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Low levels of pyrophosphate in transgenic potato plants expressing E. coli pyrophosphatase lead to decreased vitality under oxygen deficiency.

BACKGROUND AND AIMS: The aim of this study was to investigate the importance of pyrophosphate (PPi) for plant metabolism and survival under low oxygen stress. Responses of roots of wild-type potato plants were compared with roots of transgenic plants containing decreased amounts of PPi as a result of the constitutive expression of Escherichia coli pyrophosphatase in the cytosol. METHODS: For the experiments, roots of young wild-type and transgenic potato plants growing in nutrient solution were flushed for 4 d with nitrogen, and subsequently metabolite contents as well as enzyme activities of the glycolytic pathway were determined. KEY RESULTS AND CONCLUSIONS: In roots of transgenic plants containing 40% less PPi, UDPglucose accumulated while the concentrations of hexose-6-phosphate, other glycolytic intermediates and ATP were decreased, leading to a growth retardation in aerated conditions. Apart from metabolic alterations, the activity of sucrose synthase was increased to a lower extent in the transgenic line than in wild type during hypoxia. These data suggest that sucrose cleavage was inhibited due to PPi deficiency already under aerated conditions, which has severe consequences for plant vitality under low oxygen. This is indicated by a reduction in the glycolytic activity, lower ATP levels and an impaired ability to resume growth after 4 d of hypoxia. Interestingly, the phosphorylation of fructose-6-phosphate via PPi-dependent phosphofructokinase was not altered in roots of transgenic plants. Nevertheless, our data provide some evidence for the importance of PPi to maintain plant growth and metabolism under oxygen deprivation.

Blotting, Northern↗

Infarct sizing with technetium-99m stannous pyrophosphate scintigraphy in dogs and man; relationship between scintigraphic and praecordial mapping estimates of infarct size in patients.

The present study was performed in order to evaluate the ability of technetium-99m stannous pyrophosphate (99mTc-PYP) myocardial scintigrams to size infarcts in experimental animals and man. In 10 dogs with proximal left anterior descending coronary artery occlusion and acute anterior myocardial infarcts, there was a significant correlation between scintigraphic infarct size and histological infarct weight (P less than 0.01). In 25 patients with acute anterior or anterolateral myocardial infarcts, there was a significant correlation between relatively large infarct size determined scintigraphically and the acute development of left ventricular failure. There was some overlap, however, in 99mTc-PYP scintigraphic infarct size between patients who did and did not develop left ventricular failure with infarction. Presumably this is explained by some patients having had earlier myocardial damage and thus developing left ventricular failure with relatively small new infarcts. There was also a statistically significant, but weak, correlation in patients between scintigraphic infarct size and precordial ST segment mapping including peak ST segment elevation (P less than 0.05) and the number of praecordial sites with ST segment elevation equal to or greater than 2 mm (P less than 0.01). The data suggest that 99mTc-PYP scintigrams and praecordial mapping measure some similar but some dissimilar aspects of infarct size in patients, and that 99mTc-PYP scintigraphy does size acute anterior and anterolateral infarcts in experimental animals and patients.

Animals↗

Accumulation of technetium-99m pyrophosphate in experimental infarctions in the rat.

Cardiac accumulation patterns of pyrophosphate labelled with techetium-99m (TcPyP) in rats one hour to 7 days after coronary artery ligation were studied by light microscopy, myocardial scintigraphy, imaging the isolated heart and by direct measurement of tissue activity. Results suggest that myocardial cells taking up TcPyP are irreversibly damaged and that the disappearance of TcPyP uptake coincides with the removal of necrotic cells by phagocytes.

Animals↗

Particle size reduction and encapsulation affect the bioavailability of ferric pyrophosphate in rats.

