Metabolites of the Free-Living Amoeba Phreatamoeba balamuthi Analyzed by 13C- and 31P-NMR Spectroscopy: Occurrence of Phosphoinositol Diphosphates

ABSTRACT. Phreatamoeba balamuthi is a free-living heterotrophic amoeba that lacks mitochondria. Metabolites of axenically-grown cells were characterized by natural-abundance ¹³C-NMR and ³¹P-NMR spectroscopy on acellular perchloric acid extracts. The amoebae were found to contain glycogen and trehalose as storage carbohydrates, together with putrescine and several amino acids, most prominently proline; we propose that proline and trehalose may serve in osmoregulation. Glycerophosphocholine and glycerophosphoethanolamine were present with their phosphomonoester derivatives, phosphocholine and phosphoethanolamine. Along with inorganic phosphate, inorganic pyrophosphate, nucleoside diphosphates, nucleoside triphosphates and NAD, P. balamuthi amoebae also contained unusual phosphoinositol diphosphates in large quantities (0.5 μmol/g wet cells).     

A 31P-nuclear-magnetic-resonance study of the phosphate groups in lipopolysaccharide and lipid A from Salmonella

Untreated and partially deacylated lipopolysaccharides from various P- and P+ strains of Salmonella were studied with 31P nuclear magnetic resonance spectroscopy and by conventional analytical methods. The spectral signals were assigned to various phosphate groups in the lipid A moiety and in the oligosaccharide part. A signal at +2.3 ppm could be assigned to a phosphodiester linkage formed between 4-amino-4-deoxyl-L-arabinose linked via the glycosidic hydroxyl group to the 4'-phosphate group of the glucosamine disaccharide in the lipid A moiety. A strong pyrophosphate signal at +11 ppm in P- strains was identified as a pyrophosphoryl ethanolamine group at the glycosidic end of this glucosamine disaccharide unit. No evidence was found for phosphodiester or pyrophosphodiester bonds crosslinking lipopolysaccharide 'subunits'. A revised version of the lipid A structure of Salmonella is presented. By a combination of 31P nuclear magnetic resonance spectroscopy data and conventional analytical methods the extent to which the lipopolysaccharides are substituted by various phosphate groups on the lipid A and the oligosaccharide moiety could be estimated. It was thus shown that substantial heterogeneity, leading to several molecular species of lipopolysaccharides is caused by addition or omission of certain groups. Since changes in substitution were found to be dependent on the growth conditions, it is thought possible that the overall negative surface charge of Salmonella can be modified by addition or omission of neutralising amino groups from ethanolamine and/or 4-amino-4-deoxy-L-arabinose, and can thus be adapted to the environment.     

Effects of 2-deoxyglucose on Saccharomyces cerevisiae as observed by in vivo 31P-NMR

Saccharomyces cerevisiae cells were treated with 2-deoxyglucose (1 mM) and the effects induced in the levels of phosphorus compounds and in the internal pH were monitored using 31P-NMR. Upon incubation with 2-deoxyglucose a strong decrease in the polyphosphate level was observed and the cytoplasmic pH decreased by about 0.4 units. This shows that 2-deoxyglucose strongly interferes with the cell conditions and consequently, the results of experiments in which 2-deoxyglucose was used to obtain deenergized cells should be carefully reanalysed.     

A 31P-NMR study of structural and functional aspects of phosphate and phosphonate distribution in Tetrahymena

³¹P-NMR has been used to study the chemical nature of cytoplasmic components of live Tetrahymena in a non-invasive manner. The technique has further been used to characterize the physical behaviour of lipids extracted from this organism. In particular, we have shown the presence of large quantities of pyrophosphate and of tripolyphosphate in acid extracts of the organism. These are not detectable in the live cell due to the motionally rigid nature of the storage granules. We have characterized the distribution of phosphonic acids in the organism and followed the phase behaviour of the extracted cell lipids. Aqueous dispersions of extracted lipid show both bilayer and non-bilayer behaviour in the range of the growth temperature. The phosphonolipid in Tetrahymena appears to play a role similar to that of phosphatidylethanolamine in regulating the phase behaviour of the membrane. The high degree of unsaturation in the fatty acids of Tetrahymena is most likely responsible for the polymorphic phase behaviour observed near the growth temperature.     

