Nucleotide Triphosphate Concentrations Research Articles

Prodrugs of imidazotriazine and pyrrolotriazine C-nucleosides can increase anti-HCV activity and enhance nucleotide triphosphate concentrations in vitro

A number of prodrugs of HCV-active purine nucleoside analogues 2′-C-methyl 4-aza-9-deaza adenosine 1, 2′-C-methyl 4-aza-7,9-dideaza adenosine 2, 2′-C-methyl 4-aza-9-deaza guanosine 3 and 2′-C-methyl 4-aza-7,9-dideaza guanosine 4 were prepared and evaluated to improve potency, selectivity and liver targeting. Phosphoramidate guanosine prodrugs (3a–3k and 4a, b) showed insufficient cell activity for further profiling. Striking enhancement in replicon activity relative to the parent was observed for phosphoramidate imidazo[2,1-f][1,2,4]triazine-4-amine adenosine prodrugs (1a–1p), but this was accompanied by an increase in cytotoxicity. Improved or similar potency without a concomitant increase in toxicity relative to the parent was demonstrated for phosphoramidate pyrrolo[2,1-f][1,2,4]triazine-4-amine adenosine prodrugs (2a–2k). Carbamate, ester and mixed prodrugs of 2 showed mixed results. Selected prodrugs of 2 were analysed for activation to the triphosphate, with most demonstrating much better activation in hepatocytes over replicon cells. The best activation was observed for a mixed phosphoramidate-3′ester (11) followed by a simple 3′-ester (10).

Insights into the Regulatory Landscape of the Lysine Riboswitch

A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors, these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main-chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, we have determined the relative contributions of the polar functional groups to binding affinity and the regulatory response. Our results reveal that the lysine riboswitch has >1000-fold specificity for lysine over other amino acids. The aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main-chain functional groups in order to achieve this specificity. At low nucleotide triphosphate (NTP) concentrations, we observe good agreement between the half-maximal regulatory activity (T(50)) and the affinity of the receptor for lysine (K(d)), as well as many of its analogs. However, above 400 μM [NTP], the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that, under physiologically relevant conditions, riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell.

Early response of plant cell to carbon deprivation: in vivo31P‐NMR spectroscopy shows a quasi‐instantaneous disruption on cytosolic sugars, phosphorylated intermediates of energy metabolism, phosphate partitioning, and intracellular pHs

• In plant cells, sugar starvation triggers a cascade of effects at the scale of 1-2 days. However, very early metabolic response has not yet been investigated. • Soluble phosphorus (P) compounds and intracellular pHs were analysed each 2.5 min intervals in heterotrophic sycamore (Acer pseudoplatanus) cells using in vivo phosphorus nuclear magnetic resonance ((31)P-NMR). • Upon external-sugar withdrawal, the glucose 6-P concentration dropped in the cytosol, but not in plastids. The released inorganic phosphate (Pi) accumulated transiently in the cytosol before influx into the vacuole; nucleotide triphosphate concentration doubled, intracellular pH increased and cell respiration decreased. It was deduced that the cytosolic free-sugar concentration was low, corresponding to only 0.5 mM sucrose in sugar-supplied cells. • The release of sugar from the vacuole and from plastids is insufficient to fully sustain the cell metabolism during starvation, particularly in the very short term. Similarly to Pi-starvation, the cell's first response to sugar starvation occurs in the cytosol and is of a metabolic nature. Unlike the cytoplasm, cytosolic homeostasis is not maintained during starvation. The important metabolic changes following cytosolic sugar exhaustion deliver early endogenous signals that may contribute to trigger rescue metabolism.