Particle size is an important determinant of Fe absorption from poorly soluble Fe compounds in foods. Decreasing the particle size of elemental iron powders increases their absorption. The effect of a reduction in particle size on the bioavailability of ferric pyrophosphate (FePP) is unclear. Encapsulation of iron compounds for food fortification may protect against adverse sensory changes, but at the same time may reduce bioavailability. The hemoglobin (Hb) repletion method in weanling Sprague-Dawley rats (n = 100) was used to compare the relative bioavailability (RBV) of 4 forms of FePP: 1) regular FePP [mean particle size (MPS) approximately 21 microm]; 2) MPS approximately 2.5 microm; 3) MPS approximately 2.5 microm encapsulated in hydrogenated palm oil; and 4) MPS approximately 0.5 microm with emulsifiers. The RBV compared with ferrous sulfate was calculated by the slope-ratio technique. The RBV was 43% for encapsulated MPS approximately 2.5 microm, significantly lower than the other FePP compounds (P < 0.05), 59% for the regular FePP, and 69% for MPS approximately 2.5 microm, not different from each other but significantly lower than ferrous sulfate (P < 0.05), and 95% for emulsified MPS approximately 0.5 microm, comparable to ferrous sulfate. Encapsulation of FePP with hydrogenated palm oil at a capsule:substrate ratio of 60:40 decreased RBV. Particle size reduction increases the RBV of FePP and may make this compound more useful for food fortification.

Animals↗

Salt dual-fortified with iodine and micronized ground ferric pyrophosphate affects iron status but not hemoglobin in children in Cote d'Ivoire.

Deficiencies of iron and iodine are common in West Africa, and salt is one of very few food vehicles available for fortification. Salt dual-fortified with iodine and micronized ground ferric pyrophosphate (FePP) was tested for its efficacy in rural, tropical Côte d'Ivoire. First, salt and iron intakes, and iron bioavailability were estimated using 3-d weighed food records in 24 households. Local iodized salt was then fortified with 3 mg Fe/g salt as ground FePP (mean particle size = 2.5 mum), and stability, sensory and acceptability trials were done. The dual fortified salt (DFS) was distributed to households and its efficacy compared with that of iodized salt (IS) in a 6-mo, double-blind trial in 5- to 15-y-old iron-deficient children (n = 123). All children were dewormed at baseline. After 6 mo, serum ferritin (SF) and transferrin receptor (TfR) concentrations as well as body iron stores improved significantly in the DFS group but not in the IS GROUP (P < 0.05). Body iron increased from 4.6 +/- 2.7 to 5.9 +/- 2.7 mg/kg (mean +/- SD) in the DFS group; concentrations before and after treatment in the IS group were 5.5 +/- 2.9 and 5.6 +/- 3.1 mg/kg, respectively. The hemoglobin concentration and the prevalence of anemia did not change in either group. The prevalences of malaria, soil-transmitted helminths, and riboflavin deficiency were 55, 14, and 66%, respectively. In tropical West Africa, low-grade salt fortified with micronized ground FePP increased body iron stores but not hemoglobin in children. Iron utilization may have been impaired by the high prevalence of malaria and concurrent nutrient deficiencies.

Adolescent↗

Influences of amino acid, ATP, pyrophosphate and tRNA on binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

Aminoalcohol-AMP esters, structurally related to the assumed intermediates of the amino acid activation reaction, behave as competitive inhibitors both with respect to the amino acid and ATP, when tested in the ATP-(32P) PPi-exchange or the tRNA-charging reaction. However, closer investigation of the binding of norvalinyl adenylate to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 by an equilibrium method shows that only the amino acid is a true competitor, while ATP cannot displace the ester from binding. Pyrophosphate enhances the stability of the ester-enzyme complex whereas tRNA is without detectable influence.

Adenosine Monophosphate↗

Lower mevalonate pyrophosphate decarboxylase activity is caused by the reduced amount of enzyme in stroke-prone spontaneously hypertensive rat.

