Concentrations Of Adenine Research Articles

Investigations on bioanalytical chemistry part V. adsorption voltammetry of adenine

The adsorptive and voltammetric behaviour of adenine on a hanging mercury drop electrode was investigated in 0.1 mol/1 acetic acid-sodium acetate buffer (pH 3.6). The influences of the preconcentration potential and preconcentration time on the peak current as well as the optimum experimental conditions were discussed. The linear relationship between the peak current and the concentration of adenine was tested. To decrease the detection limit, the 1.5th order derivative technique was used a detection limit for adenine of 1.8 × 10 −9 mol/1 was reached when the preconcentration time was only 60 s. This method was applied to determine the adenine content in the hydrolytic product of yeast RNA with satisfactory results.

赤血球ＭＡＰに含まれるアデニンによる副作用発現の可能性の検討

One unit of red cells preserved in mannitol-adenine-phosphate solution (RC-MAP) contains 7mg of adenine. Exogenous adenine is metabolized rapidly into adenine nucleotide pool by adenine phosphoribosyltransferase (APRT). This enzyme defect results in an inability to salvage the purine base adenine, which is oxidized to 2, 8-dihydroxyadenine (2, 8-DHA) by oxanthine oxidase. Excessive amounts of 2, 8-DHA is insoluble, and produces the formation of kidney stones. We examined the possibilities of adenine-toxicity using RC-MAP.The concentration of adenine stored for 5 days reduced to 10% in RC-MAP with normal APRT activity and heterozygous APRT deficiency. In that with homozygous APRT deficiency, adenine still remained more than 80% after the storage of 6 weeks. The frequency of heterozygosity for APRT deficiency in Japanese had been estimated to be 1.2%, although we could not discover APRT deficiency among 500 blood donors. It is, therefore, considered to be a rare chance that both blood donor and the patient are homozygous APRT deficiency. It seems to be infeasible of the occurrence of adeninetoxicity using RC-MAP.

Changes of nucleotide content in human and rat heart during cardiac surgery and ischemia.

The influence of ischemia on purine nucleotide and their catabolite concentration in human myocardium was investigated during surgery of acquired and congenital heart defects. This was compared with the influence of ischemia on rat heart. Concentrations of adenine and guanine nucleotides and their catabolites were measured in the extracts of heart biopsies taken at the onset of ischemia and at the time of reperfusion. The content of myocardial ATP in human heart decreased from the initial value of 22.3 +/- 1.1 to 14.6 +/- 1.5 nmol/mg protein and total adenine nucleotide pool decreased from 34.2 +/- 1.8 to 27.6 +/- 1.5 nmol/mg protein during the operation. Significant increases in myocardial concentrations of purine catabolites were also observed with the most prominent rise in inosine from below 0.5 at the onset of the ischemia to 3.0 +/- 0.5 nmol/mg protein at the time of reperfusion. A positive correlation was demonstrated between the concentration of purine catabolites in the heart at the end of ischemia with the decrease of both ATP and the total nucleotide pool. An interesting metabolic specificity of the ischemic human heart appeared to be only a small accumulation of inosine monophosphate (IMP). The increase of IMP in the rat heart after ischemia was several-fold higher. Thus, cardiac surgery of congenital and acquired heart defects was associated with a significant decrease in myocardial adenylate pool and a single biopsy collected at the end of ischemia seems to be sufficient to evaluate the extent of this metabolic and possibly functional impairment of the heart.

Concentrations of nucleotides, nucleosides, purine bases and urate in cerebrospinal fluid of children with meningitis

The release of agents mediating inflammation in meningitis may bring about neuronal hypoxia, under which circumstances ATP concentrations decrease and its degradation products increase and are released into the cerebrospinal fluid. In this study of alterations in neuronal energy metabolism in meningitis, AMP, IMP, inosine, adenosine, guanosine, adenine, guanine, hypoxanthine, xanthine and urate were determined by high performance liquid chromatography in the cerebrospinal fluid of 54 children aged between 1 month and 13 years suffering from meningitis (25 viral, 24 bacterial and 5 tuberculous cases) and 63 controls. Compared to the controls, patients with viral meningitis exhibited high concentrations of IMP, adenosine, guanosine, adenine, guanine and xanthine; patients with bacterial meningitis exhibited high concentrations of IMP, inosine, guanosine, adenosine, hypoxanthine, xanthine and urate; and patients with tuberculous meningitis exhibited high concentrations of AMP, guanosine, xanthine and uratc. Viral and bacterial cases did not differ significantly for any of the metabolites studied. AMP and urate concentrations were significantly higher in patients with tuberculous cases compared with viral or bacterial meningitis cases.

