstrated in 3 patients. This substantiates the nonspecificity of the localization of 99mTc-phosphorus compounds, and underlines the need for close clinical correlation in the accurate interpretation of such scans.
Amiloride-sensitive, epithelial Na(+) channel (ENaC)-mediated, active absorption of Na(+) is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na(+) and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and (22)Na(+) flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives.
Modulation of cell proliferation by vasoactive hormones and growth factors involves also changes in the activity of pH-regulatory transporters. In a preceeding paper (1) we examined the influence of such factors on cellular pH Here the influence of the same factors, diadenosine polyphosphates (ApnA), atrial natriuretic peptide, the growth factor PDGF and the Ca2+-ATPase inhibitor thapsigargin, on proliferation of cultured rat mesangial cells was examined by quantification of [3H]-thymidine incorporation. Mesangial cells were synchronised and growth reduced (0.5% FCS for 24 h) before experiments were started, Incubation with Ap3A, Ap4A, Ap5A or Ap6A (all 10 microM) for 24 h all reduced cell proliferation by 30 to 45%. At 0.1 and 1 microM the effects of Ap4A, Ap5A and Ap6A did not reach significance The antimitogenic effect of Ap5A was not significantly different when cells were incubated for 24, 48 or 72 h. In addition there was no significant difference between the antiproliferative effect of Ap5A in cells of the second, sixth or thirteenth passage. The growth factor PDGF-BB (0.25 nM) resulted man approximately 3-fold increase in [3H]-thymidine incorporation. This increase in proliferation could be significantly reduced by coincubation with 10 microM Ap5A. The mitogenic effect of PDGF was completely abolished in the presence of the Ca2-ATPase inhibitor thapsigargin (1 nM), which also significantly reduced basal cell proliferation by approximately 40%. Incubation of mesangial cells with 10 nM ANP for 24 h reduced basal [3H]-thymidine incorporation slightly by approximately 20% and decreased the PDGF-induced stimulation. The antimitogenic effects of these agonists is especially pronounced when cells are stimulated.
The effects of diadenosine tetraphosphate (AP4A), diadenosine pentaphosphate (AP5A), and diadenosine hexaphosphate (AP6A) on the cytosolic free calcium concentration ([Ca2+]i) were evaluated in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent dye technique. A concentration-dependent increase of [Ca2+]i by AP4A, AP5A, and AP6A was observed in VSMC. Additions of 10 micromol/l AP4A, AP5A, and AP6A significantly increased [Ca2+]i in VSMC by 224 +/- 98 nmol/l (n = 6; p < 0.01, 205 +/- 27 nmol/l (n = 14; p < 0.01), and 269 +/- 98 nmol/l (n = 5; p < 0.05), respectively. Additions of AP4A, AP5A, and AP6A only 120 s prior to angiotensin II (Ang II) administration significantly attenuated the Ang-II-induced changes of [Ca2+]i in VSMC from 1,053 +/- 174 nmol/l to 283 +/- 42 nmol/l, 591 +/- 112 nmol/l, and 477 +/- 79 nmol/l, respectively (each p<0.01) as compared to the control). The AP6A-induced changes of [Ca2+]i were inhibited by the blockers of P2 purinoceptors, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, but not by the inhibitor of P2y purinoceptors, reactive blue. Adenosine triphosphate (ATP) also increased [Ca2+]i in VSMC, whereas the purinoceptor P2x agonist, alpha,beta-methylene-ATP, had no effect on [Ca2+]i in VSMC. Therefore diadenosine polyphosphates may induce changes of [Ca2+]i by interacting with purinoceptors and may be involved in local regulation of vascular resistance evoked by the Ca(2+)-dependent contractile response of VSMC.
In our search for potential inhibitors of casein kinase II (CKII) in Artemia, we have shown that dinucleoside polyphosphates are a novel class of effectors for this ubiquitous protein kinase. P1,P4-di(guanosine-5')-tetraphosphate (Gp4G) is a better CKII inhibitor than P1,P4-di(adenosine-5')-tetraphosphate (Ap4A). The inhibition by both effectors is more potent when GTP is used as phosphate donor instead of ATP. The inhibition of CKII increases with the number of phosphates linking the guanosine/adenosine moieties (for n = 2-6). Ap4A does not compete with the protein substrate and causes an increase in the apparent KmATP and a decrease in the apparent VmATP, indicating a mixed type of inhibition with respect to ATP.
