Free nucleotides in bromodeoxyuridine-treated neuroblastoma cells

Free nucleotides in bromodeoxyuridine-treated neuroblastoma cells were investigated. The separation and purification of nucleotides were carried out by anion exchange and paper chromatography. After bromodeoxyuridine treatment, the distribution of free nucleotides with the same base, when referred to total nucleotides, shifted toward a distribution pattern closer to that of mature neuronal cells. There was an enhancement of adenylic nucleotides and a decrease of uridylic and cytidylic nucleotides. The guanylic nucleotides displayed no changes. This nucleotide distribution is in agreement with other biochemical data from differentiated neuroblastoma cells.Dr. Ciesielski-Treska is stagiaire de Recherche à l'INSERMDr. Wintzerith is Chargée de Recherche au CNRSThis paper is part of the Doctorat d'Etat thesis of A. Dierich.     

Free nucleotides in the primary root of maize

Free nucleotides of the primary root of maize were extracted with 5% HClO₄ and separated on a column of Dowex 1×8 ion exchanger in HCOO^? cycle. A two-step elution gradient (HCOOH, HCOOHNH₄) was used for the elution of the nucleotides. The incorporation of³²P into the nucleotides was followed at different time intervals and also in young and more mature root tissues. The nucleotides AMP, GMP, ADP, GDP, and ATP were identified. Labelled phosphorus was found in ATP after 30 s, in ADP after 3 min, and in AMP after 5 min incubation of the roots. More mature roots (18 days) contained higher amounts of AMP than the young ones (3 days).     

Free pyrimidine nucleoside and nucleotides from Cicer arietinum

5-β-d-Ribofuranosyluracil (pseudouridine) and the new nucleotides uracil 5-β-d-fructofuranosyl-1′-monophosphate and uracil 5-β-d-ribofuranosyl-2′,3′-cyclic monophosphate have been isolated in the free state from Cicer arietinum seeds and characterized by spectroscopic methods.     

Quantitative histochemistry of nicotinamide adenine nucleotides in retina of monkey and rabbit

The levels of NADP⁺, NADPH, NAD⁺ and NADH were measured in the different layers of retinas from rabbit and monkey. Samples (0.1 μg) were dissected from frozen-dried sections. The sum of oxidized and reduced forms was obtained by analysis of samples diluted several thousand fold in 0.02 n -NaOH at 0°. The reduced forms were measured by analysis of the same alkaline preparation after heating to destroy NADP⁺ and NAD⁺. All assays were made at 1:100,000 tissue dilution by enzymic cycling, which is capable of measuring 10⁻¹⁴ moles of nucleotides. Profiles of nicotinamide adenine nucleotide levels werecomparable in monkey and rabbit. Both total NADP and NAD were lowest in the outer segments of the retina and highest in the inner layers. NADP of the outer layers (1-2b) was oxidized to a high degree. This was particularly striking for layer 2b, which is rich in mitochondria. In the inner layers the fraction of NADPH rose to 0.7 of the total NADP. NAD on the contrary was highly oxidized in all ten layers of the retina. Three aspects of these results seem significant: (1) The profile for NADP was not related to the distribution of any of four major NADP-requiring dehydrogenases or their sum; (2) the ratio of total NADP/NADPH in the mitochondrial layer was much higher than expected from studies with isolated mitochondria; and (3) the amount of total NADP was surprisingly high in non-mitochondrial layers.     

The influence of cyclic nucleotides on macromolecular synthesis in cultured neural retina cells

1. The effect of cyclic nucleotides on aggregates of dispersed embryonic neural retina cells was examined in order to study their influence upon macromolecular synthesis, i.e. protein and DNA. 2. Cyclic AMP, dibutyryl cAMP, cyclic GMP and dibutyryl cGMP were used at various concentrations (5 x 10(-4) -5 mM). 3. The incorporation of labeled precursors into DNA and protein were used to monitor the effect of cyclic nucleotides on cultured aggregates. 4. All nucleotides exhibited a stimulatory effect at 5 x 10(-4) and 5 x 10(-3) mM on macromolecular synthesis, with resulting growth and proliferation of chick neural retina cells. 5. High concentrations (5 x 10(-1) and 5 mM) of cyclic nucleotides exhibited an inhibitory effect upon macromolecular synthesis and a marked cytotoxic effect.     

Cyclic nucleotides and protein kinase systems in the developing chick retina and pigment epithelium

During embryonic development of the chick neural retina, cyclic nucleotide levels are relatively uniform but rise abruptly at the time of hatching. The rise is thus not temporally correlated with features of morphological development such as outer segment elongation but rather with the onset of actual visual function. In the pigment epithelium, the cyclic AMP level declines throughout the embryonic period studied and does not rise at hatching. Cyclic GMP levels are much lower in both retina and pigment epithelium but rise several-fold at hatching. A binding protein si observed for cyclic AMP in the retina prior to outer segment development; cyclic GMP binding is considerably lower. Retinal ATP-kinase activity is high throughout the embryonic period studied and is stimulated up to 6-fold by 1 μM cyclic AMP and by 100 μM cyclic GMP. The major rise in GTP-kinase activity correlates temporally with photoreceptor outer segment sevelopment and may be involved intimately in the visual process.     

Cyclic nucleotides and protein kinase systems in the developing chick retina and pigment epithelium.

During embryonic development of the chick neural retina, cyclic nucleotide levels are relatively uniform but rise abruptly at the time of hatching. The rise is thus not temporally correlated with features of morphological development such as outer segment elongation but rather with the onset of actual visual function. In the pigment epithelium, the cyclic AMP level declines throughout the embryonic period studied and does not rise at hatching. Cyclic GMP levels are much lower in both retina and pigment epithelium but rise several-fold at hatching. A binding protein is observed for cyclic AMP in the retina prior to outer segment development; cyclic GMP binding is considerably lower. Retinal ATP-kinase activity is high throughout the embryonic period studied and is stimulated up to 6-fold by 1 muM cyclic AMP and by 100 muM cyclic GMP. The major rise in GTP-kinase activity correlates temporally with photoreceptor outer segment development and may be involved intimately in the visual process.     

Interaction of guanine nucleotides with photoreceptor membranes of rod outer segments of frog retina

Rod outer segments (ROS) of the frog retina are shown to contain high affinity binding sites to guanylic nucleotides. Concentration of the binding sites comprises several per cent of rhodopsin concentration in our ROS preparations. These sites possess high affinity to GDP (Kd less than 10(-6) M) in dark-adapted preparations, and in the presence of bleached rhodopsin they effectively bind the non-hydrolizable analog of GTP--GPP (NH) P (Kd less than 10(-6) M). It is shown that one bleached rhodopsin molecule can induce the binding of up to 100 molecules of GPP (NH) P at low rhodopsin photolysis. Qur experimental results raise serious doubts as to the applicability of nucleotide exchange scheme by Fung and Stryer (1980).