Acid-soluble Pool Research Articles

Dominant glacial landforms of the lower Great Lakes region exhibit different soil phosphorus chemistry and potential risk for phosphorus loss

Abstract Phosphorus (P) losses from agricultural soils are a growing economic and water-quality concern in the Lake Erie watershed. While recent studies have explored edge-of-field and watershed P losses related to land-use and agricultural management, the potential for soils developed from contrasting parent materials to retain or release P to runoff has not been examined. A field-based study comparing eight agricultural fields in contrasting glacial landscapes (hummocky coarse-textured till-plain, lacustrine and fine-textured till-plain) showed distinct physical and geochemical soil properties influencing inorganic P (Pi) partitioning throughout the soil profile between the two regions. Fields located on the coarse-textured till-plain in mid-western Ontario, Canada had alkaline calcareous soils with the highest Total-Pi concentrations and the majority of soil Pi stored in an acid-soluble pool (up to 91%). In contrast, loosely to moderately soluble Pi concentrations were higher in soils of the lacustrine and fine-textured till-plain in southwestern Ontario, northeast Indiana and northwestern Ohio, US. Overall, soils on the lacustrine and fine-textured till-plain had a greater shrink swell-capacity, likely creating preferential flow to minimize Pi interaction with the more acidic, lower carbonate and lower sorption capacity soils. These differences in soil Pi retention and transport pathways demonstrate that in addition to management, the natural landscape may exert a significant control on how Pi is mobilized throughout the Lake Erie watershed. Further, results indicate that careful consideration of region-specific hydrology and soil biogeochemistry may be required when designing appropriate management strategies to minimize Pi losses across the lower Great Lakes region.

Glycerol uptake is by passive diffusion in the heart but by facilitated transport in RBCs at high glycerol levels in cold acclimated rainbow smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) is a small fish that accumulates glycerol at low winter seawater temperatures. In laboratory-held fish, glycerol concentration typically reaches 225 mM in plasma and in all cells. Glycerol uptake by the heart and red blood cells (RBCs) was assessed by tracking [(14)C(U)]glycerol into the acid-soluble pool. In fish acclimated to 9-10°C a decrease in perfusion/incubation temperature from 8 to 1°C resulted in a decrease in glycerol uptake with a Q(10) of 3.2 in heart and 2.4 in RBCs. Acclimation to ∼1.5°C did not result in an adaptive enhancement of glycerol uptake as rates were unchanged in heart and RBCs. Glycerol uptake at 1°C was by passive diffusion in heart as evidenced by a linear relationship between glycerol uptake and extracellular glycerol concentration and a lack of inhibition by phloretin. In contrast, in RBCs, glycerol uptake with respect to glycerol concentration showed two linear relationships with a transition point around 50 mM extracellular glycerol. The slope of the second phase was much steeper and eliminated with the inclusion of phloretin. In RBCs from Atlantic salmon (Salmo salar), a related species that does not accumulate glycerol, glycerol uptake showed only a single linear curve and was not inhibited by phloretin. The data imply a strong facilitated component to glycerol uptake in rainbow smelt RBCs at high glycerol concentrations. We propose this is related to cyclic changes in RBC glycerol content involving a loss of glycerol at the gill and a reaccumulation during passage through the liver.

AMP Deaminase Activation Contributes to Accelerated Adenine Nucleotide Pool Depletion during Periods of Energy Imbalance in Sickle Cell Erythrocytes.

