Characterization of lipid-protein interactions in acetylcholinesterase lipoprotein extracted from bovine erythrocytes.

1. The activation of human peripheral blood lymphocytes by phytohaemagglutinin in vitro was accompanied by striking increases in the concentrations of the natural polyamines putrescine, spermidine and spermine. 2. The enhanced accumulation of polyamines could be almost totally abolished by dl-alpha-difluoromethylornithine, a newly discovered irreversible inhibitor of l-ornithine decarboxylase (EC 4.1.1.17), or by methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidene)dinitrilo]diguanidine}, an inhibitor of S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). The inhibition of polyamine accumulation was associated with a marked suppression of DNA synthesis, which was partially or totally reversed by low concentrations of exogenous putrescine, spermidine, spermine and cadaverine and by higher concentrations of 1,3-diaminopropane. 3. In contrast with some earlier studies, we found that methylglyoxal bis(guanylhydrazone), at concentrations that were sufficient to prevent polyamine accumulation, also caused a clear inhibition of protein synthesis in the activated lymphocytes. Similar results were obtained with difluoromethylornithine. The decrease in protein synthesis caused by both compounds preceded the impairment of DNA synthesis. The inhibition of protein synthesis by difluoromethylornithine was fully reversed by exogenous putrescine, spermidine and spermine, and that caused by methylglyoxal bis(guanylhydrazone) by spermidine and spermine. In further support of the idea that the inhibition of protein synthesis by these compounds was related to the polyamine depletion, we found that difluoromethylornithine caused a dose-dependent decrease in the incorporation of [(14)C]leucine into lymphocyte proteins which closely correlated with the decreased concentrations of cellular spermidine. 4. Difluoromethylornithine and methylglyoxal bis(guanylhydrazone) also elicited a variable depression in the incorporation of [(3)H]uridine and [(14)C]adenine into total RNA. The apparent turnover of lymphocyte RNA remained essentially unchanged in spite of severe polyamine depletion brought about by difluoromethylornithine. 5. The present results, as well as confirming the anti-proliferative action of the inhibitors of polyamine biosynthesis, suggest that polyamine depletion may interfere with reactions at different levels of gene expression.     

Leaf pretreatment with senescence retardants as a basis for oat protoplast improvement

Protoplasts obtained from oat leaves floated on buffer for 18hr show high nuclease activity, low rates of incorporation ofamino acids and nucleosides into macromolecules, and high ratesof spontaneous lysis. Addition to the leaf flotation mediumof the senescence retardants cycloheximide or kinetin, of thedibasic amino acids L-lysine or L-arginine, or of the diaminesputrescine or cadaverine reduces the rise in nuclease activityand spontaneous lysis of protoplasts, and increases the rateor extent of presumptive protein and nucleic acid synthesis.The diamines, which also retard chlorophyll degradation in theexcised leaves, appear to act both on the membrane and on systemscontrolling macromolecular synthesis and breakdown. By contrast,the senescence promoter L-serine hastens chlorophyll degradationfrom excised leaves and does not improve protoplasts derivedfrom those leaves. (Received July 4, 1977; )     

Effect of cycloheximide on protein and ribonucleic acid synthesis in cultured human lymphocytes

1. Phytohaemagglutinin stimulates the transformation into blast cells of human lymphocytes incubated in vitro. This transformation is accompanied by an increase in the incorporation of [(14)C]leucine into protein and [(3)H]uridine into RNA. 2. The incorporation of [(14)C]leucine by cultures grown in the presence or absence of phytohaemagglutinin is inhibited to the same extent by cycloheximide, a known inhibitor of protein synthesis. 3. Lymphocytes grown without phytohaemagglutin synthesize mainly non-ribosomal RNA. [(3)H]Uridine incorporation by these cells was increased by cycloheximide. 4. Lymphocytes incubated with phytohaemagglutinin begin to synthesize substantial quantities of ribosomal RNA. Under these conditions [(3)H]uridine incorporation was partially inhibited by cycloheximide. This inhibition is shown to be largely a result of inhibition of the synthesis of ribosomal RNA.     

Synthesis and content of polyamines in bloodstream Trypanosma brucei

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Polyamines in photosynthetic eubacteria and extreme-halophilic archaebacteria

Qualitative and quantitative determinations of polyamines have been done in 4 photosynthetic eubacteria and 6 extreme-halophilic archaebacteria. For comparison, 5 moderate-halophilic eubacteria were also analyzed to determine their polyamine contents. Not only putrescine and spermidine but also homospermidine were found in the photosynthetic eubacteria, especially in the N2-fixing species, Rhodospirillum and Chromatium. Norspermidine, norspermine, and spermine were not detected in the phototrophic eubacteria. No appreciable amount of any polyamine was found in extreme-halophilic archaebacteria, Halobacterium and Halococcus, while moderate-halophilic eubacteria contained quite high concentrations of putrescine and spermidine and cadaverine. When arginine was incubated with cell lysates of these two archaebacteria, appreciable amounts of agmatine were produced; neither putrescine nor cadaverine was formed in the presence of ornithine or lysine. No detectable amount of spermidine was produced by the lysates on incubation with putrescine.     

