Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.     

Regulation of catecholamine synthesis in rat brain synaptosomes

Abstract— Catecholamine synthesis in synaptosomal preparations of rat striatum, cortex and brain stem was investigated. The striatum had much higher activity than either the cortex or brain stem. Equilibration of labelled tyrosine between tissue and incubation medium was completed within 2 min. The apparent K_m of tyrosine hydroxylase (EC 1.14.3a) and of the overall catecholamine synthetic pathway were both approximately 5 ± 10^?6m for tyrosine. The following amines were found to inhibit striatal dopamine synthesis: dopamine, 25% inhibition at 5 ± 10^?7m ; noradrenaline, 25% inhibition at 5 ± 10^?6m ;and serotonin, 30% inhibition at 10^?5m . The catecholamine-induced inhibition of synthesis was antagonized by pre-incubation with cocaine. Increasing the potassium concentration from 5 to 55 mm caused a release of amines into the medium which was accompanied by a 40% increase in dopamine synthesis, when synthesis was measured during the first 5 min of exposure to elevated potassium. These results indicate that synaptosomal catecholamine synthesis is inhibited by increases in intra-synaptosomal amine levels, and that short-term exposure to depolarizing concentrations of potassium can increase synthesis.     

Reversible inhibition of RNA synthesis and irreversible inhibition of protein synthesis by D-galactosamine in isolated mouse hepatocytes

Summary The inhibition of RNA synthesis of isolated mouse liver parenchymal cells caused by 10 mM D-galactosamine was reversible, while the inhibition of protein synthesis remained unaltered after the removal of galactosamine. 10^–5 M epinephrine and 10^–7 M glucagon have been shown to decrease aminoglycogen formation and thus to reduce the inhibitory effect of galactosamine on protein synthesis (11). However, these hormones did not decrease the inhibition of RNA synthesis. 10 mM D-galactosamine did not effect the nucleoside and amino acid incorporation of isolated non-parenchymal mouse liver cells. The predominant role of aminoglycogen in the inhibition of protein synthesis in galactosamine induced liver injury is discussed.     

Nuclear protein synthesis in regenerating rat liver : Selective histone inhibition by α-amanitin

Nuclear protein synthesis has been studied in regenerating rat hepatocytes after partial hepatectomy and α-amanitin treatment. The toxin induced a marked and precocious inhibition of histone synthesis without affecting the acidic nuclear proteins. This inhibition preceded the inhibition of DNA synthesis. The modification of polyribosome profile and of [¹⁴C]lysine incorporation on synthesized polypeptides were consistent with a reduction of specific mRNAs.     

Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of (¹⁴C)-glycine. In contrast when (¹⁴C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the (¹⁴C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrialδ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.     

Inhibition of protein synthesis by glucose 6-phosphate and fructose 1,6-diphosphate in lysed rabbit reticulocytes and the reversal of inhibition by NAD+.

Glucose 6-phosphate and fructose 1,6-diphosphate inhibit protein synthesis when added to lysed rabbit reticulocytes. Protein synthesis is inhibited 47% with 6 mM fructose 1,6-diphosphate and 86% with 6 mM glucose 6-phosphate. With 0.125 mM NAD⁺, the inhibitory effect of glucose 6-phosphate and fructose 1,6-diphosphate becomes stimulatory. The stimulation of protein synthesis in those assays with NAD⁺ and the phosphorylated sugars is 50% above those assays that contain NAD⁺ alone. The inhibition of protein synthesis by glucose 6-phosphate and the reversal of this inhibition by NAD⁺ occurs at a step before the synthesis of the initial dipeptide, methionyl-valine. These data illustrate the importance of NAD⁺ and the activation of glycolysis in regulating protein synthesis in lysed rabbit reticulocytes.     