Mature erythrocytes are unable to synthesize AMP from IMP due to the developmental loss of adenylosuccinate synthetase, which also effectively prevents the synthesis of adenine nucleotides from circulating hypoxanthine, the major salvageable purine compound. Consequently, AMP deaminase (AMPD) activity must be tightly regulated in order to avoid irreversible depletion of the erythrocyte adenine nucleotide pool during periods of energy imbalance. However, increased intracellular calcium promotes activation of erythrocyte AMPD (AMPD3) through a protein-protein interaction with calmodulin (Mahnke and Sabina, Biochemistry 44:5551, 2005). Therefore, we hypothesized that Sickle Cell Disease (SCD) erythrocytes would more rapidly accumulate IMP and deplete the adenine nucleotide pool during periods of energy imbalance. In order to test this hypothesis, acid-soluble pools of these metabolites were quantified by anion-exchange HPLC in SCD and control erythrocytes during autoincubation and glucose starvation, both at 37C. Data in the table show that SCD erythrocytes accumulate more IMP during a 6-hour autoincubation compared to control cells, and this is accompanied by a decrease in AMP.Effect of Autoincubation on Erythrocyte AMP and IMP PoolsGROUPHOURSAMPaIMPIMP/AMPControl (N=4)0.29±.08.15±.06.58±.286.29±.01.33±.11†1.15±.35†SCD (N=5)0.22±.04.08±.03.36±.106.14±.03†.74±.24†‡5.15±1.25†‡a, metabolite concentrations reported as μmol/g Hb; †, p<.05 when compared to 0 time in a paired student's t-test; ‡, p<.05 when compared to control in an unpaired student's t-testData in the figure show an accelerated depletion of ATP and increased accumulation of IMP in glucose-starved SCD erythrocytes compared to control cells (*, p<.05 when compared to control in an unpaired student's t-test). [Display omitted] These combined results demonstrate AMPD3 enzyme activation and accelerated adenine nucleotide pool depletion during periods of energy imbalance in SCD erythrocytes. This metabolic imbalance in SCD erythrocytes could contribute to 1) an increased rate of hemolysis, 2) cation channel dysregulation, 3) modulation of adhesive membrane components, and 4) diminished protein phosphorylation. Furthermore, strategies directed against this metabolic imbalance, e.g., diffusible AMPD inhibitors and adenine supplementation, may have a therapeutic benefit in SCD.

Cerebral protein synthesis in a genetic mouse model of phenylketonuria.

Local rates of cerebral protein synthesis (lCPS(leu)) were measured with the quantitative autoradiographic [1-(14)C]leucine method in a genetic mouse model (Pah(enu2)) of phenylketonuria. As in the human disease, Pah(enu2) mice have a mutation in the gene for phenylalanine hydroxylase. We compared adult homozygous (HMZ) and heterozygous (HTZ) Pah(enu2) mice with the background strain (BTBR). Arterial plasma concentrations of phenylalanine (Phe) were elevated in both HMZ and HTZ mutants by 21 times and 38%, respectively. In the total acid-soluble pool in brain concentrations of Phe were higher and other neutral amino acids lower in HMZ mice compared with either HTZ or BTBR mice indicating a partial saturation of the l-amino acid carrier at the blood brain barrier by the elevated plasma Phe concentrations. In a series of steady-state experiments, the contribution of leucine from the arterial plasma to the tRNA-bound pool in brain was found to be statistically significantly reduced in HMZ mice compared with the other groups, indicating that a greater fraction of leucine in the precursor pool for protein synthesis is derived from protein degradation. We found reductions in lCPS(leu) of about 20% throughout the brain in the HMZ mice compared with the other two groups, but no reductions in brain concentrations of tRNA-bound neutral amino acids. Our results in the mouse model suggest that in untreated phenylketonuria in adults, the partial saturation of the l-amino acid transporter at the blood-brain barrier may not underlie a reduction in cerebral protein synthesis.

Impact of 9-(2-phosphonylmethoxyethyl)adenine on (deoxy)ribonucleotide metabolism and nucleic acid synthesis in tumor cells

Following exposure to 9-(2-phosphonylmethoxyethyl)adenine (an inhibitor of the cellular DNA polymerases α, δ and ϵ), human erythroleukemia K562, human T-lymphoid CEM and murine leukemia L1210 cells markedly accumulated in the S phase of the cell cycle. In contrast to DNA replication, RNA synthesis (transcription) and protein synthesis (mRNA translation) were not affected by 9-(2-phosphonylmethoxyethyl)adenine. The ribonucleoside triphosphate pools were slightly elevated, while the intracellular levels of all four deoxyribonucleoside triphosphates were 1.5–4-fold increased in 9-(2-phosphonylmethoxyethyl)adenine-treated K562, CEM and L1210 cells. The effect of 9-(2-phosphonylmethoxyethyl)adenine on de novo (thymidylate synthase-mediated) and salvage (thymidine kinase-mediated) dTTP synthesis was investigated using radiolabelled nucleoside precursors. The amount of thymidylate synthase-derived dTTP in the acid soluble pool was 2–4-fold higher in PMEA-treated than in untreated K562 cells, which is in accord with the 3–4-fold expansion of the global dTTP level in the presence of 9-(2-phosphonylmethoxyethyl)adenine. Strikingly, 2-derived dTTP accumulated to a much higher extent (i.e. 16–40-fold) in the soluble dTTP pool upon 9-(2-phosphonylmethoxyethyl)adenine treatment. In keeping with this finding, a markedly increased thymidine kinase activity could be demonstrated in extracts of 9-(2-phosphonylmethoxyethyl)adenine-treated K562 cell cultures. Also, in the presence of 200 μM 9-(2-phosphonylmethoxyethyl)adenine, 14-fold less thymidylate synthase-derived but only 3-fold less thymidine kinase-derived dTTP was incorporated into the DNA of the K562 cells. These data show that thymidine incorporation may be inappropriate as a cell proliferation marker in the presence of DNA synthesis inhibitors such as 9-(2-phosphonylmethoxyethyl)adenine. Our findings indicate that 9-(2-phosphonylmethoxyethyl)adenine causes a peculiar pattern of (deoxy)ribonucleotide metabolism deregulation in drug-treated tumor cells, as a result of the metabolic block imposed by the drug on the S phase of the cell cycle.