Changes in Cerebral Oxygen Consumption and High-Energy Phosphates During Early Recovery in Hypoxic-Ischemic Piglets: A Combined Near-Infrared and Magnetic Resonance Spectroscopy Study

Near-infrared spectroscopy (NIRS) offers the ability to assess brain function at the bedside of critically ill neonates. Our group previously demonstrated a persistent reduction in the cerebral metabolic rate of oxygen (CMRO(2)) after hypoxia-ischemia (HI) in newborn piglets. The purpose of this current study was to determine the causes of this reduction by combining NIRS with magnetic resonance spectroscopy (MRS) to measure high-energy metabolites and diffusion-weighted imaging to measure cellular edema. Nine piglets were exposed to 30 min of HI and nine piglets served as controls. Proton and phosphorous MRS spectra, apparent diffusion coefficient (ADC) maps, and CMRO(2) measurements were collected periodically before and for 5.5 h after HI. A significant decrease in CMRO(2) (26 +/- 7%) was observed after HI. Incomplete recovery of nucleotide triphosphate concentration (8 +/- 3% <controls) and reduced ADC (16 +/- 5%) suggested mitochondrial dysfunction. However, CMRO(2) did not correlate with any metabolite concentration during the last 3 h of the recovery period, and no significant changes were found in phosphocreatine and lactate levels. Therefore, the CMRO(2) decrease is likely a combination of impaired mitochondrial function and reduced energy demands during the acute phase, which has been previously observed in the mature brain.

Multiple mechanisms are used for growth rate and stringent control of leuV transcriptional initiation in Escherichia coli.

Expression of the Escherichia coli leuV operon, which contains three tRNA(1)(Leu) genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the leuV promoter occurs in the absence of guanosine 3', 5'-bisdiphosphate. To determine the mechanisms of the leuV promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of the leuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to heparin challenge than those of rrnBp(1). The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuV promoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in leuV promoter activity was seen. It therefore appears that transcription initiation from the leuV promoter is less sensitive to changes in intracellular NTP concentration than that from rrnBp(1). Comparative analysis of regulation of the leuV promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of leuV promoter mutations, which abolished GRDC of the leuV core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type leuV promoters containing UAS showed improved GRDC in a fis mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the leuV promoter.

Amino Acid Substitutions in the Two Largest Subunits of Escherichia coli RNA Polymerase That Suppress a Defective Rho Termination Factor Affect Different Parts of the Transcription Complex

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.

Delayed (“Secondary”) Cerebral Energy Failure after Acute Hypoxia-Ischemia in the Newborn Piglet: Continuous 48-Hour Studies by Phosphorus Magnetic Resonance Spectroscopy

Phosphorous (31P) spectra from the brains of severely birth-asphyxiated human infants are commonly normal on the first day of life. Later, cerebral energy failure develops, which carries a serious prognosis. The main purpose of this study was to test the hypothesis that this delayed ("secondary") energy failure could be reproduced in the newborn piglet after a severe acute reversed cerebral hypoxic-ischemic insult. Twelve piglets were subjected to temporary occlusion of the common carotid arteries and hypoxemia [mean arterial PO2 3.1 (SD 0.6) kPa]. Mean cerebral phosphocreatine concentration [PCr]/inorganic orthophosphate concentration [Pi] decreased from 1.40 (SD 0.29) to 0.01 (SD 0.02), and nucleotide triphosphate concentration [NTP]/exchangeable phosphate pool concentration [EPP] decreased from 0.19 (SD 0.02) to 0.06 (SD 0.04) (p < 0.001 for each decrease). On reperfusion and reoxygenation of the brain, mean [PCr]/[Pi] and [NTP]/[EPP] returned to baseline. Observations continuing for the next 48 h showed that [PCr]/[Pi] again decreased, in spite of normal arterial PO2, mean arterial blood pressure, and blood glucose, to 0.62 (SD 0.61) at 24 h (p < 0.01) and 0.49 (SD 0.37) at 48 h (p < 0.001). [NTP]/[EPP] also decreased, but to a lesser degree. Intracellular pH remained unchanged. These findings appeared identical with those seen in birth-asphyxiated human infants. No changes in cerebral metabolite concentrations took place in six control piglets. The severity of secondary energy failure, as judged by the lowest [PCr]/[Pi] recorded at 24-48 h, was directly related to the extent of acute energy depletion, obtained as the time integral of reduction in [NTP]/[EPP] (p < 0.0001). This animal model of secondary energy failure may prove useful for testing cerebroprotective strategies.

