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At least 1,297 records · Page 72Linked to original sources
PubMed1990

Anti-proliferative activity of L-651,582 correlates with calcium-mediated regulation of nucleotide metabolism at phosphoribosyl pyrophosphate synthetase.

L-651,582, 5-amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-1, 2,3-triazole-4-carboxamide, is an antiproliferative and antiparasitic agent which inhibits nucleotide metabolism in mammalian cells. The drug equivalently inhibited 3H-hypoxanthine, 14C-adenine, and 14C-formate incorporation into nucleotide pools in Madin-Darby bovine kidney (MDBK) cells, suggesting depletion of the supply of phosphoribosyl pyrophosphate, (PRPP), required for each of these independent pathways. Inhibition of nucleotide metabolism correlated with inhibition of proliferation for three cell types with differing sensitivities toward the drug. L-651,582 inhibited incorporation of 3H-hypoxanthine into nucleotide pools with either glucose, uridine, or ribose as carbon source suggesting a block at PRPP synthetase, rather than a block in a pathway supplying ribose-5-phosphate. PRPP synthetase was not inhibited directly by the compound, indicating regulation of the enzyme in intact cells. Drug treatment did not kill cells but reduced the fraction of cells in S and G2/M while increasing the population in G1. Inhibition of uptake of 45Ca was demonstrated at concentrations identical to those required for inhibition of nucleotide metabolism or proliferation. Inhibition of cellular PRPP biosynthesis rates were also observed using EGTA to lower calcium levels. These data suggest a previously unrecognized link between calcium entry, the regulation of nucleotide biosynthesis at PRPP synthetase, and the rate of proliferation of mammalian cells.

Aminoimidazole Carboxamide↗
PubMed2006

Expression of metalloproteinase-7 (matrilysin) in human blood and bronchoalveolar gamma/delta T-lymphocytes. Selective upregulation by the soluble non-peptidic mycobacterial phosphoantigen (isopentenyl pyrophosphate).

Human gammadelta T-lymphocytes are believed to regulate local immune defense and enhance resistance against invading microbes, although their precise function remains unknown. Herein, we addressed the question whether gammadelta T-lymphocytes mediate these processes via synthesis of MMP-7, a protease closely associated with both epithelial repair and mucosal defense. Blood and bronchoalveolar gammadelta T-lymphocytes were cultured in the absence and presence of isopentenyl pyrophosphate (IPP) or TGF-beta1/IL-15 for 24 h, and assessed for the expression and synthesis of MMP-1, MMP-7, and MMP-9. Resting human gammadelta T-lymphocytes constitutively expressed MMP-9 mRNA, a marginal or no MMP-7 and MMP-1 mRNA. In the presence of IPP (3 microg/ml), expression of MMP-7 mRNA significantly increased, whereas TGF-beta1/IL-15 had no effect. Further, quiescent gammadelta T-lymphocytes obtained from bronchoalveolar lavage (BAL) fluid showed a weak or no MMP-7 mRNA signal which was raised significantly following stimulation with IPP. In Western blot analysis, a 28-kDa pro-matrilysin could be detected both in cell lysates (2 days) and supernatants (5 days) with a four- to sevenfold increased signal following IPP-stimulation of the gammadelta T-lymphocytes. In conclusion, the data demonstrate for the first time that both human blood and BAL gammadelta T-lymphocytes express MMP-7 mRNA and synthesize MMP-7-protein. This unfolds a new perspective for the understanding of gammadelta T-lymphocyte function.

Antigens, CD↗
PubMedDate not supplied

Evaluation of inorganic pyrophosphate in amniotic fluid as a mode of prenatal diagnosis of osteogenesis imperfecta.