Basis of Relative Insensitivity of HIV-Infected JM Cells to AZT

The basis of azidothymidine (AZT) insensitivity in the human JM T-cell line has been investigated. It was shown that uptake of radiolabelled thymidine or AZT into cellular acid-soluble pools of JM cells was about 10-fold lower than that seen in AZT-sensitive HeLa cells. Thymidylate kinase, however, was apparently not inhibited by AZTMP in JM cells to the extent observed in most cells and, as a result, AZTTP formation proceeded at a greater rate than in HeLa cells, which exhibited accumulation primarily of AZTMP. Thus the deficit in phosphorylation in JM cells cannot solely account for the decreased AZT sensitivity. Instead, it was shown that JM cells excreted AZTMP into the culture medium and that, whereas HeLa cells continued to accumulate AZT nucleotides over time, JM cells did not. This excretion of AZTMP in JM cells also led to a failure to sustain lowered competing TTP pools. It is concluded that it may not be appropriate to use the JM cell line for testing of novel anti-HIV nucleotides designed to circumvent requirements for phosphorylation.

Root-induced solubilization of phosphate in the rhizosphere of lowland rice.

Lowland rice (Oryza sativa L., cv IR74) was grown in cylinders of a P-deficient reduced Ultisol separated into upper and lower cells by a fine nylon mesh so that the roots formed a planar layer above the mesh. This enabled changes in soil P fractions and other root-induced changes in the soil near the root plane to be measured. In both P-fertilized and unfertilized soil, the quantity of readily plant-available P was negligible in comparison with the quantity of P extracted by the plants, and the plants therefore necessarily induced changes in the soil so as to solubilize P. After 6 wk of growth, 90 % of the P taken up was drawn from acid-soluble pools. The remainder was from an alkali-soluble inorganic pool which was on balance depleted, although its concentration profile contained zones of accumulation corresponding to zones of Fe(III) accumulation. There was also a small accumulation of alkali-soluble organic P. There were no changes in the more recalcitrant soil P pools. The zone of P depletion was 4-6 mm wide, increasing with P addition, and coincided with a zone of acidification in which the pH fell from near 6 in the soil bulk to less than 4 near the roots. The acidification was due to H+ generated in oxidation of Fe2+ by root-released O2 , and to H+ released from the roots to balance excess intake of cations over anions. With increasing P deficiency there were increases in the ratio of root: shoot d. wt; the ratio of shoot d. wt to total P in the plant; the excess intake of cations over anions per unit plant d. wt and corresponding release of H+ to the soil; and the quantity of Fe oxidized per unit plant d. wt and corresponding release of H+ to the soil. Independent, in vitro measurements confirmed that acid addition increased the P concentration in the soil solution and the quantity of P that could be desorbed per gram of soil. A mathematical model of the diffusion of acid away from the roots, acid reaction with the soil in solubilizing P, and the diffusion of P back to the absorbing roots showed that, under the conditions of the root-plane experiments, solubilization by acidification accounted for at least 80% of the P taken up in both P-fertilized and unfertilized soil, but that less than 50% of the P solubilized could be taken up by the roots.

