Regulation of urea uptake by ammonia in the cyanobacterium Anabaena doliolum

Abstract The urea uptake system was studied with regard to its repression and derepression in the cyanobacterium, Anabaena doliolum . The uptake of urea was energy-dependent and was repressed in ammonia grown cells. Repression of the urea uptake by ammonium did not require ammonium assimilation or de novo protein synthesis, suggesting that ammonium itself was the repressor signal. The derepression of the urea uptake system, however, required de novo protein synthesis and glutamine synthetase activity.     

Glucose transport in a methylotrophic yeast Hansenula polymorpha

Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha . Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions. Glucose-repressed cells exhibited a low-affinity transport system ( K _m for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport system ( K _m for glucose 0.05–0.06 mM). The high- and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide- m -chlorophenyl-hydrazone. The kinetic rearrangement of the glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis.     

Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. II. Effect of the nitrogen source on the nitrite reductase levels

The effect of different inorganic nitrogen sources on the cellular levels of nitrite reductase (NiR. EC 1.7.7.1) activity has been studied in the filamentous non-N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl,). Nitrate-grown cells gave the highest NiR in cell-free extracts [ca 165 nmol of nitrite reduced (mg protein)-1 min-], whereas no activity could be detected in extracts from ammonium-grown cells. The in vivo effect of ammonium on NiR was similar to that exerted by chloramphenicol, suggesting that de novo synthesis of protein was probably repressed by this ion. When ammonium was removed from the culture medium, a rapid increase of de novo synthetized NiR occurred, and the appearance of the enzyme was slightly stimulated by the presence in the medium of either nitrate or nitrite. However, remarkably high levels of NiR [around 1.2 μmol of nitrite reduced (mg protein) ⁻¹min⁻¹] could be routinely measured in nitrogen-deficient cells, indicating that the enzyme was ammonium-repressible rather than nitrate- or nitrite-inducible.     

Purine Nucleotide Synthesis in Adrenal Chromaffin Cells

Abstract: The synthesis of purine nucleotides from the salvage precursors adenine and adenosine, and from the de novo precursors formate and glycine, was studied in isolated adrenal chromaffin cells. Both [8-¹⁴C]adenine and [8-¹⁴C]adenosine from extracellular medium are effectively incorporated into intracellular nucleotides. [¹⁴C]Formate and [U-¹⁴C]glycine are also incorporated, but de novo synthesis is clearly lower than synthesis from salvage precursors, although similar to de novo synthesis in liver. The enzymes responsible for adenine and adenosine salvage, adenine phosphoribosyltransferase and adenosine kinase, were purified about 1,500-fold. Both enzymes are mainly cytosolic and exhibit a similar molecular weight of around 42,000. The results suggest that chromaffin cells can replenish their intracellular nucleotides lost during the secretory event by an active synthesis from salvage and de novo precursors.     

Characterization of ammonium and methylamine transport systems in phototrophic sulfur bacteria

Abstract Transport of ammonium and methylamine into the cells of green sulfur bacterium Chlorobium limicola and purple sulfur bacterium Thiocapsa roseopersicina is carried out by a common transport system. This system has (for C. limicola and T. roseopersicina , respectively) pH optimum 7.0 and 7.5; V _max 0.6 and 4.2 nmol min⁻¹ (mg protein)⁻¹; K_m 5.9 × 10⁻⁵ M and 1.3 × 10⁻⁵ M, and is capable of forming 120- and 600-fold methylamine gradients. The methylamine transport can be energized by the artificially imposed transmembrane K⁺ diffusive potential and is inhibited by tetraphenylphosphonium or valinomycin and K⁺. The data presented indicate that methylamine transport in both studied species is exclusively driven by the membrane potential gradient (ΔΨ).     

Bacteria starved for prolonged periods develop increased protection against lethal temperatures

Abstract A marine Vibrio sp. DW1 and two Escherichia coli strains, K165 ( htpR ⁻) and Sc122 ( htpR ⁺) were submitted to heat stress after different times of starvation. All three bacterial strains developed starvation-mediated cross protection against heat. While two hours ( Vibrio sp. DW1) and 24 hours ( E. coli ) of starvation gave near maximal protection, prolonged periods of non-growth offered increased protection. Chloramphenicol was added, at different times of starvation, to investigate the dependence on de novo protein synthesis for survival after heat stress during prolonged starvation. An obvious de novo protein synthesis mediated induction of protection against heat stress during starvation was not found. Starvation-induced cross protection against heat may be dependent on protein synthesis in the initial phase of starvation while after prolonged starvation the continuous protection offered is suggested not to be mediated by de novo protein synthesis at these times.     