Using a modified procedure by Solomons and Styner (1969), an evaluation of inorganic pyrophosphate (PPi) was performed on the amniotic fluid of two fetuses at risk for osteogenesis imperfecta (OI) at 14 1/2 weeks gestation. The parents of both cases had a previous child with OI, Type II. The normal control group at 14-16 weeks gestation had PPi values ranging from 22.0-59.2 micrograms/100 ml, with a mean of 38.6 +/- 9.51 micrograms/100 ml. In each at-risk fetus, the amniotic fluid PPi value was within normal range. The first baby was born phenotypically normal at term. Intrauterine radiographic and fetal sonograms were done on the second fetus at approximately 19 weeks gestation. Both showed evidence of OI, Type II. The pregnancy was terminated at 21 weeks. Radiologic studies of the aborted fetus were consistent with OI, Type II. Our results indicate that the evaluation of PPi levels in amniotic fluid is not the method of choice for prenatal diagnosis of OI.

Amniocentesis↗
PubMed1993

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase and fructose 2,6-bisphosphate in the bundle sheath of maize leaves.

The aim of this work was to discover whether the cells of the bundle sheath of the leaves of maize (Zea mays) contained pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) and fructose 2,6-bisphosphate (Fru-2,6-P2). Physiologically active preparations of bundle sheath cells from leaves of 4- to 6-week-old plants showed activities of PFP, 6-phosphofructo-2-kinase (6-PF-2-K), and fructose-2,6-bisphosphatase (Fru-2,6-Pase) of 38, 1.8, and 15 nmol min-1 mg-1 chlorophyll, respectively, and contained 75 pmol mg-1 chlorophyll Fru-2,6-P2. For the above enzymes, and marker enzymes for the bundle sheath and for mesophyll cells, the ratios of the activities in leaf extracts to those in bundle sheath extracts were determined. The ratios for PFP, 6-PF-2-K, Fru-2,6-Pase, and Fru-2,6-P2 were intermediate between those found for the mesophyll markers and bundle sheath markers. The distribution of PFP activity after nonaqueous fractionation of leaves differed from that of the bundle sheath and mesophyll marker enzymes. It is argued that maize bundle sheaths can contain significant activity of PFP and amounts of Fru-2,6-P2.

Carbon Dioxide↗
PubMed1993

Inorganic pyrophosphate gives a membrane potential in yeast mitochondria, as measured with the permeant cation tetraphenylphosphonium.

Evidence for a membrane potential (delta psi) generating capacity of inorganic pyrophosphate (PPi) with inorganic pyrophosphatase in mitochondria from the yeast Saccharomyces cerevisiae is presented. Addition of PPi increases the accumulation of the permeant cation tetraphenylphosphonium (TTP+) in mitochondria, as measured with a TPP(+)-selective electrode or using [3H]TPP+. This accumulation is strongly inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) but not by oligomycin, whereas accumulation of TPP+ induced by ATP is strongly inhibited by both FCCP and oligomycin. The values of delta psi obtained upon addition of PPi were similar to those obtained when yeast mitochondria were energized by NADH or ATP. Energization of the wild type yeast mitochondria with ATP resulted in higher delta psi values than those achieved when PPi was hydrolyzed, whereas the effect of PPi was more pronounced in mitochondria from a pyrophosphatase (PPase) overproducing yeast strain. These results clearly indicate that PPi is significant as an energy donor and that the yeast mitochondrial PPase, the gene for which we have recently cloned and sequenced, may participate in energy transfer reactions.

Adenosine Triphosphate↗
PubMed1994

Potato tuber pyrophosphate-dependent phosphofructokinase: effect of thiols and polyalcohols on its intrinsic fluorescence, oligomeric structure, and activity in dilute solutions.

The effect of dilution of homogeneous potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90; PFP) on the enzyme's intrinsic fluorescence, activity, and oligomeric structure has been examined. A rapid decrease in PFP's intrinsic fluorescence occurred in response to dilution. The decay follows double-exponential kinetics and was accompanied by a reduction in catalytic activity (measured in the glycolytic direction). Gel filtration-HPLC indicated a concomitant deaggregation of the native alpha 4 beta 4 heterooctamer into the inactive free alpha- and beta-subunits, followed by random aggregation of the subunits into an inactive, high M(r) conglomerate. The addition of 2 mM dithiothreitol, 2 mM 2-mercaptoethanol, or 5% (w/v) polyethylene glycol, but not any of the substrates, Mg2+, or fructose 2,6-bisphosphate, prevented this process. When purified PFP was stored for 1 week at -20 degrees C in the presence of 50% (v/v) glycerol partial degradation of its alpha-subunit occurred. This resulted in a labile enzyme that was more susceptible to subunit dissociation. The intrinsic fluorescence of the degraded PFP could be stabilized by 5% (w/v) polyethylene glycol, but not by 2 mM dithiothreitol or 2-mercaptoethanol. It is proposed that the current assay procedures for PFP, which normally involve considerable dilution in the absence of added sulfhydryl reducing agents or polyhydroxy compounds, may underestimate the actual activity of the enzyme. This has important implications for the assessment of the functions and regulation of PFP in vivo.