Rough mutants of Salmonella typhimurium: immunochemical and structural analysis of lipopolysaccharides from rfaH mutants

Lipopolysaccharides, extracted by phenol/chloroform/petroleum ether, from two rough mutants of Salmonella typhimurium of class rfaH were studied by passive haemagglutination inhibition and by methylation analysis. The structural and immunochemical analyses showed that (i) formation of the galactose I unit of the core is defective, but the defect is not complete, and (ii) of those core chains which do receive the galactose I residue, many are not continued to form complete core, but instead terminate at intermediate points. This suggests that the rfaH gene, though involved in formation of the galactose I unit, is not the structural gene for the galactosyltransferase which adds this unit. The rfaH product may be a positive regulator for several rfa genes specifying glycosyltransferases, or it may be a protein needed for the efficient action of several such transferases.     

Metabolite Changes in the Cerebral Cortex of Treated and Untreated Infant Hydrocephalic Rats Studied Using In Vitro 31P-NMR Spectroscopy

Abstract: The effect of hydrocephalus on cerebral energy metabolites and on intermediates of membrane phospholipid metabolism has been studied in H-Tx rats with inherited infantile hydrocephalus. Hydrocephalic rats and rats with shunts placed at 4–5 days or at 10 days after birth were subjected to magnetic resonance imaging in vivo before 21 days of age to determine the dimensions of the ventricles and cortex. At 21 days, the brains from the three groups of rats, together with age-matched control littermates, were frozen in situ, and chloroform/methanol extracts of cerebral cortex were prepared for high-resolution ³¹P-NMR spectroscopy. Hydrocephalus resulted in modest decreases in most metabolites quantified. Levels of phosphocreatine, ATP, and diphosphodiesters plus NAD were significantly reduced by 23–32%, and inorganic phosphate content was reduced but not significantly. Levels of the membrane phospholipid intermediates phosphorylethanolamine, glycerophosphorylethanolamine, and glycerophosphorylcholine were also significantly reduced by 30–33%, indicating changes in membrane metabolism. These general decreases are consistent with a loss of cell contents, possibly due to changes in dendrite structure in hydrocephalus. Rats shunt-treated at 4–5 days were similar to control rats for all energy metabolites, but those treated later at 10 days had reduced phosphocreatine and ATP levels. Shunt-treated rats also had reductions in levels of membrane phospholipids, some of which occurred in sham-operated rats. It is concluded that hydrocephalus leads to reductions in levels of energy metabolites and in levels of membrane phospholipids and that the changes in energy metabolites can be reversed by early, but not by later, shunt treatment.     

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.     

A 31P-NMR study of the intact liver fluke Fasciola hepatica

³¹P-NMR techniques offer a useful method of studying changes in the metabolism of intact parasitic worms. The liver flukes, Fasciola hepatica, provide good quality ³¹P high resolution NMR spectra for at least 6 h under anaerobic conditions. The levels of ATP remain constant throughout this period. There is no signal for phosphocreatine or phosphoarginine. In contrast to the findings in mammalian tissues, there is a distinct peak for the terminal phosphate of ADP. A number of signals are observed in the phosphodiester region of the spectrum the largest of which is identified as l-α-glycerophosphoryl choline. Serotonin (5-hydroxytryptamine) causes an appreciable increase in the levels of sugar phosphates when the flukes are incubated in the absence of glucose. The addition of glucose also causes a marked increase in the signals for the hexose phosphate.     

The synthesis and use of thionphospholipids in 31P-NMR studies of lipid polymorphism

(1) Dipalmitoyl- and dioleoylthionphosphatidylcholine, which are phosphatidylcholine analogues in which the double bonded oxygen of the phosphate group is replaced by a sulfur atom, have been synthesized in 50–60% yields by condensation of diacylglycerol with phosphorus thionchloride in the presence of choline toluene-sulfonate. Dioleoylthionphosphatidylethanolamine has been prepared by the phospholipase D-catalyzed base exchange reaction. (2) Freeze-fracturing of aqueous dispersions of the thionphospholipids reveals that the thionphosphatidylcholines are organized in extended bilayers whereas dioleoylthionphosphatidylethanolamine above 0°C forms the hexagonal H_II phase similar to dioleoylphosphatidylethanolamine. The gel → liquid crystalline phase transition of the dipalmitoylthionphosphatidylcholine occurs at 44°C which is only slightly higher than the transition temperature of dipalmitoylphosphatidylcholine which together with other data demonstrates that the thionphospholipids closely resemble the natural phospholipids in physicochemical behaviour. (3) Proton decoupled ³¹P-NMR spectra of aqueous dispersions of thionphosphatidylcholines have the characteristic asymmetrical line-shape with a low-field shoulder and a high-field peak typical of phospholipids organized in extended bilayers in which the phosphate group can undergo fast axial rotation. The ³¹P-NMR spectrum of the thionphosphatidylethanolamine in the hexagonal H_II phase has a line-shape with a reversed asymmetry and an effective chemical shift anisotropy half of that of thionphospholipids organized in bilayers which is caused by fast lateral diffusion of the lipids around the cylinders of the hexagonal H_II phase as has been observed for the corresponding phosphatidylethanolamines. (4) Since the ³¹P-NMR resonance of the thionphospholipids is completely separated from that of natural phospholipids, these lipids can be used to study by ³¹P-NMR the motional and structural properties of individual lipids in mixed systems. This is demonstrated for various lipid mixtures in which non-bilayer lipid structures have been induced by variations in composition, temperature and presence of divalent cations. It is shown that bilayer → non-bilayer transitions can be modulated by gel → liquid crystalline phase transitions and that typical bilayer forming lipids can be incorporated into non-bilayer structures such as the hexagonal H_II phase.     

