Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. IV. Nucleoside transport in cells depleted of nucleotides by treatment with KCN

Incubation of Novikoff rat hepatoma cells in glucose-free basal medium containing 2 mM KCN results in a rapid and almost complete loss of uracil and adenine nucleotides. By following the fate of radioactivity from ³H-nucleoside pulse-labeled cells during incubation with KCN it was shown that the nucleotides are degraded to nucleosides and bases which are released into the culture fluid. Depletion of the cells of nucleotides by incubation with KCN allows a direct analysis of the kinetics of uridine transport into the cell, since KCN-treated cells fail to phosphorylate uridine. Uridine uptake follows normal Michaelis-Menten kinetics with an apparent K_n of about 50 μm at 18°C. Uptake is by facilitated diffusion since it does not require energy and uridine is not transported against a concentration gradient. The effects of KCN are largely prevented by the presence of 10 mM glucose in the medium. They are also rapidly reversed by resuspending the cells in fresh medium without KCN. Upon removal of KCN, the cells rapidly regenerate their nucleotide pools and resume growth at the normal rate.     

In vitro interactions between membrane, hormone, and cyclic nucleotides as revealed with aequorin

Aequorin was used to study Ca²⁺ movements in an acellular plasma membrane-rich extract from starfish oocyte cortices. Adenosine cyclic nucleotides were found to release Ca²⁺ in this system and to enhance Ca²⁺ release resulting from hormonal stimulation. The natural hormone, 1-methyladenine (1-MeAde), must act directly, independently of cyclic nucleotides, since incubating the extract with phosphodiesterase does not significantly affect the response to the hormone. Moreover, this preparation responds faster to the hormone than to cyclic nucleotides.     

Metabolic coupling between cultured pancreatic B-cells

The transfer of [³H]uridine nucleotides from donor (uridine-loaded) to recipient (thymidine-prelabelled) pancreatic endocrine islet cells in monolayer culture was qualitatively and quantitatively assessed by light and electron microscope autoradiography. Recipient cells showed a labelled cytoplasm only when they were in contact with donor cells or positive recipients. Controls indicated that the labelling was not due to the incorporation of [³H]uridine, ³H nucleotides or nucleic acids lost by donor cells in the medium. Quantitation showed that cytoplasmic labelling of positive recipient cells was higher than cellular background, but lower than the cytoplasmic labelling of donor cells. These data indicate that label in recipient cells was derived from donors by direct intercellular transfer of ³H nucleotides. Differentiated insulin-producing cells (B cells) were involved in the exchange.     

Characterization of long patch excision repair of DNA in ultraviolet-irradiated Escherichia coli: An inducible function under rec-lex control

Summary Excision repair in ultraviolet-irradiated wild-type Escherichia coli produces a bimodal distribution of repair patch sizes in the DNA. Approximately 99% of the repair events result in short patches of 20–30 nucleotides produced by a constitutive repair system. The remaining 1% result in patches which are at least 1,500 nucleotides in length. This long patch repair is shown to be a damage-inducible process under control of the rec-lex regulatory circuit. The kinetics of the two processes differ; short patch synthesis begins immediately after irradiation and is virtually completed prior to synthesis of the majority of the long patches. Long patch repair synthesis is a linear function of UV dose up to a plateau at 60 J/m², and hence each long patch event is the consequence of a single UV-induced lesion. Long patch repair does not appear to be nessarily error-prone, since no alteration in repair synthesis occurs as a result of a mutation umuC ^- which renders cells nonmutable by UV. Evidence is presented suggesting that DNA polymerase I is responsible for both long and short patch synthesis in wild type cells under inducing conditions. In the absence of polymerase I the constitutive patch size averages 80–90 nucleotides, and this distribution is unchanged by induction.     

Tightly bound nucleotides of the energy-transducing ATPase,and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations.2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300 000. An inhibitor protein is bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment.3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one.4. Under de-energised conditions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate ³²P_i. ³²P_i is incorporated into the β and γ positions of the bound nucleotides, but β-labelling probably does not occur on the coupling ATPase.5. Uncouplers inhibit the exchange of the free nucleotides or ³²P_i into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange.6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.     