Alcohols↗
PubMed1999

Expression, characterization, and crystallization of the pyrophosphate-dependent phosphofructo-1-kinase of Borrelia burgdorferi.

The two genes for the putative pyrophosphate-dependent phosphofructokinases (PPi-PFKs) of Borrelia burgdorferi were cloned by PCR and expressed in Escherichia coli, and their protein products were purified to near homogeneity. The larger of the two gene products, a 62-kDa protein, is an active PPi-PFK and exists in solution as a dimer. It has apparent K(m) values for fructose 6-P and PPi of 109 and 15 microM, respectively, and a pH optimum of 6.4 to 7.2. The 62-kDa protein was crystallized and subjected to preliminary diffraction analysis. The smaller gene product, a 48-kDa protein, exists in solution as a higher polymer and shows no ATP- or PPi-dependent activity, despite having a secondary structure as estimated by circular dichroism that is not significantly different from that of other PFKs.

Animals↗
PubMed2000

Site-directed mutagenesis of the fructose 6-phosphate binding site of the pyrophosphate-dependent phosphofructokinase of Entamoeba histolytica.

Attempts to define the active site of pyrophosphate-dependent phosphofructokinase (PPi-PFK) using homology modeling based on the three-dimensional structure of the ATP-dependent PFKs from bacteria have been frustrated by low sequence identity between PPi- and ATP-PFKs in their carboxyl terminal halves. In the current study, alanine scanning mutagenesis of residues in the carboxyl terminal half of the PPi-PFK of Entamoeba histolytica coupled with comparative sequence analysis and computational modeling is used to identify residues that contribute to fructose 6-phosphate (fructose 6-P) binding. Of seven alanine mutants that were generated by site-directed mutagenesis, Arg377, Ser392, Arg405, Lys408, His415, His416, and Arg423, only the last mutant, Arg423Ala, was found to have dramatically lower affinity for fructose 6-P. Mutation of Arg 423 decreased k(cat) by 10,000-fold and decreased apparent affinity for fructose 6-P by 126-fold, while the K(m) for PPi increased only 4-fold. The second greatest effect was seen with Arg377Ala, which had a nearly 10-fold decrease in apparent affinity and an approximate 60-fold decrease in maximal activity. Another residue, Tyr420, was chosen for mutagenesis by its complete identity in all other PPi-PFK. This residue and its homologue in Escherichia coli ATP-PFK, His249, were mutated and shown to be very important for substrate binding in both enzymes.

Alanine↗
PubMed1996

Real-time DNA sequencing using detection of pyrophosphate release.

An approach for real-time DNA sequencing without the need for electrophoresis has been developed. The approach relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (Nyrén, P. (1987) Anal. Biochem. 167, 235-238). The PPi formed in the DNA polymerase reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the sequencing procedure, immobilized single-stranded template was used in a repeated cycle of deoxynucleotide extension. Real-time signals in the ELIDA, proportional to the amount of incorporated nucleotide, were observed when complementary bases were incorporated. An increased signal-to-noise ratio was obtained by substitution of deoxyadenosine alpha-thiotriphosphate (dATP alpha S) for the natural deoxyadenosine triphosphate, dATP alpha S is efficiently used by the DNA polymerase, but is not recognized by the luciferase. As a model, 15 bases of a single-stranded PCR product were sequenced. The possibility for parallel processing of many samples in an automated manner is discussed.

Adenosine Triphosphate↗
PubMed1997

Determination of farnesyl pyrophosphate in dog and human plasma by high-performance liquid chromatography with fluorescence detection.