Tolerance of Low Intracellular pH During Hypercapnia by Rat Cortical Brain Slices: A 31P/1H NMR Study

Metabolic tolerance of low intracellular pH (pH(i)) was studied in well-oxygenated, perfused, neonatal, rat cerebrocortical brain slices (350 microns thick) by inducing severe hypercapnia. In each of 17 separate experiments 80 brain slices (approximately 3.2 g wet weight) were suspended in an NMR tube, perfused with artificial CSF (ACSF), and studied at 4.7 T with 31P and 1H NMR spectroscopy. Spectra obtained every 5 min monitored relative concentrations of lactate or high-energy phosphate metabolites, from which pH(i) and extracellular pH were determined. Unperturbed slice preparations were metabolically stable for > 10 h, with no significant changes occurring in pHi, ATP, phosphocreatine (PCr), inorganic phosphate, or lactate. Different levels of hypercapnia were produced by sequentially perfusing slices with the following different ACSF batches, each having previously been equilibrated with a specific mixture of CO2 in oxygen: (a) 10% CO2, 15 min of perfusion; (b) 30% CO2, 15 min of perfusion; (c) 50% CO2, 15 min of perfusion; (d) 70% CO2, 30 min of perfusion; (e) 50% CO2, 15 min of perfusion; (f) 30% CO2, 15 min of perfusion; and (g) 10% CO2, 15 min of perfusion. At the completion of this protocol slices were again perfused with fresh ACSF that was equilibrated with a 95% O2/5% CO2 gas mixture. In each of five separate 1H and 31P experiments, brain slices were recovered within 2 h after termination of exposure to high CO2. The pHi was determined from measurements of the chemical shift difference between phosphoethanolamine and PCr, using a calibration curve obtained for our preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-component regulatory system with a possible role in virulence.

We isolated spontaneous mutations (pmrA) in the smooth strain Salmonella typhimurium LT2 that show increased resistance to the cationic antibacterial proteins of human neutrophils and to the drug polymyxin B. The mutation in one strain, JKS5, maps to 93 min on the S. typhimurium chromosome, near the proP gene and the melAB operon. The mutation, designated pmrA505, confers a 1,000-fold increase in resistance to polymyxin B and a 2- to 4-fold increase in resistance to neutrophil proteins. We cloned both the pmrA505 and pmrA+ alleles and found that the pmrA+ gene is partially dominant over pmrA505. DNA sequence analysis of the pmrA505 clone revealed three open reading frames (ORFs). The deduced amino acid sequences indicated that ORF1 encodes a 548-amino-acid (aa) protein with a putative membrane-spanning domain and no significant homology to any known protein. ORF2 and ORF3, which encode 222- and 356-aa proteins, respectively, show strong homology with the OmpR-EnvZ family of two-component regulatory systems. ORF2 showed homology with a number of response regulators, including OmpR and PhoP, while ORF3 showed homology to histidine kinase-sensor proteins EnvZ and PhoR. Genetic analysis of the cloned genes suggested that ORF2 contained the pmrA505 mutation. Comparison of the pmrA505 and pmrA+ ORF2 DNA sequences revealed a single G-A transition, which would result in a His-to-Arg substitution at position 81 in the ORF2 mutant protein. We therefore designate ORF2 PmrA and ORF3 PmrB. The function of ORF1 is unknown.     