PURPOSE: To determine the role of polyphosphate kinase 1 (PPK1) in the ocular virulence of Pseudomonas aeruginosa. METHODS: Using a mouse model of infection, P. aeruginosa strains PAO1, PAOM5 (an isogenic mutant of PAO1 deficient in PPK1), and PAOM5+PPK1 (the mutant complemented with PPK1 on plasmid pHEPAK11) were compared for ocular virulence. These strains were also characterized with respect to traits associated with survival and pathogenicity in an ocular environment. RESULTS: The PPK1-deficient strain PAOM5 was significantly less virulent than either wild-type PAO1 or the complemented mutant (P <0.016). Loss of virulence was not associated with serum sensitivity or diminished adherence to the cornea. However, PAOM5 has an increased susceptibility to oxidative stress and was cleared from corneal tissue significantly better (P <0.006) than either the wild-type or restored strain. Furthermore, the PPK1-deficient mutant produced significantly less (P <0.022) pyocyanin. CONCLUSIONS: PPK1 is essential for a successful ocular infection by P. aeruginosa. The loss of ocular virulence is probably due to the dysregulation of multiple genes, including those responsible for stress response.
PURPOSE: To analyze the levels of the diadenosine polyphosphates Ap4A and Ap5A in tears, in a set of control subjects and in groups of symptomatic and nonsymptomatic persons with dry eye. METHODS: Ninety-seven subjects participated in the study. The subjects were divided into five experimental groups: control subjects; symptomatic patients with normal tear secretion; symptomatic patients with low tear secretion; forced blink; and corneal mechanical stimulation provided by a gas esthesiometer. The Schirmer I test was used to measure and collect tear secretions from each subject. All samples were processed by high pressure liquid chromatography (HPLC) and their Ap4A and Ap5A levels determined. RESULTS: The levels of Ap4A and Ap5A in tears were greater in all symptomatic patients than in control subjects, especially in symptomatic subjects with low tear secretion. Within the symptomatic subjects with normal tear secretion, significant differences in concentrations of Ap4A and Ap5A were found between men and women. In the forced blink experiments, concentrations of the Ap4A and Ap5A rose with increasing blink frequency. When the cornea was mechanically stimulated, the levels of Ap4A and Ap5A rose significantly during both moderate and high-flow rate tests. CONCLUSIONS: The increased levels of Ap4A and Ap5A in tears of patients with dry eye allow these dinucleotides to be used as objective biomarkers in dry eye conditions.
PURPOSE: To investigate the influence of diadenosine polyphosphates on the rate of corneal epithelial cell migration. METHODS: Primary corneal epithelial cell cultures were obtained from New Zealand White rabbits. Immunocytochemical experiments were performed by fixing the cells with 4% paraformaldehyde (PFA) and incubated with cytokeratin 3 primary antibody, which was subsequently incubated with a secondary IgG mouse labeled with FITC, and the cells were observed under confocal microscopy. Migration studies were performed by taking confluent monolayers that were wounded with a pipette tip and challenged with different di- and mononucleotides with or without P2 antagonist (n = 8 each treatment). For concentration-response analysis, compounds were tested in doses ranging from 10(-8) to 10(-3) M (n = 8). The stability of the dinucleotides was assayed by HPLC, with an isocratic method (n = 4). RESULTS: Cells under study were verified as corneal epithelial cells via the immunocytochemical analysis. Cell migration experiments showed that Ap4A, UTP, and ATP accelerated the rate of healing (5, 2.75, and 3 hours, respectively; P < 0.05; P < 0.001), whereas Ap3A, Ap5A, and UDP delayed it (6.5, 10, and 2 hours, respectively; P < 0.05). ADP did not modify the rate of migration. Antagonists demonstrated that Ap4A and Ap3A did activate different P2Y receptors mediating corneal wound-healing acceleration and delay. Concerning the possible degradation of the dinucleotides, it was almost impossible to detect any products resulting from their cleavage. CONCLUSIONS: Based on the pharmacological profile of all the compounds tested, the two main P2Y receptors that exist in these corneal cells are a P2Y(2) receptor accelerating the rate of healing and a P2Y6 receptor that delays this process.
Abnormal femoral head activity in Legg-Calve-Perthes' disease has been measured using 99Tcm -polyphosphate and a gamma camera/computer data collection system. A reference point on the data matrix, which is unaffected by the diease, is used for deriving comparative uptake ratios in each femoral head. The reference point remains unaltered after surgical procedures or short intervals between follow-up studies. Early abnormality can be measured in both unilateral and bilateral femoral head pathology.