Regulation of glutamine uptake in the cyanobiont Nostoc ANTH

Abstract Glutamine uptake in the cyanobiont Nostoc ANTH was energy-dependent and repressed in ammonia-grown cells. l -Methionine- dl -sulphoximine (MSX), a glutamate analogue and an inhibitor of glutamine synthetase (GS), did not affect glutamine uptake whereas azaserine, an inhibitor of glutamate synthase (GOGAT) did, suggesting that GS activity is not necessarily involved in the glutamine uptake system and that increased intracellular glutamine level regulates its own uptake. Repression of glutamine uptake by ammonia did not require de novo protein synthesis but required GS activity, suggesting that ammonia itself was not the repressor signal. The derepression of the glutamine uptake system did not require GS activity but required de novo protein synthesis.     

Assimilation of nitrate and ammonium by the Scots pine (Pinus sylvestris) seedling under conditions of high nitrogen supply

Seedlings of Scots pine ( Pinus sylvestris L.) were grown on perlite for 21 days under controlled conditions. Apart from the water control, KNO₃ (15 m M ), (NH₄)₂SO₄ (7.5 m M ), and NH₄NO₃ (15 m M ) were offered to study the effects of a high nitrogen supply on nitrogen assimilation. In some experiments 1.3 m M potassium was added to the basic ammonium solutions. In labelling studies nitrate and ammonium were 2.3 atom%¹⁵N-enriched. It was found that over the 21-day period approximately three times more ammonium-N was taken up than nitrate-N. However, nitrate and ammonium, applied simultaneously, were taken up to the same extent as if they were applied separately (additivity). The presence of K⁺ in the medium did not affect N-uptake. Among the soluble N-containing compounds nitrate, ammonium and 8 amino acids were quantified. It was found that assimilation of nitrate can cope with the uptake of NO⁻₃ under all circumstances. Neither free nitrate nor ammonium or amino acids accumulated to an extent exceeding the values of water-grown seedlings. On the other hand, in case of high ammonium supply considerably more nitrogen was taken up than could be incorporated into nonsoluble N-containing substance ('protein'). The remaining nitrogen was found to accumulate in intermediary storage pools (free NH₄⁺, glutamine, asparagine, arginine). Part of this accumulated N could be incorporated into protein when potassium was offered in the nutrient solution. It is concluded that potassium is a requirement for a high rate of protein synthesis not only in crop plants but also in conifers.     

Common nitrogen control of caesium uptake, caesium toxicity and ammonium (methylammonium) uptake in the cyanobacterium Nostoc muscorum

Abstract Studies were carried out to examine the role of ammonium transport activity in the control of caesium uptake and toxicity in Nostoc muscorum . The results showed a definite specific role of the ammonium-repressible/derepressible ammonium transport system of the cyanobacterium in caesium uptake, accumulation and toxicity. Furthermore, the results showed that N. muscorum can acquire resistance against diazotrophically-associated caesium toxicity when supplied with ammonium as a nitrogen source. In addition, alternatively, a mutant strain was Cs-resistant in the absence of any effect on NH₄⁺-transport, suggesting that Cs⁺ resistance may be determined at more than one cellular site.     

Nitrogen fixation by the unicellular cyanobacterium Gloeothece. Nitrogenase synthesis is only transiently repressed by oxygen

Abstract The extent of recovery of nitrogenase activity of Gloeothece transferred from an atmosphere of O₂ to air depended on the duration of exposure to O₂. Activity recovered at increasing rates after up to 24 h exposure to O₂ and a lag before detection of activity, present after short (1 h) exposure times, disappeared with longer exposures. Synthesis of nitrogenase de novo was implicated, since chloramphenicol, tetracycline, or repressive levels of NH⁺₄, prevented recovery of activity. Specific radioimmunoassay of the rate of synthesis of the MoFe protein of nitrogenase under O₂ correlated well with the activity measurements, and indicate that a shift from air to O₂ only transiently represses nitrogenase synthesis.     