Concentrations of nucleotides, nucleosides, purine bases and urate in cerebrospinal fluid of children with meningitis.

The release of agents mediating inflammation in meningitis may bring about neuronal hypoxia, under which circumstances ATP concentrations decrease and its degradation products increase and are released into the cerebrospinal fluid. In this study of alterations in neuronal energy metabolism in meningitis, AMP, IMP, inosine, adenosine, guanosine, adenine, guanine, hypoxanthine, xanthine and urate were determined by high performance liquid chromatography in the cerebrospinal fluid of 54 children aged between 1 month and 13 years suffering from meningitis (25 viral, 24 bacterial and 5 tuberculous cases) and 63 controls. Compared to the controls, patients with viral meningitis exhibited high concentrations of IMP, adenosine, guanosine, adenine, guanine and xanthine; patients with bacterial meningitis exhibited high concentrations of IMP, inosine, guanosine, adenosine, hypoxanthine, xanthine and urate; and patients with tuberculous meningitis exhibited high concentrations of AMP, guanosine, xanthine and urate. Viral and bacterial cases did not differ significantly for any of the metabolites studied. AMP and urate concentrations were significantly higher in patients with tuberculous cases compared with viral or bacterial meningitis cases.

Determination of adenine by chronopotentiometric stripping analysis with nafion‐modified electrode

AbstractThe determination of adenine by chronopotentiometric stripping analysis with Nafion‐modified electrode is reported. Various conditions for the determination of adenine were explored. In the Britton‐Robinson (BR) buffer solution (pH 3.3), preconcentration was carried out at −0.2 V (vs. SCE) for 90 seconds by chronopotentiometric stripping analysis. The peak height was proportional to the adenine concentration between 2.5 × 10−7 and 8.0 × 10−6 mol/L. For 10 repetitive measurements, the relative standard deviation for 5 × 10−6 mol/L, adenine was 2.2%. The method was fairly free of interference, and the results of real samples were satisfactory.

A Macintosh software package for simulation of human red blood cell metabolism

We have developed a computer software package for Macintosh to simulate the metabolism and hemoglobin binding affinity of human red blood cell. The model is capable of simulating hemoglobin binding of ligands, metabolite concentrations, and metabolic fluxes at physiological steady state and in response to extracellular parameter variations, such as pH, osmolarity, glucose, and adenine concentrations. The kinetic parameters of enzymes, extracellular conditions, and initial intracellular metabolite concentrations can be specified by the user in order to model a particular situation. The software is use friendly, utilizing menu, window, and mouse to interact with the user. It also provides a pathway map of the red cell, which allows a direct access to enzyme kinetics by clicking the enzymes in the map.

INFLUENCE OF CULTURE AGE, CYTOKININ LEVEL IN CULTURE, AND “RETIPPING” ON GROWTH AND THE INCIDENCE OF VARIATION IN TISSUE-CULTURED RHODODENDRONS

Significant occurrences of phenotypic variation have been noted in micropropagated Rhododendron. Studies were undertaken to determine what aspects of micropropagation lead to variation. Rhododendron `Molly Fordham' was used to evaluate growth parameters and the incidence of variation in plants that originated from 3 month and 54 month old cultures. Plants from 3-month-old cultures were significantly wider than plants from 54-month-old cultures. Rhododendron `Aglo', `Molly Fordham', and `Scintillation' were used to evaluate growth and the incidence of variation in plants grown from microcutting bases and rerooted microcutting tips (retips). Three-month-old retips were significantly taller and wider than bases of the same age, but possessed fewer branches. The influence of in vitro N6-[2-isopentenyl]adenine (2-iP) concentration on the growth and phenotype of regenerated plants of `Aglo', `Molly Fordham', and `Scintillation' was examined. Data taken 3 months post-acclimation indicate that growth and the incidence of variation in response to 2-iP concentration is cultivar dependent.

Factors affecting morphogenesis from immature cotyledons of Phaseolus coccineus L.

Direct somatic embryogenesis as well as somatic embryogenesis and organogenesis mediated by small glossy calluses were obtained from immature cotyledon explants of bean (P. coccineus) cv Streamline 770 on a modified half-strength MS medium (Murashige & Skoog 1962) containing various concentrations of (2-isopentenyl)adenine and 2-naphthoxyacetic acid. Substitution of sucrose with glucose gave, in the range of concentrations tested, the strongest enhancement of the morphogenic process. Further improvement regarding the number of morphogenic cotyledons, the number of regenerations per cotyledon and the quality of the embryos was observed when 2,3,5-triiodobenzoic acid or abscisic acid were added to the medium. After cycles of micropropagation on MS medium plus 4.4 μM 6-benzyladenine and rooting in the absence of growth factors, plantlets were adapted to ex vitro conditions and grown to maturity.