Spontaneously hypertensive rat (stroke-prone) (SHRSP) has a low serum cholesterol level as compared with the normotensive Wistar Kyoto rat (WKY). We previously indicated that the lower activity of mevalonate pyrophosphate decarboxylase (MPD) was responsible for the reduced cholesterol biosynthesis in the liver of SHRSP [Sawamura et al. (1992) J. Biol. Chem. 267, 6051-6055]. To elucidate the mechanism of the reduced activity, we purified liver MPD from SHRSP treated with cholestyramine and pravastatin in this study. We compared its enzymatic properties with those of the enzyme from WKY, and also measured the amounts of MPD in the crude extract of various tissues in WKY and SHRSP by Western blot analysis. Results indicated that (i) MPD of SHRSP has essentially the same properties as MPD of WKY, except for a difference in the dependency on divalent cations. (ii) The amount, as well as the activity, of MPD in the crude extract of brain and liver was reduced in SHRSP. (iii) There was no difference between SHRSP and WKY, in the ratio of the enzyme activity to the amount of MPD in the crude extract. These data led us to conclude that the lower activity of MPD was caused by the reduced amount of this enzyme in SHRSP.

Animals↗

The mobility of gizzard myosin in pyrophosphate gel electrophoresis is increased by its light chain phosphorylation.

Phosphorylation of the 20,000 Mr light chain (L20) of gizzard myosin reversibly increased the mobility of myosin in pyrophosphate polyacrylamide gel electrophoresis (PP1 PAGE). Gizzard heavy meromyosin (HMM) with phosphorylated L20 also moved faster than that with unphosphorylated L20. This mobility increase of HMM is large enough to account for that of intact myosin. Scallop myosin, desensitized by removing its regulatory light chain, was combined with L20 and subjected to PPi PAGE. Hybrid myosin with the phosphorylated light chain moved faster than that with the unphosphorylated light chain. No such effect of light chain phosphorylation was observed with phosphorylatable light chain from breast or ventricular myosin. Thus, gizzard, but not breast or ventricular phosphorylatable light chain is furnished with the 'regulatory' property that is phosphorylation increases myosin mobility in PPi PAGE.

Animals↗

Native pyrophosphate gel analysis of smooth muscle myosin phosphorylated with protein kinase C.

We have shown that the phosphorylation of smooth muscle regulatory myosin light chain (L20) with myosin light chain kinase (MLCK) produces faster moving bands (GMP1: heterodimer myosin with 1 unphosphorylated L20 and 1 mono-phosphorylated L20, GMP2: homodimer myosin with 2 mono-phosphorylated L20S) on native pyrophosphate polyacrylamide gel electrophoresis (PP1 PAGE) (J. Biochem. 100, 259-268, 1986; J. Biochem. 100, 1681-1684, 1986). However, the mobility of the myosin phosphorylated, at its L20, with protein kinase C (PK-C) was the same that of the unphosphorylated myosin (GM) on PPi PAGE. When the myosin prephosphorylated with MLCK was further phosphorylated with PK-C, PPi PAGE analysis showed only one band comigrating with GM, i.e., GMP1 and GMP2 migrated to the same position as GM. Conversely, when the myosin prephosphorylated with PK-C was further phosphorylated with MLCK, GMP1 and GMP2 were not produced. Thus the effect of L20 phosphorylated with PK-C is quite the opposite of that with MLCK, and the former predominated over the latter. We speculate that phosphorylation of L20 with PK-C "freezes" myosin in the inactive state.

Animals↗

Rat liver phosphoribosyl pyrophosphate synthetase: existence of the purified enzyme as heterogeneous aggregates and identification of the catalytic subunit.