In principal cells of rat cortical collecting ducts (CCD) cellular pH (pHi) is regulated by basolateral Na+/H+ exchange. The influence of various agonists on pHi and cellular Ca2+ activity ([Ca2+]i) in freshly isolated CCD cells was examined with BCECF and fura-2 fluorescence ratios. The recovery of pHi per minute (delta pH/min) after an acid load was 0.26 +/- 0.03 (N = 53) in control conditions and was increased by the diadenosine polyphosphates Ap4A, Ap5A, Ap6A, the phorbol ester phorbol 12-myristat 13-acetate (PMA) (each 5 mumol/L) and angiotensin II (100 nmol/L) by 0.05 +/- 0.02 (N = 10), 0.11 +/- 0.05 (N = 13), 0.09 +/- 0.02 (N = 24), 0.10 +/- 0.03 (N = 7), and 0.09 +/- 0.03 (N = 8), respectively. Vasopressin (10 nmol/L) decreased delta pH/min by 0.11 +/- 0.03 (N = 9); ATP and Ap3A (each 5 mumol/L) had no significant effect. The increase in delta pH/min with Ap6A was abolished in the presence of an inhibitor of protein kinase C, calphostin C (0.1 mumol/l, N = 8). Fura-2 fluorescence ratio was not significantly changed with angiotensin II, Ap3A, or Ap4A but increased with vasopressin, ATP, Ap5A, and Ap6A by 0.08 +/- 0.02 (N = 13), 0.04 +/- 0.02 (N = 13), 0.03 +/- 0.01 (N = 14), and 0.03 +/- 0.01 (N = 10), respectively. These data indicate that Na+/H+ exchange in rat CCD is activated by the stimulation of a Ca(2+)-independent protein kinase C and inhibited by protein kinase A.
Diadenosine polyphosphates are a group of low-weight compounds that increase after exposure to a wide variety of oxidants and have been suggested to act as "alarmones," alerting the cell to the onset of metabolic stress. We demonstrate here that glucose at concentrations that induce insulin release produce a 30- to 70-fold increase in the concentration of diadenosine triphosphate (Ap3A) and tetraphosphate (Ap4A) in beta-cells. Furthermore, Ap3A and Ap4A, at the concentrations found in glucose-stimulated cells, are effective inhibitors of the ATP-regulated K+ channels when applied to the intracellular side of excised membrane patches from cultured beta-cells. We suggest that Ap3A and Ap4A act as second messengers mediating a glucose-induced blockade of the pancreatic beta-cell ATP-regulated potassium channel.
Poly-beta-hydroxybutyrate/calcium polyphosphate (PHB-CaPolyPi) complexes exist as labile quasi-crystalline structures in bacterial plasma membranes. The composition, structure, and distribution of the complex suggest it may play a role in the regulation of intracellular calcium and in calcium signaling. The importance of these functions led to this investigation of the occurrence of PHB-CaPolyPi complexes in eukaryotes. A variety of plant and animal systems were analyzed and all were found to contain PHB associated with CaPolyPi. The intracellular location of the complex in bovine liver was primarily the mitochondria and microsomes, with smaller amounts in the plasma membranes. Eukaryotic PHB had the same narrow range of chain lengths (120-200 subunits) as PHB in bacterial membranes, and was associated with PolyPi of somewhat greater length (170-220) than the bacterial counterpart (130-170).
Broiler chick diets and drinking water were supplemented with two sources of vitamin C: crystalline L-ascorbic acid (AsA) or L-ascorbyl-2-polyphosphate (APP) to provide 0, 25, 50, 100, 200, 400, 800, 1,600, and 3,200 ppm (mg/kg) AsA. The bioavailability of APP relative to AsA, as estimated by the change in plasma AsA concentration, was evaluated during 24-h periods of supplementation. When provided in the feed, no differences in dietary AsA content were attributed to vitamin source. In contrast, APP administration at 25 and 50 ppm, resulted in higher (P < .001) AsA values in drinking water when compared with AsA supplementation. Plasma AsA values were elevated (P < .05) above baseline when either AsA or APP were supplemented in the feed or water at a level of 400 ppm or greater. Plasma AsA concentrations, following supplementation of the diets, were higher (P < .05) in AsA-treated (800 ppm) chicks when compared with APP-supplemented chicks. During water supplementation, AsA (800 ppm) and APP (3,200 ppm) administration resulted in higher plasma AsA values when compared with their alternate vitamin source. At all other levels of water supplementation, no differences in plasma AsA were associated with vitamin source. The absence of a consistent difference in plasma AsA, relative to vitamin source, suggests that the isolated differences observed may be due to chance. It was concluded that APP was of similar bioavailability to that of AsA, as estimated by the ability to elevate plasma AsA concentrations in broiler chicks.