Author index

The effect of the accumulation of β-galactosides on the uptake of P_i into cells and cell nucleotides was examined in ML strains of Escherichia coli. Nonmetabolizable sulfur analogs of lactose, which are accumulated only in the presence of the product of y gene of the Lac operon, inhibited the uptake of P₁ into whole cells and into cell nucleotides. This inhibition was most pronounced in starved cells, those with a low rate of ATP production. When the cell membrane was disrupted by sonication or detergents, the inhibition was lost. No significant inhibition was seen in y^? strains or in inducible y⁺ strains which were not induced. Hence. inhibition of the uptake of phosphate into nucleotides is dependent on the presence of the product of the y gene and a β-galactoside.A technique using ³²P_i and ³³P₁ was developed for simultaneously measuring the turnover and level of nucleotides. β-Galactosides inhibited ATP synthesis in aerobic cells, but stimulated ATP synthesis in anaerobic cells, indicating that an intermediate of oxidative phosphorylation was the source of energy for β-galactoside accumulation.     

Studies on the Active Transport of Calcium in Human Red Cells

The Ca⁺⁺ transport mechanism in the red cell membrane was studied in resealed ghost cells. It was found that the red cell membrane can transport Ca⁺⁺ from inside the cell into the medium against great concentration gradient ratios. Tracing the movement of ⁴⁵Ca infused inside red cells indicated that over 95% of all Ca⁺⁺ in the cells was transported into media in 20 min incubation under the optimum experimental conditions. The influence of temperature on the rate constant of transport indicated an activation energy of 13,500 cal per mole. The optimum pH range of media for the transport was between 7.5 and 8.5. As energy sources, ATP¹, CTP, and UTP were about equally effective, GTP somewhat less effective, and ITP least effective among the nucleotides tested. The Ca⁺⁺ transport does not appear to involve exchange of Ca⁺⁺ with any monovalent or divalent cations. Also, it is not influenced by oligomycin, sodium azide, or ouabain in high concentrations, which inhibit the Ca⁺⁺ transport in mitochondria or in sarcoplasmic reticulum. In these respects, the Ca⁺⁺ transport mechanism in the red cell membrane is different from those of mitochondria and the sarcoplasmic reticulum.     

Endogenous extracellular purine nucleotides redirect α2-adrenoceptor signaling

Many receptors coupled to inhibitory G_o/G_i-type G proteins often also produce stimulatory signals like Ca²⁺ mobilisation. When expressed in CHO cells the α₂-adrenoceptor subtypes α_2A, α_2B and α_2C mobilised Ca²⁺. These responses were strongly reduced by the P_2Y-purinoceptor antagonist suramin. A large proportion of the total pool of purine nucleotides was found extracellularly. Removal of extracellular nucleotides with apyrase or by constant perfusion had a similar effect as suramin. These treatments did not affect the α₂-adrenoceptor-mediated inhibition of cAMP production. This indicates that cells may be primed or their signaling pathways redirected towards Ca²⁺ mobilisation by `autocrine' release of nucleotides.     

Divergent action mechanism of cAMP and dibutyryl cAMP on cell proliferation and macromolecular synthesis in HeLa S3 cultures

Summary In HeLa S3 cultures, exogenous cAMP and its dibutyryl derivative (DBcAMP) produced divergent effects with respect to glycogen levels as well as to incorporation of labeled precursors into nucleic acids, concentration of nucleotide triphosphates, and the extent of cell proliferation inhibition.The actions of exogenous cAMP were unspecific, as they could be imitated by various adenine nucleotides. Evidence is presented which shows that all effects seen with exogenous cAMP were brought about by adenosine set free continuously by the action of extracellular enzymes.Adenosine, when provided continuously by infusion or enzymically from exogenous adenine nucleotides, caused a depletion of pyrimidine nucleotides and a subsequent inhibition of net nucleic acid formation and of cell proliferation. The increased incorporation rates of (³H)uridine and (³H)thymidine into nucleic acids seen under these conditions were also consequences of the altered precursor pool sizes.Continuous provision of adenosine leading to effective cytostasis could also be achieved in tumor-bearing animals when high doses of adenine nucleotides like cAMP or NAD were injected.DBcAMP was able to imitate intracellular cAMP only after conversion to N⁶-monobutyryl cAMP which accumulated inside the cells and which had a high affinity to a muscle protein kinase. N⁶-MBcAMP also inhibited HeLa cAMP phosphodiesterases (low and high K_m) competitively and to a similar degree as DBcAMP and theophylline. This inhibition, however, does not seem to contribute significantly to the effects of DBcAMP in HeLa cultures.The actions opposite to DBcAMP of exogenous cAMP disappeared when its extracellular degradation was prevented by theophylline or by heat-inactivation of the serum used in the culture medium. Its effects under these conditions, however, were still less pronounced than the alterations caused by DBcAMP.with the assistance of Ulrike FUHRMANN and Barbara WAGENHALS     