Disturbance in the metabolism of 5′-methylthioadenosine and adenine in patients with neoplastic diseases, and in those with a deficiency in adenine phosphoribosyltransferase

5′-Methylthioadenosine (MTA) produced during the synthesis of polyamines is degraded to adenine by MTA phosphorylase. This pathway is considered to be the main source of endogenous adenine. We determined the concentrations of MTA and adenine in control subjects and in those with a pathological disorder. In patients with active leukemias, as well as with other types of malignancies, the concentrations of MTA and adenine in the urine were elevated. These changes seemed to be the result of an accelerated production of MTA due to an accelerated biosynthesis of polyamine. In patients with adenine phosphoribosyltransferase (APRT) deficiency, the concentrations of adenine in the urine were elevated, presumably due to a disturbance in the catabolism of adenine. Although adenine is a potent inhibitor of MTA phosphorylase, APRT-deficient patients did not excrete MTA into urine in concentrations significantly larger than noted for control subjects. However, the amount of MTA excreted positively correlated with that of adenine in these patients, hence that accumulated adenine probably had a slight, but positive, inhibitory effect on the degradation of MTA.

The effect of halide ions on the condensation of adenine at the mercury-water interface

The interface between mercury and concentrated aqueous solutions of adenine exhibits a well-studied capacitance pit, A. In the presence of I −, Br − or Cl −, a second pit region, B, can be observed at more positive potentials. In the presence of Br − anions, the formation of this second capacitance pit is relatively slow, and can be interrupted by switching the potential from region B to region A, in which case the as yet unused fraction of the mercury surface will be covered by an adenine film of type A, coexisting with the earlier-formed film B. Thus it is possible to prepare surfaces fully covered by a controlled mixture of two different condensed phases. This coexistence is metastable because, on a much longer timescale, film B will dissolve at potentials in region A. We also report on some capacitance oscillations at the negative limit of the adenine pit, A, at low temperatures and/or high adenine concentrations.

Inhibition of oral contraceptive steroid—metabolizing enzymes by steroids and drugs

The major 17α-ethinyl estradiol 2-hydroxylase is humans is the hepatic enzyme cytochrome P-450 IIIA4 (P-450NF), which is known to be inducible by rifampicin or barbiturates. The literature indicates that 17β-estradiol, progesterone, and norgestrel are competitive inhibitors and that primaquine and tolbutamide are rather weak noncompetitive inhibitors. Recent experiments in this laboratory indicate that gestodene is a relatively potent mechanism-based inactivator of cytochrome P-450 IIIA4 in vitro. Inhibition requires incubation with the reduced form of nicotinamide adenine dinucleotide phosphate, is time and concentration dependent, and can be partially blocked by the presence of noninhibitory cytochrome P-450 IIIA4 substrates. The in vitro activation by gestodene provides a possible explanation for the increase in plasma estrogen levels reported in women administered gestodene along with 17α-ethinyl estradiol.

Adenylate deaminase deficiency in a mutant murine T cell lymphoma cell line.

From a population of wild type S49 cells, a clone, DTB6, was isolated in a single step from selective medium containing thymidine and dibutyryl cyclic AMP that exhibited a 60% deficiency in AMP deaminase (AMP-D) activity. The AMP-D deficiency conferred to the DTB6 cells a striking susceptibility to killing by low concentrations of either adenine or adenosine, the latter in the presence of an inhibitor of adenosine deaminase activity. This growth supersensitivity of DTB6 cells toward adenine could be ameliorated by the addition of hypoxanthine to the culture medium. Immunoprecipitation of AMP-D from wild type and mutant cells revealed that the DTB6 cell line contained markedly diminished amounts of the AMP-D isozyme which reacts with antisera to the predominant isoform expressed in adult kidney. The quantities of the AMP-D isozyme immunoprecipitated by antisera raised to the predominant isoform prepared from adult heart were equivalent in the two cell lines. Although Northern blot analyses revealed no alterations in mRNA sizes or levels encoded by either of the AMP-D genes, Southern blots of genomic DNA hybridized to a cDNA specific for the ampd2 gene revealed the presence of a new BamHI restriction fragment in the DNA of DTB6 cells. These data suggested that a point mutation has occurred in the ampd2 gene of DTB6 cells which encodes the AMP-D isozyme recognized by the kidney antisera. The DTB6 cells also possessed a virtual complete deficiency in thymidine kinase activity. The two enzyme deficiencies were distinguishable. The ability to isolate single step mutants with two seemingly independent biochemical abnormalities raises the speculation that there may be some link between cellular functions responsible for purine nucleotide and thymidine metabolism.