The effects of supplemental ascorbyl-2-polyphosphate (APP) on adrenocortical function and underlying fearfulness in broiler chickens were assessed in a number of test situations. Chicks pretreated for a minimum of 24 h with APP (1,000 ppm equivalents of L-ascorbic acid) in their drinking water or with no APP (tap water controls; CON) had blood samples taken immediately following water treatment and again after exposure to a capture and cooping stressor for 10 min. First, although the cooping stressor markedly increased plasma corticosterone concentrations, pretreatment with APP failed to attenuate this adrenocortical response. Second, APP-treated chicks showed less freezing and vocalized sooner in an open field (novel environment) than did controls. They also showed nonsignificant tendencies toward accelerated and enhanced ambulation. Third, supplementation with APP reduced the duration of the birds' tonic immobility fear reactions. Collectively, these behavioral effects are indicative of dampened fear. The apparent reduction of nonspecific, underlying fearfulness by APP treatment may have important implications for poultry welfare and performance.
FcgammaR-mediated phagocytosis is accompanied by the generation of tissue-damaging products such as inflammatory cytokines and reactive oxygen species. Hence, the phagocytic response must be a tightly regulated process. Recent studies have established that clustering FcgammaR on human myeloid cells causes tyrosine phosphorylation of Src homology 2 domain-containing inositol polyphosphate phosphatase (SHIP). However, it is not known how these immunoreceptor tyrosine-based activation motif (ITAM)-bearing phagocytic FcgammaR activate SHIP, or whether the activation of SHIP by ITAMs has any functional relevance. Experiments addressing the mechanism of SHIP association with ITAMs have been done in in vitro systems using phosphopeptides. In this study we undertook to dissect the molecular mechanism by which SHIP associates with the native ITAM-FcgammaR and becomes phosphorylated. In this report we provide evidence that first, SHIP is indeed phosphorylated by ITAM-FcgammaR, using cell systems that lack FcgammaRIIb expression; second, coimmunoprecipitation experiments demonstrate that SHIP associates with native ITAM-bearing FcgammaRIIa in vivo; and third, phosphorylation of SHIP by FcgammaRIIa is inhibited by overexpressing either the SHIP Src homology 2 domain or a dominant negative mutant of Shc. In contrast, SHIP phosphorylation was not inhibited by a dominant negative mutant of Grb2. We extend these observations to show that SHIP activation by ITAM-FcgammaR down-regulates NF-kappaB-induced gene transcription. These findings both provide a molecular mechanism for SHIP association with native ITAM-bearing receptors and demonstrate that SHIP association with ITAM-FcgammaR serves to regulate gene expression during the phagocytic process.
Polyphosphate (polyP) is a conserved inorganic polymer traditionally viewed as a stress-induced phosphate and energy reserve. In cyanobacteria, however, polyP granules are constitutively present and frequently observed in proximity to carboxysomes, the bacterial microcompartments that mediate CO2 fixation. Here we show that polyP functions as a spatially organized regulator of the photosynthetic cytoplasm in Synechococcus elongatus. PolyP granules localize to the nucleoid and are periodically arranged along the cell axis, independently of the McdAB carboxysome positioning system. Despite this independence, polyP and carboxysomes associate non-randomly, and this association is enhanced when active carboxysome positioning by the McdAB system is disrupted. Loss of polyP synthesis leads to nucleoid expansion, an increased number of smaller carboxysomes with high mobility, and severe defects in growth under ambient CO2. Perturbation of polyP turnover further reveals structural connections to both carboxysomes and thylakoid membranes. Together, these findings identify polyP as an architectural integrator that couples chromosome organization, metabolic compartmentalization, and photosynthetic fitness.
The study was conducted to determine whether living or killed cell polyphosphate bodies (PPB) would sequester more of several heavy metals. Living and heat- or glutaraldehyde-killed cells were exposed to 20 ppm of Zn, Pb, Mn and Al. Air-dried cells on Formvar-coated grids were first observed in the transmission electron microscope. The unit was then switched to the scanning transmission mode of operation with the spot setting. X-rays were collected on an energy dispersive X-ray spectrometer and mass fractions of the metals were determined. In all cases live cells sequestered a larger amount of the metal than cells killed by boiling or with glutaraldehyde. In all cases the cells killed by glutaraldehyde sequestered more of the heavy metals than cells killed by boiling. The results of the investigation show that PPB in living cells with active uptake systems take up and sequester more of the metals Zn, Pb, Mn and Al than killed cells.
Joint imaging with 99m-Tc-Polyphosphate was carried out in 21 patients having inflammatory or degenerative diseases of the joints of the hands. A comparison was made between 99m-TcPP, xeroradiography and radiography. In a total of 630 different joints 237 positive scans, 132 positive roentgenograms and 183 positive xeroradiograms were obtained. In the early stage of inflammatory joint disease only the scintigraphic results are positive, in the later stage 99mTcPP-imaging was less sensitive than xeroradiography and radiography. The reason for the increased concentration at the site of acute joint inflammation is probably the increased uptake in the soft tissues of the joints. The best time for scanning after the application of 99m-TcPP seemed to be at 3 hours.