The plasma membrane of yeast protoplasts exposed to hypotonicity becomes porous but does not disintegrate in the presence of protons or polyvalent cations

Protoplasts of Saccharomyces cerevisiae swelled, lysed and disintegrated when exposed to hypotonic solutions at neutral pH. At pH 4.5 or lower the hypotonically treated protoplasts did not disintegrate and they retained their intracellular proteins, nucleic acids and nucleotides. However, they became leaky for K+ and Ca2+, indicating that pores had been created in the surface membrane, relaxing the osmotic stress. Upon readjustment of pH to neutral, the hypotonically treated protoplasts released the intracellular content and disintegrated. Also, at low pH, protoplasts did not swell in isotonic ammonium acetate and were refractory to the permeabilizing effect of nystatin and to lysis with low concentrations of detergents. Protoplasts were similarly protected against lysis and disintegration by hypotonic treatment or by detergents, even at neutral pH, if the incubation media contained polyvalent cations, especially Zn2+, La3+, spermine, and Ca2+ chelated with EDTA. The protoplasts exposed to hypotonic stress at low pH did not respire and could not regenerate into viable cells. Effects of H+ and polyvalent cations on intramembrane forces acting between molecules of membrane phospholipids are considered along with possible changes in interactions between membrane proteins.     

Influence of diamines and polyamines on the senescence of plant suspension cultures

The diamines putrescine and cadaverine and the polyamines spermine and spermidine inhibited the senescence of nonphotosynthetic cultures of Paul's Scarlet rose. Response was observed when the media of stationary phase cultures was adjusted to either 1 mM of cadaverine or putrescine; or 0.1 μM of either spermine or spermidine along with 2% sucrose in all cases. Senescence of the cultures was followed by microscopic examination of cell aliquots removed at 10 day intervals and treated with the vital stain, fluorescein diacetate.     

Control of development of amylolytic and proteolytic activities in cotyledons of germinating black gram seeds

The effect of polyamines and related metabolites on several parameters of leaf senescence was followed in detached radish ( Raphanus sativus L. var. radicular cv. "Giant Butter") leaves floated on test solutions in darkness. Leaf senescence was accompanied by a marked loss of chlorophyll, which started at 24–48 h of incubation. The polyamines, spermine and spermidine, and the diamines, putrescine and cadaverine, were highly effective in arresting chlorophyll loss over a period of at least 96 h. l -arginine, and especially l -ornithine, were less active. Polyaminens prevented the marked chlorophyll loss in dark-incubated leaves, but did not compensate for the moderate chlorophyll loss when the leaves were aged in light. Polyamines were also highly effective in retarding earlier events of leaf senescence, prior to chlorophyll loss: both protein degradation and ribonuclease activity were inhibited by spermidine. Chlorophyll and protein loss in dark-or light-incubated suspensions of either "intact" or disrupted chloroplasts was not affected by polyamines. – It is concluded that polyamines are highly effective in preventing chlorophyll loss from detached leaves, possibly by controlling early senescence-linked events which occur in darkness rather than by direct inhibition of chlorophyll degradation.     

Transport of putrescine across duodenal,jejunal and ileal brush-border membrane of chicks (Gallus domesticus)

Luminal polyamines and their absorption are essential for proliferation of the enterocytes and, therefore, nutrition, health and development of the animal. The transport systems that facilitate the uptake of putrescine were characterized in chick duodenal, jejunal and ileal brush-border membrane vesicles prepared by MgCl2 precipitation from three-week-old chicks. An inwardly-directed Na+ gradient did not stimulate putrescine uptake and, therefore, putrescine transport in chick intestine. In the duodenum, jejunum and ileum, kinetics of putrescine transport fitted a model with a single affinity component plus a non-saturable component. The affinity (Kt) for [3H]putrescine transport across the brush-border membrane increased along the length of the small intestine. A model of intermediate affinity converged to the data obtained for [3H]putrescine transport with Kt approximating 1.07 and 1.05 mM or duodenum and jejunum, respectively; and high affinity with a Kt of 0.35 mM for the ileum. The polyamines cadaverine, putrescine, spermidine and spermine strongly inhibited the uptake of [3H]putrescine into chick brush-border membrane vesicles, more so for the jejunum and ileum than the duodenum. The kinetics of cadaverine, spermidine and spermine inhibition are suggestive of competitive inhibition of putrescine transport. These uptake data indicate that a single-affinity system facilitates the intestinal transport of putrescine in the chick; and the affinity of transporter for putrescine is higher in the ileum than in the proximal sections of the small intestine. In addition, this study shows that the ileum of chicks plays an important role in regulating cellular putrescine concentration.     