A nonradioisotopic method has been developed for the determination of all-trans-farnesyl pyrophosphate (FPP), the common intermediate at the branch point of the biosynthesis of cholesterol and nonsterol end products, in dog and human plasma. FPP was cleaved to the parent alcohol, farnesol, by the direct addition of alkaline phosphatase to plasma. Farnesol extracted from plasma was converted into a fluorescent derivative with 9-anthroylcyanide. After the excess reagent was removed using an NH2-bonded phase cartridge, the derivative was separated by a column-switching high-performance liquid chromatographic system, followed by fluorescence detection. A linear response was obtained over the range of 2-18 ng/ml, when FPP was added to dog plasma in which the endogenous FPP concentrations had been lowered to undetectable levels by treatment with an HMG-CoA reductase inhibitor. The endogenous plasma FPP levels in the morning in dog and human detected for the first time by our method were 5.2 and 6.6 ng/ml, respectively. The method was utilized to examine the circadian rhythm of FPP in dog plasma, and different rhythms were observed between feeding and fasting dogs.

Adult↗
PubMed2002

Geranylgeranyl pyrophosphate counteracts the cataractogenic effect of lovastatin on cultured rat lenses.

Statins are commonly prescribed cholesterol-lowering agents which inhibit the rate-limiting enzyme of the cholesterol biosynthetic pathway. In addition to inhibiting cholesterol synthesis, statins also inhibit the synthesis of other sterol and non-sterol compounds produced by the pathway including the isoprenoids, farnesyl (FP) and geranylgeranyl pyrophosphate (GGP). Certain proteins, most notably small GTP-binding proteins of the Ras superfamily, must be post-translationally modified by addition of a farnesyl or geranylgeranyl moiety in order to be properly targeted to membranes and to be active. Statins have been shown to affect cellular processes such as proliferation, signaling and apoptosis and it is likely that these effects are due, at least in part, to decreased isoprenoid synthesis. Certain statins have been shown to produce cataracts in experimental animals. We have previously demonstrated that lenses exposed to lovastatin during organ culture may develop cataracts as well, and we proposed that this resulted from decreased prenylation of small GTP-binding proteins. To test our hypothesis, rat lenses were exposed to lovastatin in organ culture with concomitant supplementation of the medium with GGP and/or FP. The results clearly demonstrated that GGP strongly inhibited lovastatin-induced lens opacification in this system while FP had little effect. GGP also markedly reduced the histological changes and the increased epithelial cell apoptosis induced in the cultured lenses by lovastatin. The data indicate that inhibition of protein prenylation, perhaps of Rho GTPases, is an important factor in the lovastatin-induced cataract in vitro.

Animals↗
PubMed1980

Changes in liver inorganic pyrophosphate content during ethanol metabolism.

The injection of ethanol (70 mmol/kg body weight) into male Wistar rats fed ad libitum caused an increase within 15 min in the liver inorganic pyrophosphate (PPi) content from 0.012 +/- 0.001 mumol/g (wet weight) to 0.029 +/- 0.001 mumol/g. The injection of acetate (20 mmol/kg body weight) increased the liver PPi to 0.157 +/- 0.075 mumol/g in 15 min. The alcohol dehydrogenase inhibitor 4-methylpyrazole blocked the accumulation of acetate and the increase in PPi was prevented. Disulfiram, an inhibitor of aldehyde dehydrogenase, did not prevent changes in redox state, but the accumulation of acetate in the liver was decreased and the increase in liver PPi content was diminished (0.019 +/- 0.003 mumol/g). These data suggest that the increase in PPi observed after injection of ethanol may be due to the activation of the acetate produced during ethanol and acetaldehyde oxidation.

Animals↗
PubMed1995

Structure of a functional geranylgeranyl pyrophosphate synthase gene from Capsicum annuum.

A geranylgeranyl pyrophosphate synthase (GGPPS) gene from Capsicum annuum (bell pepper) was cloned. The nucleotide sequence shows that this gene, like the capsanthin/capsorubin gene but unlike the phytoene synthase gene from C. annuum, is not interrupted by an intron. Southern blot analysis of C. annuum genomic DNA suggests the presence of a single gene highly similar to the cDNA and also of additional related sequences. The present data suggest that this cloned gene is functional.