ATP-sensitive potassium channels in adult mouse skeletal muscle: characterization of the ATP-binding site.

Single K+-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160 mM KCl solutions were blocked reversibly by 10 microM internal ATP and thus identified as ATP-sensitive K+ channels. The channels were also blocked reversibly by mM concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K+ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5 mM chloramine-T, 50 mM hydrogen peroxide or 2 mM N-ethylmaleimide (NEM). Large-conductance Ca-activated K+ channels were not affected by these reagents. The presence of 1 mM internal ATP prevents the irreversible inhibition of ATP-sensitive K+ channels by NEM. The results suggest that internal Na+ ions increase the affinity of the ATP-sensitive K+ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.

Adenine and pyridine nucleotide levels during calcium-induced conidiation in Penicillium notatum.

Concentrations of adenine and pyridine nucleotides and the associated charge values were examined in extracts of mycelium of Penicillium notatum during vegetative growth and reproductive development promoted by the addition of Ca2+ (10 mmol dm-3). The significant increase in adenylate energy charge promoted by Ca2+ was due to a fall in intracellular AMP and a concomitant rise in ATP concentration. Intracellular concentrations of NADH and NAD fell within 1 h of the addition of Ca2+. The catabolic reduction charge was unchanged by Ca2+ whilst the anabolic reduction charge increased in Ca2+-induced mycelium due to lowered intracellular NADP concentration. Reduced concentration of NADPH in Ca2+-induced mycelium, relative to the vegetative controls, lowered the phosphorylated nucleotide fraction. The results are discussed in relation to metabolic economy during morphogenesis in P. notatum.

Biosynthesis of Cytokinins in Soil

AbstractCytokinin biosynthesis in soil by rhizosphere microorganisms may increase upon the addition of physiological precursors and affect plant growth. This study was conducted to establish the effects of the purine, adenine (ADE), the isoprenoid, isopentyl alcohol (IA), and a cytokinin‐producing bacterium, on cytokinin biosynthesis in an Arlington soil (coarse‐loamy, mixed thermic Haplic Durixeralf). Various concentrations of ADE were applied, separately and in combination with 8.81 × 10−2 g IA kg−1 soil, and an inoculum of Azotobacter chroococcum (ATCC 9043), as soil treatments. Cytokinin bioactivity, isolation, and quantification of soil extracts were monitored over a period of 7 d using the radish cotyledon bioassay and HPLC‐UV spectrometry. Cytokinin production was evident as early as 2 d and reached a maximum at 5 d post‐treatment. An application of 1.35 × 10−4 g ADE kg−1 soil, 8.81 × 10−2 g IA kg−1 soil, and A. chroococcum enhanced cytokinin production of zeatin riboside and t‐zeatin in this soil up to 1.50‐ and 1.39‐fold, respectively, in comparison to controls containing no precursors. The production of zeatin riboside (78.0 µg kg−1) and t‐zeatin (22.1 µg kg−1) in the presence of ADE and IA was 1.35‐ and 2.44‐fold greater with the inoculation of A. chroococcum, when compared to the indigenous microflora alone. This study provides information on extraction and analytical detection of cytokinins in soil and reveals that microbial biosynthesis of cytokinins can be enhanced by applying physiological precursors to soil.

Factors in the liquid portion of stored blood inhibit the proliferative response in mixed lymphocyte cultures

The transfusion of blood may suppress the immune responses of patients with renal transplants and with malignant disorders. To study the in vitro suppressive effects of banked blood, 4 units of blood were stored in CPDA-1 and ADSOL at 4 degrees C for 14 days. Lymphocytes and plasma or ADSOL supernatants were harvested on Days 0, 4, 7, 10, and 14. Subpopulations of lymphocytes were enumerated by flow cytometry. Recalcified and heat-treated plasma and supernatants from the units of blood were added to mixed lymphocyte cultures (MLC) composed of cells from normal individuals. No significant changes were noted in the proportions of T or B cells from blood stored under these conditions. A 60 +/- 3 percent inhibition in the proliferative response was observed when plasma from CPDA-1 units was added to MLCs (p less than 0.02). Supernatants from ADSOL units demonstrated a 29 +/- 4 percent inhibition (p less than 0.10) of the proliferative response, and this inhibition of response was observed on all 14 days of the study. When appropriate concentrations of dextrose or adenine were added to other MLCs, adenine (at the concentration found in ADSOL) caused a significant inhibition of the proliferative response. This inhibition was not, however, as marked as that observed with recalcified, heat-treated plasma from CPDA-1 units. We conclude that adenine plus some additional factor(s) found in the liquid portion of stored blood inhibits the proliferative response of normal lymphocytes. It is possible that these factors contribute to the immune suppression observed in vivo in some patients who receive blood transfusions.