Effect of Blood Transfusion on Immune Function: IX. Effect on Lymphocyte Metabolism

Blood transfusions have been repeatedly shown to be immunosuppressive in nature. The intracellular mechanisms of this immunosuppression have not been extensively investigated. We investigated the effect of blood transfusions on lymphocyte intracellular metabolism of glucose and amino acids, as well as levels of adenosine deaminase activity and nucleotide triphosphate concentrations. Blood transfusions were found to increase the rate of glucose and glutamine metabolism, to increase nucleotide triphosphate concentrations, and to increase the level of adenosine deaminase activity. This increased level of lymphocyte metabolism in the face of immunosuppression would appear to indicate that the transfusion-induced immunosuppression is an active dynamic process.

Adrenergic Regulation of Erythrocyte Oxygen Affinity, pH, and Nucleotide Triphosphate/Hemoglobin Ratio in the Mummichog,Fundulus heteroclitus

The effect of epinephrine in regulating erythrocytic nucleotide triphosphate (NTP) concentrations was examined in blood and erythrocytes from Fundulus heteroclitus. Epinephrine decreased the NTP/hemoglobin (NTP/Hb) ratio by 44% over the Pco₂ range of 1.5-7.6 mmHg. Decreases in erythrocytic pH, caused by increases in Pco₂, mediated the extent to which reduced NTP concentrations increased Hb O₂ afinity. Both the Na⁺/K⁺ pump and Cl⁻/HCO⁻₃ exchange were involved in lowering erythrocyte NTP concentrations. Epinephrine stimulation, alone or in the presence of amiloride or ouabain, had no effect on erythrocyte pH. A marked intracellular alkalinization occurred in the presence of SITS (4acetamide-4-isothyocyanato-stilbene). The data suggest that the Cl⁻/HCO⁻₃ and Na⁺/H⁺ exchange are tightly coupled and responsible for maintenance of transmembrane pH. Finally, the decrease in erythrocyte NTP concentration following epinephrine stimulation was not accompanied by an increase in intracellular pH, which indicates that Donnan equilibrium was not maintained. This suggests that the Cl⁻/HCO⁻₃ exchange may be active in F. heteroclitus erythrocytes.

Fidelity ofThermococcus litoralisDNA polymerase (VentTM) in PCR determined by denaturing gradient gel electrophoresis

DNA synthesis fidelities of two thermostable DNA polymerases, Thermus aquaticus (Taq) and Thermococcus litoralis (Tli, also known as Vent), and a non-thermostable enzyme, a modified T7 DNA polymerase (Sequenase), were determined by analyzing polymerase chain reaction (PCR) products using denaturing gradient gel electrophoresis (DGGE). The error rates were 4.4, 8.9, and 2.4 x 10(-5) errors/bp for modified T7, Taq, and Tli polymerase, respectively. Reducing the nucleotide triphosphate concentration for Tli polymerase during PCR did not alter the fidelity. The ability of DGGE to detect a mutant present at several percent in a wild type population is related to the polymerase fidelity. To examine the sensitivity of mutant detection, human genomic DNA containing a 1% fraction of a known base pair substitution mutant was PCR-amplified with the three enzymes using primers that flank the mutant sequence. The PCR products were analyzed by DGGE. The signal from the mutant present at 1% was visible in the samples amplified with modified T7 and Tli polymerase, but the higher error rate of Taq polymerase did not permit visualization of the signal in DNA amplified with Taq polymerase.

Absolute quantitation of phosphorus metabolites in the cerebral cortex of the newborn human infant and in the forearm muscles of young adults using a double-tuned surface coil

The application of a double-tuned surface coil with strong coupling for both 31P and 1H to the in vivo measurement of metabolite concentrations by NMR spectroscopy is demonstrated. It is shown that sample loading, although important for a coil tuned to a single frequency, does not necessarily have a significant effect on absolute quantitation results if the coil is strongly coupled to the sample for both nuclei. For the coil used in the present study, the spectrometer calibration coefficient is almost independent of loading and the 1H and 31P flip angles at the coil center produced by fixed length pulses could be arranged to be nearly equal over a range of loading conditions. In seven normal infants, of gestational plus postnatal age 35 to 37 weeks, the cerebral cortex nucleotide triphosphate concentration was 3.7 ± 0.6 m M/liter wet (mean ± SD). Metabolite concentrations were low in the cerebral cortex of a severely birth asphyxiated infant. The adenosine triphosphate concentration in the resting, fresh forearm muscles of six young adults was 6.3 ± 0.8 m M/liter wet.