Calcium is involved in regulation of the synthesis of HSPs in suspension-cultured sugar beet cells under hyperthermia

Exposure of plants to elevated temperatures induces a complex set of changes that enable plants to adapt following heat stress. In order to test the effect of Ca²⁺ on heat shock-induced changes in cell protein synthesis the incorporation of [ 35 S]methionine into protein was studied in cultured sugar beet ( Beta vulgaris L.) cells incubated in media containing different calcium concentrations. Heat shock inhibited the synthesis of non-heat shock proteins (non-HSPs) and promoted the synthesis of a set of HSPs, typical of plants. The synthesis of non-HSPs was greatly inhibited by external Ca²⁺ removal by treatment of the cells with ethylene glycol-bis( β -aminoethylether)- N,N,N',N'- tetraacetic acid. In contrast, extracellular Ca²⁺ appeared not to be strictly required for the de novo production of HSPs, but this cation exerted different effects on the synthesis of individual HSPs. Cell injury increased if the cells were exposed simultaneously to high temperature and Ca²⁺-deficient medium. Recovery of HSP synthesis and reduced cell injury were observed after addition of exogenous calcium to Ca²⁺-depleted cells. These findings are consistent with a Ca²⁺ requirement for the survival of the cells under heat shock, and likely for the development of cell thermotolerance.     

Progression of programmed cell death in tobacco BY-2 cells is delineated by specific changes in de novo and salvage synthesis of purine nucleotides

Alterations in the pattern of purine nucleotide synthesis and degradation were investigated during programmed cell death (PCD) of tobacco BY-2 cells, induced by a simultaneous increase in the endogenous levels of nitric oxide (NO) and hydrogen peroxide. The de novo synthesis of purine nucleotides was estimated by following the metabolic fate of the [8-¹⁴C]5-aminoimidazole-4-carboxamide-1-β- d -ribofuranoside (AICAR), the salvage synthesis was investigated using [8-¹⁴C]adenine and adenosine, and the degradation pathway was studied by following the incorporation of [8-¹⁴C]inosine. The results indicated that specific changes in purine metabolism occurred during the death programme of tobacco cells. During the early phases of PCD, increases in the salvage activity of adenine and adenosine were observed, and these were related to the high activity of the two major salvage enzymes: adenine phosphoribosyltransferase (APRT) and adenosine kinase (ARK). During the following stages, a large fraction of purine nucleotide was also produced through the de novo pathway, suggesting a tight regulation between salvage and de novo synthesis. These changes were strictly associated with PCD, as they did not occur if NO or hydrogen peroxide was increased individually, or if actinomycin, which inhibits the death programme, was added to the medium in the presence of NO and hydrogen peroxide. These changes in purine nucleotide synthesis represent an early metabolic switch which may be needed to ensure the proper execution of all the high-energy demand processes characteristic of the death programme.     

Effect of acetate on growth and ammonium uptake in the microalga Scenedesmus obliquus

The effect of acetate on growth and rate of ammonium uptake in Scenedesmus obliquus (UTEX 78) was investigated under light-limiting conditions. Addition of acetate to autotrophic cells with a growth constant of 0.71 day⁻¹ resulted in an increase in the growth rate (mixotrophy, k = 1.3 day⁻¹), and in the presence of acetate, growth occurred in the dark (heterophy, k = 0.44 day⁻¹). The rate of ammonium uptake in autotrophy (17.8 amol cell⁻¹ min⁻¹) was similar to that in heterotrophy (17.4 amol cell⁻¹ min⁻¹) but was 3.7 times lower than that in mixotrophy (65.9 amol cell⁻¹ min⁻¹). In general, mixotrophic cells showed optimum ammonium uptake at the acetate concentration at which they were grown. In autotrophy, uptake of ammonium leveled off at about 12.5 μ M while no saturation was observed in mixotrophic cells. An increase in the rate of uptake of ammonium was observed in autotrophic cells within 1 h after the addition of acetate. The activity of isocitrate lyase (EC 4.1.3.1), a key enzyme for the regulation of the glyoxylate cycle responsible for acetate catabolism, showed a 3.9-fold increase in activity after 24 h in the dark in the presence of acetate. The level of isocitrate lyase activity in cells grown for 24 h in the dark in the presence of 0–20 m M acetate also increased as a function of acetate concentration.     

Protein Kinase C-Mediated Down-Regulation of Voltage-Dependent Sodium Channels in Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 12- O -tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), decreased [³H]saxitoxin ([³H]STX) binding in a concentration (IC₅₀ = 19 n M )- and time ( t _1/2 = 4.5 h)-dependent manner. TPA (100 n M for 15 h) lowered the B _max of [³H]STX binding by 53% without altering the K _D value. Phorbol 12,13-dibutyrate (PDBu) also reduced [³H]STX binding, whereas 4α-TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H-7 (an inhibitor of PKC), but not H-89 (an inhibitor of cyclic AMP-dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine-induced ²²Na⁺ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine-induced ²²Na⁺ influx by brevetoxin. These results suggest that an activation of PKC down-regulates the density of Na channels without altering their pharmacological features; this down-regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.     