Uptake, Transport and Metabolism of Exogenous Nucleosides in Intestinal Epithelial Cell Cultures

The uptake and transport of nucleosides (adenosine, cytidine, guanosine, thymidine, uridine and inosine) were examined in intestinal cell lines (Caco-2 and IEC-6). Well-differentiated Caco-2 cells took up significantly more uridine and thymidine than less-differentiated cells over 24 h. These differences were reflected in the resulting trichloroacetic acid-soluble (nucleotide, nucleoside and nucleobase) pool sizes rather than in differences in nucleic acid incorporation. IEC-6 cells have smaller soluble pools than Caco-2 cells, and the uptake of nucleosides over 60 min reflected this difference. Caco-2 cells transported significantly more thymidine, cytidine, guanosine, adenosine and inosine into acid-soluble pools than IEC-6 cells. Caco-2 cells were grown on filters to examine the transcellular transport of nucleosides and to examine the chemical form in which nucleosides appeared at the basolateral aspect of the cell monolayer. The pattern of transport varied for individual nucleosides. The transport of cytidine and guanosine was significantly greater in the apical-to-basolateral direction than in the opposite direction over 2 h. No purine nucleoside was transported intact across the Caco-2 cell monolayer in either direction, with the majority of the transported material appearing as nucleobases. Nucleobases also represented the main metabolites of pyrimidines transported. However, some cytidine and uridine were detectable, but each constituted < 20% of transported material. Nucleosides are therefore transported by enterocytes but metabolized during transport.

Effect of insulin on rat heart and skeletal muscle phenylalanyl-tRNA labeling and protein synthesis in vivo

In vivo measurement of muscle protein synthesis and its hormonal regulation is limited by the difficulty of measuring aminoacyl-tRNA specific activity (SA). We assessed the kinetics of heart and skeletal muscle phenylalanyl-tRNA labeling during continuous infusion of L-[ring-2,6-3H]phenylalanine (Phe) to fasted anesthetized rats. We measured Phe SA in arterial and femoral venous plasma, the tissue acid-soluble pool and muscle protein hydrolysates after 5 min (n = 7), 30 min (n = 6), and 90 min (n = 7). We also assessed insulin's effect on labeling of the tRNA pool and muscle protein synthesis during a hyperinsulinemic clamp (2 mU.kg-1.min-1; n = 7). Labeling of tRNA in heart reached 59 +/- 5, 67 +/- 3, and 83 +/- 3% of arterial SA at 5, 30, and 90 min of saline infusion, respectively, but only 10 +/- 5, 34 +/- 2, and 48 +/- 2% in skeletal muscle at those times (P < 0.01 vs. heart). The tRNA SA was intermediate between SA in the acid-soluble pool and arterial plasma. Femoral venous SA was 32 +/- 2% lower (P < 0.001) than arterial SA. Skeletal muscle tRNA SA was also 29 +/- 3% lower (P < 0.001) than femoral venous SA. Insulin did not alter tRNA labeling and neither heart (9.8 +/- 1.1%/day for saline vs. 8.4 +/- 1.0%/day for insulin) nor skeletal muscle (6.7 +/- 1.5%/day vs. 4.2 +/- 0.4%/day) protein synthesis. Thus labeling of phenylalanyl-tRNA occurs more rapidly in heart than in skeletal muscle and is unaffected by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of phosphonate on the acid-soluble phosphorus components in the genus Phytophthora

The high concentrations of short-chain soluble polyphosphates, previously reported in Phytophthora palmivora, have been found in six other species of Phytophthora, and appear to be characteristic of this genus. The distribution of phosphorus among the various acid-soluble pools was similar in all species except P. infestans, which accumulated less polyphosphate than other species when grown in high phosphate medium, and contained several unique and as yet unidentified organic phosphorus compounds. When grown in the presence of potassium phosphonate sufficient to reduce growth by 50%, all species showed an increased accumulation of phosphorus in both pyrophosphate and polyphosphate, without parallel increases in sugar phosphate or nucleotide phosphorus pools. The internal concentration of phosphonate required to produce 50% inhibition varied with species, ranging from 4·6 to 52 μmol (g dry wt)-1. Assimilation of orthophosphate was only slightly reduced in the presence of phosphonate, resulting in an increased concentration of phosphorus per unit mass. Metalaxyl, which also inhibits growth of Phytophthora spp. did not cause accumulation of either pyro- or polyphosphate. Phosphonate treatment also resulted in the formation of a compound identified as isohypophosphate, a metabolite of phosphonate not previously reported in Phytophthora. Taken together, these observations suggest that the primary site of phosphonate inhibition in Phytophthora spp. lies in the metabolism of pyrophosphate.