Phosphohydrolases and the production of 5′-nucleotides by direct fermentation

A major problem involved in the direct fermentation of nucleotides is their breakdown by phosphohydrolases. Thus, adenine auxotrophs of most microorganisms produce hypoxanthine and/or inosine rather than inosine 5′-monophosphate (IMP) while guanine auxotrophs excrete xanthosine rather than xanthosine 5′-monophosphate (XMP). Examination of a Bacillus subtilis mutant producing hypoxanthine plus inosine revealed at least four phosphohydrolases, three of which could attack nucleotides. Even when the extracellular nucleotide phosphohydrolase was inhibited by Cu⁺² and its surface-bound alkaline phosphohydrolase was repressed and inhibited by inorganic phosphate, or removed by mutation, the breakdown products were still the only products of fermentation. Under these conditions, the third enzyme, a surface-bound non-repressible nucleotide phosphohydrolase was still active. It appears, at least in B. subtilis, that excretion is dependent upon breakdown by this enzyme and if hydrolysis does not occur, excretion of purine nucleotides is feedback inhibited by the resultant high intracellular IMP concentration. Corynebacterium glutamicum mutants, on the other hand, can excrete intact nucleotides, and direct fermentations for IMP, XMP, and GMP have been described. An examination of phosphohydrolases in a GMP-producing culture revealed no extracellular or surface enzymes. Disruption of the cells resulted in liberation of cellular phosphohydrolase activity with a substrate specificity remarkably similar to the flavorenhancing properties of the 5′-nucleotides. The order of decreasing susceptibility was GMP, IMP, XMP; AMP was not attacked.     

The regulation of DNA replication during senescence and rejuvenation of Dunaliella tertiolecta by factors other than available metabolic energy

The unicellular green alga, Dunaliella tertiolecta undergoes a 4-fold reduction in DNA while progressing from early to late log phase of culture. During the period when the reduction in DNA occurs, the cells continue to divide at the maximal rate. Pulse labelling indicates little incorporation of [³H]thymidine into DNA during late log phase. Stationary phase cultures diluted with fresh medium undergo a lag period during which there is a 4-fold increase in DNA and a rapid incorporation of [³H]thymidine into DNA into DNA before division. The evidence indicates that the differences in the levels of DNA are not attributable to tetraploidy, multinucleated cells or a high level of redundancy of G-C-rich satellite DNA in early log phase cells. During stationary phase there is an increase in cellular starch and a decrease in free nucleotides. Electron microscopy reveals that stationary phase cells are distorted by many granules proven to be starch by their susceptibility to amylase treatment. The production of starch by stationary phase cells indicates that DNA replication does not cease because of a deficit of metabolic energy but because of some direct function of culture density.     

INvestigation of the basis of reduced metabolic cooperation in mec- cells.

The basis for the apparent loss of metabolic co-operation in a line of mec^? cells derived by selection from a mec⁺ line has been investigated. The results indicate that the loss of pyrimidine deoxyribonucleotide co-operation in these cells is due neither to the dilution of transferred nucleotides by enlarged endogenous pools nor to the failure of distinct internal pools to equilibrate, but rather to a deficiency of intercellular junctions. This deficiency can be reversed by treatment of the cells with db-cAMP and theophylline which results in the restoration of co-operation for TdR nucleotides. The results also indicate that the residual junctional sites in mec^? cells can transfer TdR nucleotides under conditions where the mec⁺ donor pools are expanded.     

THYMIDINE TRIPHOSPHATE SYNTHESIS IN TETRAHYMENA : I. Studies on Thymidine Kinase

The amount of thymidine-H³ converted to thymidine-H³ monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena.     

Intracellular formation of analogues of cyclic AMP: Studies with brain slices labeled with radioactive derivatives of adenine and adenosine

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N⁶-ethano[¹⁴C]adenosine nor 1,N⁶-ethanol[¹⁴C]adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-³H]adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chlorocyclic AMP was not obtained. N⁶-Benzyl[¹⁴C]adenosine was converted only to intracellular monophosphates and significant formation of radioactive N⁶-benzylcyclic AMP was not detected during a subsequent incubation. 2′-Deoxy-[8-¹⁴C] adenosine was converted to both intracellular radioactive 2′-deoxyadenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2′-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

Purine reutilization in phytohemagglutinin-stimulated human T-lymphocytes.