METHYL MERCURY INHIBITION OF SYNAPTOSOME AND BRAIN SLICE PROTEIN SYNTHESIS: IN VIVO AND IN VITRO STUDIES

Subacute methyl mercury (MeHg) intoxication was induced in adult rats following the daily intragastric administration of 1 mg MeHg/100 g body weight. Decreased [¹⁴C]leucine incorporation into cerebral and cerebellar slice protein was found. Weight loss occurred during the latent and neurotoxic phases but pair feeding did not reveal a significant defect in amino acid incorporation into slice protein. There was no decline in synaptosome protein synthesis in vitro during the latent phase but a significant decline in cerebellar and cerebral synaptosome synthesis was found during the neurotoxic phase. MeHg in vitro inhibited cerebral slice and synaptosome protein synthesis at half maximal concentrations of 7.5 and 12.5 μM respectively. Inhibition of synthesis in synaptosomes was non-competitive with K₁ of 4 × 10^?6M. MeHg had no effect on [¹⁴C]leucine or [¹⁴C]proline uptake into synaptosomes. There was no significant inhibition of synaptosome basal ATPase or Na + K ATPase at concentrations of MeHg (12 μM) giving half maximal inhibition of protein synthesis. No preferential inhibition of the chloramphenicol (55S) or cycloheximide sensitive components of synaptosome fraction protein synthesis was found, suggesting that the inhibition is common to both mitochondrial and extramitochondrial protein synthesizing systems. Addition of nucleotides and/or atractylate failed to influence protein synthesis and did not reverse the MeHg inhibition. Mannitol, as a replacement for the predominant cation species of the incubation medium, gave 40% inhibition of protein synthesis in the control but protected against further inhibition by MeHg.     

Effect of calcium on synthesis of dipicolinic acid inPenicillium citreoviride and its feedback resistant mutant

Dipicolinic acid synthesis inPenicillium citreoviride strain 3114 was inhibited by Ca²⁺ ions, but not by Ba²⁺, Cu²⁺or Fe²⁺. Among the metals tested, only Zn²⁺ inhibited the synthesis of dipicolinic acid and promoted sporulation. None of these metals reversed the inhibition by Ca²⁺ or Zn²⁺. A mutant 27133-dpa-ca selected for resistance to feedback inhibition by dipicolinic acid: Ca²⁺ complex showed cross-resistance to inhibition by dipicolinic acid: Zn²⁺. Both 3114 and271 33-dpa-ca excreted a number of aliphatic and amino acids during secondary metabolism of dipicolinic acid. In the presence of 1000 ppm of Ca²⁺, accumulation of citric acid and α-aminoadipic acid was completely inhibited under conditions of inhibition of dipicolinic acid in parent strain 3114 but not in the mutant. Citric acid with or without Ca²⁺ did not inhibit thede novo synthesis of dipicolinic acid in the strain 3114. In fact, citric acid in the presence of Ca²⁺ improved significantly rate of dipicolinic acid synthesis. Apart from resistance to feed back inhibition by dipicolinic acid: Ca²⁺ complex, mutant differed from the parent in three other aspectsviz. (i) dipicolinic acid synthesis was not subject to catabolite repression by glucose, (ii) sporulation as well as dipicolinic acid synthesis was dependent on the presence of Ca²⁺ ions in the medium and (iii) Mg²⁺ requirement for the mutant increased three fold. Higher requirement of the Mg²⁺ could be partially relieved by Ca²⁺ during secondary metabolism. The results support the inference thatde novo synthesis of dipicolinic acid is regulated through feedback inhibition by dipicolinic acid: Ca²⁺complex.     

Synthesis of purines in human lymphoblast cells deficient in methylthioadenosine phosphorylase activity

Two human lymphoblastic cell lines, deficient in methylthioadenosine phosphorylase (MTAP) activity, were found to have increased rates of de novo purine synthesis. These MTAP⁻ cell lines were K562, an undifferentiated leukemic line and CCRF-CEM, a leukemic line of T-cell origin. Another T-cell line, CCRF-HSB-2 was found to be deficient in activity. However, this line did not demonstrate elevated rates of purine synthesis. Purine metabolism in the above cell cultures was compared with MTAP⁺ human B-cell lines and two human T-cell lines (MOLT-3 and MOLT-4). In all the MTAP⁺ cell lines, the rate of de novo purine synthesis was inhibited by the presence of methylthioadenosine in the assay medium (10 μM concentration produced more than 90% inhibition). However, purine synthesis in the MTAP⁻ cells was resistant to inhibition by methylthioadenosine. Adenine in the assay medium inhibited de novo purine synthesis in MTAP⁺ and MTAP⁻ cells to a similar degree. This inhibition was dose dependent and was elicited by concentrations similar to those of methylthioadenosine. Growth of the cell lines in culture was not affected by either methylthioadenosine or adenine at the concentrations which produced inhibition of purine synthesis. These results suggest that purine synthesis in MTAP⁺ cells is inhibited by adenine formed from the phosphorolytic cleavage of methylthioadenosine by methylthioadenosine phosphorylase.     