Effects of axotomy on protein synthesis in the rat hypoglossal nucleus: examination of the influence of local recycling of leucine derived from protein degradation into the precursor pool.

The quantitative autoradiographic L-[1-14C] leucine method for the determination of regional rates of cerebral protein synthesis (lCPSleu) requires knowledge of the degree of recycling of leucine derived from protein degradation into the precursor pool for protein synthesis, which can be evaluated by measuring lambda i, the steady-state ratio of the leucine-specific activity in the precursor amino acid pool (tRNA-bound leucine) to that of the arterial plasma. To define the changes in lCPSleu during regeneration of the hypoglossal nerve, we examined the effects of axotomy on the value of lambda i. Because the concentration of tRNA-bound leucine in the hypoglossal nucleus is too low to measure, we measured the equivalent ratio for the total acid-soluble pool (psi i) and applied the linear relationship between lambda and psi found in the whole brain to calculate a value of lambda i in the ipsilateral and contralateral hypoglossal nuclei of 22 adult female rats 2, 18, 35, and 60 days after unilateral hypoglossal axotomy. Statistically significant but quantitatively inconsequential effects of axotomy on values of psi i and lambda i were found. Therefore, the mean value for lambda i (0.64) of the left and right hypoglossal nuclei in all 22 axotomized rats was used to calculate lCPSleu. In a separate group of 15 unilaterally axotomized rats, lCPSleu was determined by the autoradiographic technique; lCPSleu was increased on the axotomized side by 23% on day 2, 30% on day 18, and 13% on day 35. By postaxotomy day 60, lCPSleu had returned to normal.

Incorporation of substrate carbon from

Of the substrate carbon incorporated from 1.12 mM [U-14C]acetate as sole energy substrate during a 2.5-h incubation, a large proportion was isolated in the acid-soluble fraction. Although there was no significant change over time in the rate of entry into this pool for embryos, the rate of accumulation by the trophoblast and yolk sac increased as development progressed. At Days 13 and 15 of pregnancy, incorporation of acetate into the acid-insoluble fraction of embryos accounted for almost half the total label accumulated. The rate of this incorporation fell rapidly over time and by Day 19 less than 30% of carbon accumulated was in this fraction. By contrast, the rate of incorporation into this fraction by trophoblastic tissue was low at Day 13 but rose dramatically as development progressed. Incorporation by the yolk sac into acid-insoluble components also rose with time. At the early stages of pregnancy studied, lipid synthesis accounted for the majority of acetate carbon accumulated by the conceptus in the acid-insoluble pool. At later stages of development, incorporation into lipids constituted a minor pool of acetate carbon. Some acetate carbon was found in the glycogen fraction of the conceptus. The rate of incorporation into the acid-soluble glycogen fraction by embryos was constant throughout the period studied. By contrast, trophoblast increased its rate of incorporation markedly into both acid-soluble and acid-insoluble glycogen pools as did the yolk sac from Day 17.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of regional rates of cerebral protein synthesis adjusted for regional differences in recycling of leucine derived from protein degradation into the precursor pool in conscious adult rats.

The quantitative autoradiographic L-[1-14C]leucine method for the determination of regional rates of cerebral protein synthesis in vivo takes into account recycling of unlabeled leucine derived from protein degradation into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the apparent steady-state leucine specific activity in the precursor amino acid pool (tRNA-bound leucine) to that in the arterial plasma. In the whole brain of the conscious rat this ratio (lambda WB) equals 0.58. The equivalent ratio for leucine in the acid-soluble pool in whole brain (psi WB) is 0.49. A first-degree polynomial equation for lambda WB as a function of psi WB was fitted from paired determinations. To determine the degree of recycling in local regions of the brain, we have measured in individual brain regions (i) psi i and calculated lambda i assuming that the fitted equation also applies to these localized regions. Our results indicate that the degree of recycling into the precursor pool does vary regionally; lambda i in the individual regions varies from 0.62 in the hypoglossal nucleus to 0.50 in the globus pallidus. Local rates of protein synthesis were then determined by the autoradiographic technique with regional corrections for recycling of unlabeled leucine. Rates of leucine incorporation into protein averaged 6.1 nmol/g of tissue/min in the brain as a whole, with the rates in gray matter about twice those in white matter.