Capsicum↗
PubMed1992

Pyrophosphate synthesis from phospho(enol)pyruvate catalyzed by precipitated magnesium phosphate with "enzyme-like" activity.

The enzyme-like kinetic properties of precipitated magnesium phosphate as a catalyst for formation of pyrophosphate (PPi) from phospho(enol)pyruvate (PEP) are described. This synthesis occurs at a low temperature (37 degrees C) and represents a model that may help us understand the relevance to chemical evolution of minerals as ancient catalysts whose functions could have been taken over by contemporary enzymes. An insoluble Pi.Mg matrix was formed in a medium with 80% of the water replaced by dimethyl sulfoxide as a way of simulating conditions in a drying pond. Phospho(enol)pyruvate adsorbs onto the Pi.Mg surface according to a Langmuir isotherm, and the PEP concentration dependence of PPi formation follows a Michaelian-like function. A yield of 33% for transformation of the initially adsorbed PEP into PPi was attained after 4 days of incubation with equimolecular concentrations of Pi, MgCl2, and PEP. The magnesium concentration dependence for Pi and Mg precipitation, for adsorption of PEP onto solid Pi.Mg, and for PPi formation showed complex cooperative behavior. These results taken as a whole lead to the conclusion that the Pi.Mg surface not only provides a reactant for PPi formation but also catalyzes the reaction.

Diphosphates↗
PubMed1994

Case report 859: Massive calcium pyrophosphate dihydrate crystal deposition disease involving the wrist joint.

A case of massive calcium pyrophosphate dihydrate crystal deposition disease (MCPDD) is reported. A 41-year-old Japanese man presented with swelling, intermittent pain, and limitation of motion in the wrist. Radiological studies indicated amorphous calcification and bone erosion. MCPDD, although rare, should be included in the list of the differential diagnosis of periarticular calcific diseases.

Adult↗
PubMed1978

Accumulation of 99m Tc-Sn-pyrophosphate in pleural effusions.

Accumulation of 99m Tc-Sn-pyrophosphate in pleural effusions has been evaluated in 56 patients grouped as follows: 8 with bacterial effusion (Group A), 27 with malignant effusion treated by local and/or parenteral antitumor chemotherapy (Group B), 21 with malignant effusion treated only by supportive therapy (Group C). Results, expressed as effusion to plasma PPi ratio, ranged from 0.1 to 0.28 in group A, from 0.04 to 0.64 in group B and from 0.60 to 1.73 in group C, with significant differences among the three groups. In no case was uptake found in cells of the sediment. Chemical analysis (including total and ionized calcium, total protein, acid and alkaline phosphatase) of plasma and exudate in neoplastic patients showed a slight, but significant, difference between groups B and C as regards plasma-effusion gradient for total calcium and acid phosphatase. Negative correlation also exists between effusion to plasma PPi ratio and plasma-exudate gradient for ionized calcium in neoplastic patients. The data support the hypothesis that acid phosphatase content and calcium gradient are among the factors involved in the mechanism of PPi accumulation in pleural effusions.

Diphosphates↗
PubMed1989

The influence of hydroxyapatite and pyrophosphate on the formation product of calcium oxalate at different pHs.

The nucleating effect of hydroxyapatite (HAP) and the inhibitory effect of pyrophosphate (PPi) on calcium oxalate crystallization have been studied at different pH's in solution metastabely supersaturated with respect to calcium oxalate but saturated with respect to HAP. Crystallization was monitored by a decrease of calcium in the supernatant and formation products were calculated. At a pH above 6.0 already minimal HAP concentrations proved to be a suitable substrate for heterogeneous nucleation and growth of calcium oxalate. PPi showed a pronounced inhibitory effect on spontaneous as well as on HAP induced crystallization of calcium oxalate, this effect being highly pH dependent. HAP was found to neutralize the inhibitory effect of PPi in a molar ratio of 10:1.

Calcium Oxalate↗