Cd++ inhibits EGF-induced DNA synthesis but not EGF induced myc mRNA accumulation in serum starved NRK-49F cells

Cd⁺⁺ inhibits EGF-induced ³H-thymidine incorporation in serum deprived NRK-49F cells in a dose dependent pattern. The underlying mechanisms for this inhibition are largely unknown. EGF-induced myc mRNA accumulation in NRK-49F cells and the effects of Cd⁺⁺ on this response were examined under conditions that result in partial or complete inhibition of EGF-induced DNA synthesis. It was found that doses of Cd⁺⁺ that inhibit EGF-induced DNA synthesis do not inhibit EGF-induced protein synthesis and myc mRNA accumulation. Cd⁺⁺ doses of 0.5 µM and 1 µM were found actually to increase EGF-induced myc mRNA accumulation and amino acid incorporation. These results show that the effect of Cd⁺⁺ on EGF-induced DNA synthesis is not due to inhibition of entrance into G₁, but rather that Cd⁺⁺ acts on events subsequent to myc accumulation; that is, events associated with either G₁ progression, entry into S or DNA synthesis.Abbreviations EGF Epidermal Growth Factor - ³HTdr Tritium thymidine - MeAIB Methylaminoisobutyrate     

Effect of some pre-treatments on UV-sensitivity and on the course of macromolecular synthesis inEscherichia coli 15 T−, U−, his−

The UV-sensitivity ofEscherichia coli 15 T⁻, U⁻, his⁻ cells after a 45 minutes glucose, thymine uracil, or histidine pre-irradiation starvation, as well as the course of DNA, RNA, and protein synthesis during starvation and during a 60 minute post-treatment in a complete medium were investigated. An increased radioresistance was observed when starvation for some compounds resulted in a consequent inhibition of protein synthesis, as it was observed in the case of glucose, histidine, or uracil starvation. During thymine starvation, which led to a decreased resistance, no inhibition of protein synthesis was recorded. The postirradiation time-course of DNA synthesis did not show any correlation with the increased rate of resistance. The DNA synthesis after U⁻ pre-treatment was greatly delayed, however, after glucose pre-treatment no retardation was observed although both factors increased the rate of surviving cells approximately to the same extent. We assume that the factors which increase the radio-resistance could act by a similar mechanism which would take part in the inhibition of protein synthesis. This mechanism could consist in a decrease of the m-RNA turnover.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

Amiloride inhibits protein synthesis in isolated rat hepatocytes

The effects of amiloride on Na⁺ ion influx, amino acid transport, protein synthesis and RNA synthesis have been studied in isolated rat hepatocytes. The initial rate of ²²Na⁺ uptake and the amount of ²²Na⁺ taken up at later time points were decreased in hepatocytes incubated in the presence of amiloride. Amiloride inhibited by about 25% the influx of α-methylamino[1?¹⁴C]isobutyric acid, a specific substrate for the A (Alanine preferring) system of neutral amino acid transport. By contrast, the activity of system L (Leucine preferring) was not affected by amiloride. Rates of protein synthesis were determined by using high extracellular concentrations of [¹⁴C]valine in order to maintain a constant amino acid precursor pool. Amiloride inhibited protein synthesis by 85% and had no effect on RNA synthesis. Half-maximal inhibition of protein synthesis occurred with amiloride at about 150 μM. In the absence of Na⁺ in the incubation medium, the rate of protein synthesis was reduced by about 35% and no further inhibition was observed with amiloride. These results suggest that in isolated rat hepatocytes protein synthesis is partially dependent on Na⁺, and that amiloride is an efficient inhibitor of protein synthesis.     