Absolute quantitation of neonatal brain spectra acquired with surface coil localization.

Surface coils with strong coupling for both 31P and 1H were used to measure metabolite concentrations by in vivo NMR spectroscopy. Tissue water was used as an internal concentration reference and the 31P and 1H spatial sensitivities of the coils were matched. For such coils, sample loading does not necessarily have a significant effect on absolute quantitation results. The coils had proportionality constants which were almost independent of loading and the 1H and 31P flip angles at the coil centre produced by fixed length pulses were approximately equal over the range of loading conditions encountered in vivo. For 7 normal infants, of gestational plus postnatal age (GPA) 35 to 37 weeks, the nucleotide triphosphate concentration in the cerebral cortex was 3.7 +/- 0.6 mmol/L wet tissue (mean +/- SD). Further studies of normal infants down to 26 weeks GPA indicated that phosphorus metabolite concentrations increased significantly with GPA during this period. Concentrations were often low in the cerebral cortices of birth asphyxiated infants. In order to provide corroboration for the results from neonatal brain, data were acquired also from the resting, unexercised forearm muscles of 6 young adults and the measured adenosine triphosphate concentration was 6.3 +/- 0.8 Mmol/L wet tissue.

Decreased UDP-glucuronic acid in rat liver after ether narcosis: An isotachophoretic study

For the past several years our laboratory has studied the effect of protein deficiency and cytostatics on the RNA metabolism of the liver in rat. With capillary isotachophoresis a fast screening method for determination of acid-extracted nucleotides was developed [ 11. In, that work unanaesthetized rats were killed by the double hatchet method [2] and the liver fragments obtained were rapidly frozen. This method seems to be the most accurate but we did not succeed in keeping the time from hatchet stroke to freeze-clamping [3] sufficiently short and constant. A sampling time of >7 s gave low nucleotide triphosphate concentrations and the ATP/ADP ratio dropped below 2.0. Therefore, other methods of liver sampling, namely under diethylether or divinylether narcosis, were also tried primarily to obtain better reproducibility with high and constant ATP/ADP/AMP ratios. Unexpectedly these anaesthetic agents lowered the UDP-GlcUA level of the liver, a finding which prompted this publication.

Oxygen dissociation constants in haemoglobins of Helicops modestus and Liophis miliaris, two water-snakes with different morphological adaptations to their aquatic environment

1. 1.Both Helicops modestus and Liophis miliaris. Brasilian water snakes with different degrees of aquatic adaptations, present 3 haemoglobin components identified by starch-gel electrophoresis and separated by DEAE-Sephadex A-50 and CM-cellulose chromatography respectively. 2. 2.Some functional properties of their haemoglobins were determined. Both the oxygen affinity 1.0mmHg at pH 7) and the alcaline Bohr effect value (−0.07) of the stripped haemoglobins of the more aquatic species H. modestus were higher than those of L. miliaris (1.4mmHg and −0.30 respectively). 3. 3.The ATP effect on both haemoglobin-oxygen affinity and Bohr effect was more pronounced in L. miliaris haemoglobins. The values found were 17.8mmHg at pH 7 and −0.9; in H. modestus. they were 8.93 mmHg and −0.55. 4. 4.Nucleotide triphosphate concentrations were determined as 2.84 mmHg in H. modrstus and 2.65 mM in L. miliaris (whole blood); haemoglobin concentrations were 6.14mM and 4.21 mM (as haem), respectively. The physiological implications of these results are discussed.

The haemoglobins of Labeo umbratus (Smith) and Labeo capensis (Smith)

The haemoglobins of both L. umbratus and L. capensis separated into four components on polyacrylamide electrophoresis. The isoelectric points of these fractions were found to be 9.35, 6.89, 6.63 and 6.40. Organic nucleotide triphosphate concentration in the blood of L. capensis was found to be 56.72 ± 18.01 mg% and 58.72 ± 17.9 mg% in the case of L. umbratus. It is not possible to distinguish between L. umbratus and L. capensis purely on the basis of the haemoglobin electropherogram.