Mammalian phosphoribosyl pyrophosphate (PRPP) synthetase has been extensively investigated. However, considerable ambiguity remains concerning its physical and regulatory properties. We purified PRPP synthetase from rat liver and studied some of the physical properties, in parallel with cloning experiments (Taira, M. et. al. [1987] J. Biol. Chem. 262, 14867-14870). 1) The enzyme was purified to a specific activity of 7,280 milliunits/mg, the highest value in the literature for a mammalian PRPP synthetase. The apparent molecular mass was over 1,000 kDa. 2) The final preparation contained 34-kDa, 38-kDa, and 40-kDa protein species, as analyzed by SDS gel electrophoresis. 3) Further attempts at separation using conventional procedures only led to a co-purification of the components. Thus, the purified enzyme appears to exist as complex aggregates composed of heterogeneous components. 4) Gel filtration of the enzyme in the presence of 1 M MgCl2 isolated part of the 34-kDa component, free of other species. The preparation was catalytically active, indicating that this component is the catalytic subunit. 5) The amino acid composition of the 34-kDa subunit and the amino acid sequences of its N-terminal region and of two tryptic peptides were determined. The results are in accord with the results of cDNA analyses.

Amino Acids↗

Acceptor specificity of ATP:nucleoside-5'-phosphate pyrophosphotransferase from Streptomyces adephospholyticus. Synthesis of the 3'-pyrophosphates of pyrimidine nucleotides, some oligoribonucleotides, 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am.

ATP: nucleoside-5'-phosphate pyrophosphotransferase [EC 2.7.6.4] of Streptomyces adephospholyticus synthesizes not only 3'-pyrophosphates of 5'-purine ribomononucleotides but also those of pyrimidine mononucleotides, some short oligonucleotides, a variety of 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am.

NAD↗

Farnesyl pyrophosphate synthetase located in microsomes from pig liver.

The microsomes from pig liver contained farnesyl pyrophosphate synthetase and it was solubilized with Triton X-100. The microsomal enzyme had a pH optimum of 6.5-7.0 and required Mg2+ or Mn2+ for maximum activity. Dimethylallyl-transferring activity of the enzyme was much lower compared with the geranyl-transferring activity. In the presence of Triton X-100, the geranyl-transferring activity was about two-fold activated whereas the dimethylallyl-transferring activity was almost the same.

Animals↗

Cloning and functional expression of a novel geranylgeranyl pyrophosphate synthase gene from Arabidopsis thaliana in Escherichia coli.

A gene encoding a novel geranylgeranyl pyrophosphate (GGPP) synthase from Arabidopsis thaliana has been identified and termed GGPS5. The gene has been sequenced and expressed in Escherichia coli. The deduced amino acid sequence showed 64.5% and 57.5% identify with a putative GGPP synthase from Arabidopsis and Capsicum annuum, respectively. GGPP enzymatic activity was detected in E. coli cells expressing the GGPS5 gene in two different ways. One was the direct measurement of GGPP synthase activity in cell extracts and the other was the yellow color production of cells when the GGPS5 gene was co-expressed with crtB, crtI, crtY and crtZ genes derived from Erwinia uredovora.

Alkyl and Aryl Transferases↗

Testis-specific transcripts of rat farnesyl pyrophosphate synthetase are developmentally regulated and localized to haploid germ cells.

A rat farnesyl pyrophosphate (FPP) synthetase cDNA was used to study the expression of FPP synthetase mRNA levels during spermatogenesis in the rat. RNA blot analysis showed an increase in the level of FPP synthetase transcripts during postnatal development of rat testes. The increase is due to the appearance of longer testis-specific FPP synthetase transcripts as assayed by primer extension mapping. In situ hybridization analysis of adult rat testis sections with an FPP synthetase antisense probe showed that FPP synthetase mRNA levels are greatly enriched in the haploid round spermatidis at stages 7 to 8 of the seminiferous epithelium. These results show that FPP synthetase transcripts in testis are expressed at high levels in haploid male germ cells in a stage-specific manner.

Animals↗

Detection of heterotopic calcification with 99mTc-pyrophosphate in spinal cord injury patients.