Complete structure of lipid A obtained from the lipopolysaccharides of the heptoseless mutant of Salmonella typhimurium

A highly purified monophosphoryl lipid A, TLC-3 fraction obtained from the lipopolysaccharides of the heptoseless mutant Salmonella typhimurium G30/C21 was converted to the dimethyl pentatrimethylsilyl derivative and analyzed by proton NMR spectroscopy at 400 MHz. Substantial downfield shifts of the resonances for protons at the 3- and 3'-carbons of the glucosamine disaccharide to 5.06 and 5.15 ppm, respectively, occurred from the normal range of 3.5-4.1 ppm, indicating that these two positions on the sugar rings were acylated. Significant downfield shift of the resonances for protons at the 4- and 6'-carbons did not occur, indicating the absence of acyl groups at these two positions. Since positive ion fast atom bombardment mass spectrometry previously established the presence of hydroxymyristoyl and myristoxymyristoyl esters at the reducing end and distal subunits, respectively, these acyl groups must be attached to the oxygen of the corresponding 3- and 3'-carbons of lipid A. With these results, we can now describe the complete structure of the monophosphoryl lipid A, TLC-3 from S. typhimurium.     

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

Immobilization, stabilization and remobilization of nitrogen in forest soils at elevated CO2: a 15N and 13C tracer study

The fate of immobilized N in soils is one of the great uncertainties in predicting C sequestration at increased CO₂ and N deposition. In a dual isotope tracer experiment (¹³C, ¹⁵N) within a 4‐year CO₂ enrichment (+200 ppm_v) study with forest model ecosystems, we (i) quantified the effects of elevated CO₂ on the partitioning of N; (ii) traced immobilized N into physically separated pools of soil organic matter (SOM) with turnover rates known from their ¹³C signals; and (iii) estimated the remobilization and thus, the bio‐availability of newly sequestered C and N. (1) CO₂ enrichment significantly decreased NO₃^? concentrations in soil waters and export from 1.5 m deep lysimeters by 30–80%. Consequently, elevated CO₂ increased the overall retention of N in the model ecosystems. (2) About 60–80% of added ¹⁵NH₄¹⁵NO₃ were retained in soils. The clay fraction was the greatest sink for the immobilized ¹⁵N sequestering 50–60% of the total new soil N. SOM associated with clay contained only 25% of the total new soil C pool and had small C/N ratios (<13), indicating that it consists of humified organic matter with a relatively slow turn over rate. This implies that added ¹⁵N was mainly immobilized in stable mineral‐bound SOM pools. (3) Incubation of soils for 1 year showed that the remobilization of newly sequestered N was three to nine times smaller than that of newly sequestered C. Thus, inorganic inputs of N were stabilized more effectively in soils than C. Significantly less newly sequestered N was remobilized from soils previously exposed to elevated CO₂. In summary, our results show firstly that a large fraction of inorganic N inputs becomes effectively immobilized in relative stable SOM pools and secondly that elevated CO₂ can increase N retention in soils and hence it may tighten N cycling and diminish the risk of nitrate leaching to groundwater.     

Effects of N-Methyl-d-Aspartate on [Ca2+]i and the Energy State in the Brain by 19F- and 31P-Nuclear Magnetic Resonance Spectroscopy

The effects of N-methyl-D-aspartate (NMDA) on the free intracellular Ca2+ concentration [( Ca2+]i) and the energy state in superfused cerebral cortical slices have been studied using 19F- and 31P-nuclear magnetic resonance spectroscopy. [Ca2+]i was measured using the calcium indicator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA). NMDA (10 microM) in the absence of extracellular Mg2+ caused the expected rise in [Ca2+]i but produced an impairment of the energy state: the phosphocreatine (PCr) content was decreased by 42%, and the Pi/PCr ratio was increased by 55%. There was no detectable change in ATP or free intracellular Mg2+ concentration. Increasing the NMDA concentration in the superfusing medium to 100 or 400 microM caused no further increase in [Ca2+]i or further decrease in PCr content, but the Pi/PCr ratio continued to rise. The impairment of the energy state preceded the effect on [Ca2+]i, and these changes were irreversible on return to control conditions. Repeating the experiments in the presence of 1.2 mM extracellular Mg2+ resulted in similar changes in the energy state, with no change in [Ca2+]i. The possibilities that the effects were due to membrane depolarisation or to the presence of 5FBAPTA within the tissues were eliminated. The results suggest that low concentrations (10 microM) of NMDA produce an impaired energy state independent of the presence of extracellular Mg2+ and that the decreased energy state is not due to the changes in [Ca2+]i, which are seen only in the absence of extracellular Mg2+.     

Increased Binding of [3H]Muscimol and [3H]Flunitrazepam in the Rat Brain Under Hypoxia

We examined the effects of in vivo hypoxia (10% O2/90% N2) on the gamma-aminobutyric acid (GABA)/benzodiazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6-h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X-100 eliminated the increase in the binding capacity. These results may represent an up-regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.