Incubation of human peripheral blood T-lymphocytes with phytohemagglutinin (PHA) resulted in increased rates of metabolism of the purine bases adenine, hypoxanthine, and guanine. The respective rates decreased to unmeasurable levels in cells incubated without PHA. [¹⁴C]Adenine was converted predominantly into adenine nucleotides, with slight catabolism to hypoxanthine and very low conversion into guanine nucleotides. [¹⁴C]Guanine labeled predominantly the guanine nucleotide pool, but some adenine nucleotide formation also took place. From [¹⁴C]hypoxanthine, adenine nucleotides in the soluble pool were more heavily labeled than the guanine nucleotides, whereas in the nucleic acid fraction the latter contained more radioactivity. Adenosine at low concentrations was mainly phosphorylated to adenine nucleotides, but at higher concentrations this process leveled off, while deamination continued to increase linearly. PHA-stimulation resulted in an increased rate of adenosine metabolism but no qualitative differences in comparison to unstimulated cells were observed. Enzyme assays indicated that after PHA-stimulation the activities of adenine and hypoxanthine phosphoribosyltransferases, and those of adenosine deaminase and kinase, increased with a peak at 48 h, when expressed on a per cell basis, but not at all when expressed per mg of protein. We conclude that stimulation of human T-lymphocytes with PHA increases the capacity of the cells for purine nucleotide synthesis from all the directly re-utilizable catabolic products, namely the purine bases and adenosine.     

Catecholamine binding to CNS adrenergic receptors

The properties of ³H-catecholamine binding to α- and β-adrenergic receptors in CNS are reviewed. ³H-epinephrine and ³H-norepinephrine label one class of α-receptors throughout the brain, with high affinities for agonists and some antagonists. Agonist affinities at this site are increased in low temperature conditions but are reduced by guanine nucleotides and monovalent cations. Divalent cations reverse both effects. This α-receptor may be coupled to adenylate cyclase by GTP and/or sodium, and uncoupled by divalent cations. ³H-epinephrine labels β₂, but not β₁, receptors in CNS, especially in bovine cerebellum. The same β-receptor does not show agonist-specific GTP-sensitivity, but does exhibit Na⁺-sensitivity. This receptor appears to be linked to adenylate cyclase, and sodium rather than GTP may be the coupling agent.     

Failure of opiates to increase the hydrolysis of GTP in neuroblastoma-glioma 108-15 cells

It has been repeatedly demonstrated that the neuroblastoma-glioma (NG 108-15) cell line has opiate receptors that inhibit adenylate cyclase and it has been proposed that this inhibition is mediated by a naloxone reversible stimulation of a low K_m GTPase (Koski and Klee, Proc. Natl. Acad. Sci. 78:4185, 1981). The guanine nucleotides of NG cells were labeled with [³H]guanine followed by incubation with 10^?6M guanine. Etorphine (10^?6M] or vehicle were added and the incubations continued for 1–4 min. The reaction was stopped with 5 percent TCA containing nucleotides as carriers and markers for the HPLC. Marker nucleotides were detected at 254 nm and the labeled nucleotides by liquid scintillation spectrometry. In several experiments, etorphine failed to produce any measurable change in the labeled nucleotides or in the GTP/GDP ratios. To verify that the opiate receptors were functional we measured its capacity to inhibit the formation of cAMP induced by PGE₁. We also studied the effects of naloxone and PGE₁ on the formation of cAMP in opiate tolerant cells. Tolerant cells responded to naloxone with a 50 percent increase in cAMP, indicating again that the opiate receptors were functional. Our results are consistent with the idea that in intact NG108-15 cells the opiate-mediated hydrolysis of GTP observed in cell membrane preparations is of very small magnitude.     

Palindromic Nucleotide Analysis in Human T Cell Receptor Rearrangements

Diversity of T cell receptor (TCR) genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3) of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8⁺ and CD4⁺ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8⁺ naïve T cells. The naïve CD8⁺ T cell clones with P nucleotides are more highly expanded.     

Dietary Nucleotides Increase the Proportion of a TCRγδ+ Subset of Intraepithelial Lymphocytes (IEL) and IL-7 Production by Intestinal Epithelial Cells (IEC); Implications for Modification of Cellular and Molecular Cross-talk between IEL and IEC by Dietary Nucleotides

We have investigated the effects of dietary nucleotides on intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC) in weanling mice. The proportion of T-cell receptor (TCR) γδ⁺ IEL in BALB/c mice fed a diet supplemented with nucleotides (NT(+) diet) was significantly higher than that in mice fed the nucleotide-free diet, while the proportion of TCRαβ⁺ IEL in NT(+) diet-fed mice was significantly decreased. The change of the TCRαβ⁺/TCRγδ⁺ ratio was mainly observed in a CD8αα⁺ subset of IEL. IEC from NT(+) diet-fed mice produced a higher level of IL-7, which is important in the development of TCRγδ⁺ IEL, than those from control diet-fed mice. The expression levels of IL-7 and IL-2 receptors on IEL were not different between the two dietary groups. Our findings suggest that the increased population of a TCRγδ⁺ IEL subset by feeding nucleotides may be caused by the increased production of IL-7 by IEC.