Changes since 1950 of mineral pools in the upper C-horizon of Swedish deciduous forest soils

Soils from the C-horizon of deciduous forests in southern Sweden, originally sampled in 1947-52, were resampled in 1988. Air-dried soil from both periods were extracted in 1991 using M NH4Ac, pH 4.8, and 0.2 M HNO3 for analysis of exchangeable and acid soluble pools of Na, Mn, Ca, Mg, K, Sr, Zn, Fe, S, P, and Al. Using acid NH4Ac as extractant nine elements had changed significantly (p 0.90 for Na, K, Mg, and Sr), but with the exception of S, values for HNO3 were higher or much higher. It is concluded that a decided decrease of the exchangeable pools of base cations and an equally decided increase of exchangeable Al has occurred even in the C-horizon, well below the main rhizosphere. That also the acid soluble pools of base cations have decreased indicates mineralogical changes which may counteract a complete reversibility of the current soil acidification.

Synergistic inhibition of human gastric carcinoma cell growth by 1-β- d-arabinofuranosylcytosine and hydroxyurea or 2′-deoxyguanosine in vitro

The cytotoxicity of 1-β- d-arabinofuranosylcytosine (ara-C) in combination with hydroxyurea (HU) or 2′-deoxyguanosine (GdR) on human gastric carcinoma MK-1 cells and colon carcinoma HT-15 cells was studied. Synergistic interaction between ara-C and HU on MK-1 cells and HT-15 cells, or ara-C and GdR on MK-1 cells was shown using the combination index method. HU increased the accumulation of ara-C triphosphate (ara-CTP) in the acid-soluble pool and diminished the cellular deoxyCTP (dCTP) pool. HU had no effect on the incorporation of ara-C into DNA and RNA. These results indicate that HU-induced elevation in ara-CTP and decrease in dCTP are the basis for synergy among ara-C and HU in MK-1 cells. GdR diminished cellular dCTP slightly, but it decreased the accumulation of ara-CTP in the acid-soluble pool and did not increase the incorporation of ara-C into DNA. On the other hand, ara-C increased cellular deoxyGTP (dGTP) level in the presence of GdR. These results indicate that synergy between ara-C and GdR is mediated through increased cellular dGTP which might inhibit DNA synthesis directly.

Effect of Loading Doses of l‐Valine on Relative Contributions of Valine Derived from Protein Degradation and Plasma to the Precursor Pool for Protein Synthesis in Rat Brain

"Flooding" amino acid pools with high doses of labeled amino acids of low specific activity has been proposed to minimize the effects of recycling of amino acids derived from protein degradation on the specific activity of the amino acid precursor pool for protein synthesis. We have examined the influence of recycling on the precursor pool for protein synthesis under conditions in which plasma valine concentrations were normal (0.19 mM) and "flooded" (10-28 mM) by comparing the steady-state specific activity of the tRNA-bound valine with that of the plasma valine. Under normal and "flooding" conditions, the relative contributions of valine from protein degradation to the precursor pool were 63 and 26%, respectively; "flooding" with a plasma level of 28 mM raised the brain acid-soluble pool level to 3.1 mM but was no more effective in decreasing the relative contribution of valine from protein degradation to the precursor pool than "flooding" with a plasma level of 17 mM valine, which raised the brain acid-soluble level only to 2.3 mM. The results of these studies show that "flooding" amino acid pools does indeed reduce the effect of recycling on the precursor amino acid pool for protein synthesis, but it does not totally eliminate it.

Some remarks on the presence of organic phosphates in sediments

This article describes a new method developed to assess the size and nature of the organic phosphate pool. Using sediment suspensions from the Rhone, Garonne and Po rivers, inorganic P compounds, Fe(OOH) and CaCO3 were removed using mild extractants at sediment pH. The residual phosphate was then fractionated into an acid soluble organic phosphate pool and a residual organic phosphate pool by acid hydrolysis (0.5 M H+). Both pools were quantitatively important, accounting for between 16 and 54% and 16 and 51% of total phosphate respectively. Acid hydrolysis was chosen since it yielded a distinct plateau, with high reproducibility, within 30 minutes. This fractionation permits a further study of dynamics and bioavailability of sediment org-P, without interference of Fe(OOH) and CaCO3. In many studies in which changes in the organic pool were examined after extraction of inorganic phosphate, 0.5 M HCl was used to extract apatite bound phosphate. The results presented here show that this is likely to result in a considerable underestimation of the organic phosphate pool.