Effect of humic acids on the inhibition of pea choline esterase and choline acyltransferase with malathion

Humic acids in a concentration of up to 100 μg ml^-1 affect the activity of choline esterase of pea only weakly during sinapin (choline ester of sinapic acid) hydrolysis. The inhibition is stronger at higher concentrations. A similar course of inhibition with humic acids was also observed during the synthesis of sinapin catalysed with choline acyltransferase present in the same enzyme extract. The organophosphate malathion is a strong inhibitor of both the hydrolysis and the synthesis of sinapin. Thus, for example, at a 3.5 × 10^-5 M concentration it causes 85% inhibition of pea choline esterase. However, in combination with 100 μg of humic acids this inhibition decreases to about 8%. It was observed that humic acids prevent the inhibition of both the hydrolysis and the synthesis of sinapin with malathion even at low concentrations. The interaction of the organophosphate malathion with humic acids seems specific. Humic acids do not affect the inhibition of choline esterase with physostigmine or the inhibition of the hydrolysis and the synthesis of sinapin with chlorocholine chloride, alar and hexamine either. This is also true of the activation of the processes with the K⁺ ions. Humic acids represent competitive inhibitors of choline esterase. Malathion shows an acompetitive — noncompetitive character of the inhibition. The interaction of humic acids with malathion in the roots of the plants may also be assumed in the soil when malathion is applied as an insecticide.     

Hydroxyurea inhibits degradation of internalized epidermal growth factor in HeLa cells

Because epidermal growth factor stimulates DNA synthesis in cultured cells, five inhibitors of DNA synthesis were tested in HeLa cells to see whether the inhibition of DNA synthesis has any effect on the metabolism of the growth factor. Among these, only hydroxyurea inhibited the degradation of ¹²⁵I-labeled epidermal growth factor strongly. The reversal of hydroxyurea-induced inhibition of DNA synthesis by deoxyribonucleosides did not result in a recovery from the inhibition of the degradation. From these findings, it might be concluded that the inhibitory effect of hydroxyurea on the degradation is distinct from that on DNA synthesis.     

Inhibition of protein synthesis elicits early grey crescent formation in the axolotl oocyte

Summary In the axolotl (Ambystoma mexicanum Shaw), it was recently shown that cycloheximide (CH) could induce early grey crescent formation (EGC) in non-activated oocytes, maturing in vitro (Grinfeld and Beetschen 1982). Since it was not proved that EGC was a consequence of protein synthesis inhibition rather than a side-effect of the drug, experiments were performed using microinjections of a quite different inhibitor, diphtheria toxin (DT). This toxin also appeared to elicit EGC. Incorporation of (³H) leucine into oocyte proteins in the presence of increasing DT concentrations (10^–11 to 10^–6 M) was then investigated. The frequency of EGC closely parallels the level of protein synthesis inhibition, which is higher in symmetrized oocytes. The lowest CH concentration which can still elicit EGC also exerts a fairly strong inhibition of (³H) leucine incorporation into proteins. It is concluded that protein synthesis inhibition in the late maturing oocyte actually creates specific conditions which allow cytoplasmic rearrangements to occur, leading to grey crescent formation. These results support the interpretation that (a) proteinic inhibitor (s) of symmetrization could be synthesized in the normal maturing oocyte.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

Inhibition of glucose utilization for acetylcholine synthesis by cerebrospinal fluid.

Abstract— Replacement of bicarbonate-Locke incubation medium with feline CSF reduced [¹⁴C]ACh formation from [U-¹⁴C]glucose by rat brain mince approx 30%. CSF was obtained from a cannula leading to the cisterna magna of freely moving cats. The component of CSF responsible for inhibition was characterized as a dialyzable heat-stable organic anion. Choline acetyltransferase activity was not altered by CSF. [¹⁴C]ACh synthesis and ¹⁴CO₂ production from [U-¹⁴C]glucose but not from [2-¹⁴C]-pyruvate were inhibited by CSF, suggesting inhibition in the metabolism of glucose to pyruvate. The anionic fraction of human CSF was as potent as that from feline CSF in inhibiting ¹⁴CO₂ production from [U-¹⁴C]glucose. Brain hexokinase was inhibited by the anionic fraction of feline CSF. The inhibition was non-competitive with respect to glucose and uncompetitive with respect to ATP. It is suggested that inhibition of hexokinase by CSF was responsible at least in part for the inhibition of glucose metabolism which resulted in decreased [¹⁴C]ACh synthesis and ¹⁴CO₂ production.