Regulation of arginine-ornithine exchange and the arginine deiminase pathway in Streptococcus lactis

Streptococcus lactis metabolizes arginine by the arginine deiminase (ADI) pathway. Resting cells of S. lactis grown in the presence of galactose and arginine maintain a high intracellular ornithine pool in the absence of arginine and other exogenous energy sources. Addition of arginine results in a rapid release of ornithine concomitant with the uptake of arginine. Subsequent arginine metabolism results intracellularly in high citrulline and low ornithine pools. Arginine-ornithine exchange was shown to occur in a 1-to-1 ratio and to be independent of a proton motive force. The driving force for arginine uptake in intact cells is supplied by the ornithine and arginine concentration gradients formed during arginine metabolism. These results confirm studies of arginine and ornithine transport in membrane vesicles of S. lactis (A. J. M. Driessen, B. Poolman, R. Kiewiet, and W. N. Konings, Proc. Natl. Acad. Sci. USA, 84:6093-6097). The activity of the ADI pathway appears to be affected by the internal concentration of (adenine) nucleotides. Conditions which lower ATP consumption (dicyclohexylcarbodiimide, high pH) decrease the ADI pathway activity, whereas uncouplers and ionophores which stimulate ATP consumption increase the activity. The arginine-ornithine exchange activity matches the ADI pathway most probably by adjusting the intracellular levels of ornithine and arginine. Regulation of the ADI pathway and the arginine-ornithine exchanger at the level of enzyme synthesis is exerted by glucose (repressor, antagonized by cyclic AMP) and arginine (inducer). An arginine/ornithine antiport was also found in Streptococcus faecalis DS5, Streptococcus sanguis 12, and Streptococcus milleri RH1 type 2.

Salvage mechanisms for regeneration of adenosine triphosphate in rat cardiac myocytes

Since mitochondrial oxidative phosphorylation does not produce sufficient adenosine triphosphate for rapid restoration of contractile function in myocardium reoxygenated after ischaemia alternative non-oxidative routes by which purine nucleosides or bases may be used to increase the cytoplasmic adenine nucleotide pool were studied. Comparative rates of uptake and salvage of adenosine, adenine, and hypoxanthine were determined using myocytes isolated from adult rat heart. For each precursor reactions limiting the overall rate at which substrate in the extracellular fluid is incorporated into the intracellular adenosine triphosphate pool were identified. Adenosine was salvaged at twice the rate seen with adenine in the presence of ribose, whereas the rate of salvage of hypoxanthine, in the presence of ribose, was only 5% of that for adenosine. Adenine may be an advantageous substrate since high concentrations of adenine are not inhibitory to salvage and do not influence cardiac haemodynamics. Salvage of hypoxanthine appeared to be limited by the rate of adenylosuccinate synthetase, which was present at less than 1% of the adenylosuccinase rate in rat, rabbit, and beef heart. In addition, since salvage of bases is dependent on the availability of phosphoribosylpyrophosphate rates of synthesis of phosphoribosylpyrophosphate were measured in whole myocytes from ribose and in cytoplasmic extracts from ribose and from ribose-5-phosphate. Metabolic sites were identified at which interventions designed to accelerate salvage rates might best be directed.

Adenine and pyridine nucleotides in the red blood cells of subjects with solid tumors.

The concentration of adenine (ATP, ADP, AMP) and pyridine (NADP+, NADPH, NAD+, NADH) nucleotides in the erythrocytes of subjects affected by solid tumors was evaluated using a method which allows their simultaneous extraction and reverse-phase high-performance liquid chromatographic analysis. The results showed a lower level of ATP in the erythrocytes of subjects affected by solid tumors, whereas no significant modifications were observed in the other compounds. In fact, the mean value of ATP in these subjects was 27% lower than that of normal adults. This fact is discussed in relation to other enzymatic and metabolic modifications previously observed in red blood cells.