Study of the metabolism of flucytosine in Aspergillus species by 19F nuclear magnetic resonance spectroscopy.

The metabolism of flucytosine (5FC) in two Aspergillus species (Aspergillus fumigatus and A. niger) was investigated by 19F nuclear magnetic resonance spectroscopy. In intact mycelia, 5FC was found to be deaminated to 5-fluorouracil and then transformed into fluoronucleotides; the catabolite alpha-fluoro-beta-alanine was also detected in A. fumigatus. Neither 5-fluoroorotic acid nor 5-fluoro-2'-deoxyuridine-5'-monophosphate was detected in perchloric acid extracts after any incubation with 5FC. 5FC, 5-fluorouracil, and the classical fluoronucleotides 5-fluorouridine-5'-mono-, di-, and triphosphates were identified in the acid-soluble pool. Two hydrolysis products of 5-fluorouracil incorporated into RNA, 5-fluorouridine-2'-monophosphate and 5-fluorouridine-3'-monophosphate, were found in the acid-insoluble pool. No significant differences in the metabolic transformation of 5FC were noted in the two species of Aspergillus. The main pathway of 5FC metabolism in the two species of Aspergillus studied is thus the biotransformation into ribofluoronucleotides and the subsequent incorporation of 5-fluorouridine-5'-triphosphate into RNA.

Selective purine release from P388D1 macrophages injured by silica.

Silica particles are toxic to primary cultures of macrophages or the P388D1 cell line in vitro. Loss of viability in these model systems is accompanied by depletion of ATP content within 3 to 6 hours. The mechanisms responsible for ATP depletion will be explored in this paper. After prelabeling for 1 hour with 3H-adenine, silica-treated cells released 60-80% of their labeled acid-soluble pool into the culture medium. This release did not occur after phagocytosis of nontoxic titanium dioxide particles and was specific for purines. ATP depletion was accompanied by purine catabolism: inosine, hypoxanthine, xanthine, and uric acid were detected in the culture medium using thin layer or high-performance liquid chromatography. The final xanthine oxidase step in purine catabolism generates reactive oxygen metabolites. Silica toxicity was not prevented by the xanthine oxidase inhibitor allopurinol nor exogenous purines. It is concluded that adenine nucleotide depletion and purine catabolism are not solely responsible for irreversible injury in silica toxicity. It is hypothesized that purine catabolism and release from injured macrophages may lead to generation of reactive oxygen species, injury to surrounding tissue, and fibrosis.

Cellular metabolism of 2',3'-dideoxycytidine, a compound active against human immunodeficiency virus in vitro.

The nucleoside analog 2',3'-dideoxycytidine (ddCyd) has been shown to inhibit the infectivity and cytopathic effect of human immunodeficiency virus on human OKT4+ lymphocytes in vitro. Metabolism of ddCyd by human T-lymphoblastic cells (Molt 4) negative for human immunodeficiency virus and OKT4 was examined. Molt 4 cells accumulated ddCyd and its phosphorylated derivatives into acid-soluble and acid-insoluble material in a dose-dependent manner. For each concentration tested, 2',3'-dideoxycytidine triphosphate represented 40% of the total acid-soluble pool of ddCyd metabolites. Uptake of 5 microM ddCyd was linear for 4 h after addition of drug. Efflux of ddCyd metabolites from cells followed a biphasic course with an initial retention half-life of 2.6 h for 2',3'-dideoxycytidine triphosphate. DNA, but not RNA, of cells incubated with [3H]ddCyd became radiolabeled. Nuclease and phosphatase treatment of DNA followed by reverse-phase high pressure liquid chromatography showed that the nucleoside was incorporated into DNA in its original form. ddCyd was not susceptible to deamination by human Cyd-dCyd deaminase. It was a poor substrate for human cytoplasmic and mitochondrial dCyd kinases, with Km values of 180 +/- 30 and 120 +/- 20 microM, respectively. DNA polymerases alpha, beta, and gamma varied in their sensitivity to inhibition by ddCTP with Ki values of 110 +/- 40, 2.6 +/- 0.3, and 0.016 +/- 0.008 microM, respectively; however, inhibition was competitive with dCTP in each case.