Whole body 99mTc-pyrophosphate scans were obtained and correlated with skeletal radiographs for detection of heterotopic calcification in 122 spinal injury patients. There were 27 patients with recent injury (less than 6 months) and 95 with injury of 6 months to 25 years duration. Ectopic calcification was detected in 8 of the 27 patients (30%) with recent injury and in 32 of the 95 (34%) with injury of over 6 months duration. Ectopic calcification seen on radiographs were also seen on scans in all instances. The scan was a sensitive indicator of the ectopic calcification particularly at its early stage, and detected its presence before observable radiographic changes in 5 patients with injury of 1-3 months duration. In 3 of the 5 patients, the ectopic calcification mimicked deep vein thrombosis in presentation, and the scan was valuable in identifying the nature of the complication.

Adult↗

Unilateral increased radioactivity in the lower extremities on routine 99mTc-pyrophosphate bone imaging.

Unilateral increased activity in the lower extremities was noted in 5 patients who underwent routine 99mTc-pyrophosphate bone imaging for metastatic evaluation. In three patients a large pelvic mass or retroperitoneal hematoma was noted on the affected side. Clinical evidence of lymphatic obstruction was apparent in two cases with pelvic mass. In the fourth case, venous disease (thrombophlebitis) was noted and the fifth case showed osteomyelitis. The possible mechanism for this phenomenon may include obstruction of the lymphatic or venous circulation and inflammation. Other causes of soft tissue uptake of 99mTc-PYP are reviewed. The above mentioned conditions must be excluded when asymmetrical activity of 99mTc-PYP is seen on routine bone imaging.

Adult↗

Sensitivity and specificity of Tc-99m-pyrophosphate myocardial scintigraphy for the detection of acute myocardial infarction.

Tc-99m-pyrophosphate myocardial scintigraphy was performed on 1,077 patients admitted to the Coronary Care Unit. Results of scintigraphy were compared to the diagnosis as established by ECG, enzymes, and clinical findings to determine the sensitivity and specificity of scintigraphy for the diagnosis of acute myocardial infarction (AMI). Scintigrams were graded according to the intensity of myocardial radioactivity and the distribution pattern of activity as either diffuse or localized. In a coronary care unit, a scintigram of positive intensity grade is 92% sensitive and 68% specific for the diagnosis for AMI. A localized pattern of myocardial radioactivity is 66% sensitive and 93% specific for AMI. With a localized pattern of the highest intensity, the sensitivity is 28% with a specificity of 99.8% for AMI. Therefore, a myocardial scintigram of normal intensity grade excludes the diagnosis of AMI with a 92% probability. A localized pattern is a strong indicator of an AMI and a localized pattern of the greatest intensity is very specific for AMI.

Acute Disease↗

Acute subendocardial myocardial infarction: its detection by Tc-99m stannous pyrophosphate myocardial scintigraphy.

Sixty-two patients hospitalized because of prolonged chest pain and initial electrocardiographic (ECG) changes of ST depression and T-wave inversion suggestive of acute subendocardial myocardial infarction were evaluated to determine the ability of Tc-99m-stannous pyrophosphate mycardial scintigraphy to detect the presence or absence of acute subendocardial myocardial necrosis. Three groups of patients were designated. Group A consisted of eight patients (13%) who developed reduction of R-waves of more than 25% or new Q-waves broader than 0.03 seconds; of these patients with acute transmural myocardial infarction, all had well-localized, abnormal scintigrams. Group B consisted of 30 patients with ECG changes and subsequent enzymatic documentation, including elevated serum creatine kinase-B levels as determined by radioimmunoassay, of the presence of acute subendocardial myocardial infarction. Of these, 27 had abnormal scintigrams, including 18 with well-localized patterns and nine with "poorly localized" patterns. Group C consisted of 24 patients (39%) with chest pain, but without enzymatic documentation of the presence of acute myocardial infarction (acute coronary insufficiency). Eight of these had abnormal scintigrams, including one with a well-localized pattern and seven with "poorly localized" patterns. In four of the latter, the scintigrams were "persistently positive" several weeks to months after a previous myocardial infarct. Serial myocardial imaging will be necessary to identify such patients.

Adult↗