Cell Position Dependence of Labelling Thymidine Nucleotides Using the De Novo and Salvage Pathways In the Crypt of Small Intestine

Abstract. About twice as much tritiated thymidine ([³H]TdR) is taken up by cells at the bottom of the crypt of the small intestine as by the rapidly cycling mid-crypt cells. However, the uptake of tritiated deoxyuridine ([³H]UdR) is even throughout the crypt.
Exogenous thymidine is incorporated about four times and eight times more efficiently than deoxyuridine by the cells in the mid-crypt and cells at the bottom of the crypt, respectively. However all S phase cells in the crypt appear to be capable of using either precursors, i.e. either the de novo or salvage pathway.
Since methotrexate (1 or 5 mg/kg) inhibits (at 5 mg/kg completely) the uptake of [³H]UdR, but has no effect on [³H]TdR uptake, the de novo and salvage pathways appear to be independent. Within the precision of the methods used in the experiments the 3 hr inhibition of the de novo pathway of deoxythymidylic acid (dTMP) synthesis by methotrexate does not produce any increase in utilization of the salvage pathway measured by incorporation of [³H]TdR into DNA. the increased efficiency of thymidine utilization by crypt base cells is not attributable to (i) differences in accessibility of thymidine; (ii) differences in the rate of DNA synthesis or (iii) the size of the nuclei.     

Characterization of Trypanosoma cruzil-cysteine transport mechanisms and their adaptive regulation

l -Cysteine and methionine are unique amino acids that act as sulfur donors in all organisms. In the specific case of Trypanosomatids, l -cysteine is particularly relevant as a substrate in the synthesis of trypanothione. Although it can be synthesized de novo , l -cysteine is actively transported in Trypanosoma cruzi epimastigote cells. l -Cysteine uptake is highly specific; none of the amino acids assayed yield significant differences in terms of transport rates. l -Cysteine is transported by epimastigote cells with a calculated apparent K _m of 49.5 μM and a V _max of about 13 pmol min⁻¹ per 10⁷ cells. This transport is finely regulated by amino acid starvation, extracellular pH, and between the parasite growth phases. In addition, l -cysteine is incorporated post-translationally into proteins, suggesting its role in iron–sulfur core formation. Finally, the metabolic fates of l -cysteine were predicted in silico .     

Hormonal regulation of barley nuclease: investigation using a monoclonal antibody

Abstract. A monoclonal antibody prepared against barley ( Hordeum vulgare L., cv. Himalaya) nuclease (EC 3.1.30.2) was characterized with solid-state enzyme-linked immunosorbent assays and immuno-blotting. The antibody was specific for intracellular and secreted nuclease. Hormonal regulation of the synthesis and secretion of nuclease in isolated aleurone layers was investigated by immunoprecipitation of biosynthetically-labelled nuclease using polyclonal antibodies and by immunoblot analyses using the monoclonal antibody, respectively. Gibberellic acid (GA₃) induced the de novo synthesis and secretion of nuclease in a time-and concentration-dependent manner. Nuclease was detected in aleurone layers incubated in 1 mmol m⁻³ GA₃, after 24 h. The maximum rates of nuclease synthesis and secretion occurred 36–48 h after hormone treatment. A minimum concentration of 10⁻⁶ mol m⁻³ GA₃ was required for nuclease synthesis and secretion, whereas the maximum rate of nuclease secretion occurred at concentrations of 10⁻⁵ mol m⁻³ and higher. In the presence of abscisic acid, the synthesis and secretion of nuclease from GA₃-treated aleurone layers was almost completely inhibited. Based on these findings, the authors conclude that all nuclease within and secreted from aleurone layers treated with GA₃ is the result of its de novo synthesis.     

Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH-dependent glutamate dehydrogenase

Cell-free extracts of nitrate-grown as well as of ammonium-grown cells of the filamentous non-nitrogen-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate-grown cells was higher than that of ammonium-grown cells, whereas the GDH level was higher in ammonium-grown cells and depended on the external ammonium concentration. When nitrate-grown cells were transferred to an ammonium-containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia-grown cells were transferred to a nitrate-containing medium, an increase of GS and a decrease of GDH-specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate-grown cells were transferred to nitrate medium with L-methionine-DL-sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.