Polyamine oxidase in oat leaves: a cell wall-localized enzyme

The localization and activity of polyamine oxidase (PAO; EC 1.5.3.3), was investigated in leaves and protoplasts of oat seedlings. Activity of the enzyme is highest with spermine as substrate; spermidine is also oxidized, but putrescine and cadaverine are unaffected by the enzyme. Protoplasts isolated following digestion of leaves with cellulase in hypertonic osmoticum showed no PAO activity, and about 80% of the total leaf PAO activity could be accounted for in the cell wall debris. Histochemical localization experiments showed intense PAO activity in guard cells and in vascular elements whose walls are not digested by cellulase. When protoplasts were cultured in a medium suitable for regeneration of cell wall, PAO activity could be detected as the cellulose wall developed. Thus, PAO appears to be localized in cell walls.     

Ribonucleic acid synthesis in vitro in primary spermatocytes isolated from rat testis

The incorporation of [(3)H]uridine into RNA was studied quantitatively (by incorporation of [(3)H]uridine into acid-precipitable material) and qualitatively (by phenol extraction and electrophoretic separation of RNA in polyacrylamide gels) in preparations enriched in primary spermatocytes, obtained from testes of rats 26 or 32 days old. The rate of incorporation of [(3)H]uridine into RNA of isolated spermatocytes was constant during the first 8h of incubation, after which it decreased, but the decreased rate of incorporation was not reflected in a marked change in electrophoretic profiles of labelled RNA. In isolated spermatocytes, [(3)H]uridine was incorporated mainly into heterogeneous RNA with a low electrophoretic mobility. Most of this RNA was labile, as shown when further RNA synthesis was inhibited with actinomycin D. Spermatocytes in vivo also synthesized heterogeneous RNA with a low electrophoretic mobility. A low rate of incorporation of [(3)H]uridine into rRNA of isolated spermatocytes was observed. The cleavage of 32S precursor rRNA to 28S rRNA was probably retarded in spermatocytes in vitro as well as in vivo. RNA synthesis by preparations enriched in early spermatids or Sertoli cells was qualitatatively different from RNA synthesis by the spermatocyte preparations. It is concluded that isolated primary spermatocytes maintain a specific pattern of RNA synthesis, which resembles RNA synthesis in spermatocytes in vivo. Therefore isolated spermatocytes of the rat can be used for studying the possible regulation of RNA synthesis during the meiotic prophase.     

Synthesis and Content of Polyamines in Bloodstream Trypanosoma brucei*

SYNOPSIS. The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [³H]ornithine, [¹⁴C]arginine and [¹⁴C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [¹⁴C]methionine. Putrescine and spermidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous ¹⁴C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Lactose utilization in a cryptic strainEscherichia coli ML 35

The permease-negative strainE. coli ML 35 utilized lactose at 30°C 5–6 times more slowly than glucose or permease-positive strains. The rate of utilization could be raised by cultivating the cells in the presence of streptomycin, acriflavine or actinomycin C. Phenethyl alcohol and the basic proteins ribonuclease, protamine or histone stimulated lactose utilization by washed cells of the same strain not precultivated in the presence of these substances. Ribonuclease did not influence lactose utilization by the permease-positive strain ML 308. Under the given experimental conditions, the presence of basic proteins did not cause β-galactosidase to be released into the medium as a result of lysis of the cells. Streptomycin and ribonuclease did not influence TMG transport into the cells. Basic proteins increased the rate of ONPG hydrolysis by intact cells almost to the level observed in the permease-positive strain. Guanidine, spermine, spermidine, putrescine and cadaverine did not influence lactose utilization by strain ML 35. Spermine antagonized the stimulant effect of ribonuclease or histone on lactose utilization. The experiments were supplemented by comparing the effect of temperature on lactose and glucose utilization by a permease-positive and-negative strain ofE. coli.     

Transport and subcellular localization of polyamines in carrot protoplasts and vacuoles

Putrescine and spermidine uptake in carrot (Daucus carota L., cv “Tip top”) protoplasts and isolated vacuoles was studied. Protoplasts and vacuoles accumulated polyamines very quickly, with maximum absorption within 1 to 2 minutes. The insertion of a washing layer containing 100 millimolar unlabeled putrescine or spermidine did not change this pattern, but strongly reduced the uptake of putrescine and spermidine in protoplasts and in vacuoles. The dependence of spermidine uptake on the external concentration was linear up to the highest concentrations tested in protoplasts, while that in vacuoles showed saturation kinetics below 1 millimolar (K_m = 61.8 micromolar) and a linear component from 1 to 50 millimolar. Spermidine uptake in protoplasts increased linearly between pH 5.5 and 7.0, while there was a distinct optimum at pH 7.0 for vacuoles. Preincubation of protoplasts with 1 millimolar Ca²⁺ affected only surface binding but not transport into the cells. Nonpermeant polycations such as La³⁺ and polylysine inhibited spermidine uptake into protoplasts. Compartmentation studies showed that putrescine and spermidine were partly vacuolar in location and that exogenously applied spermidine could be recovered inside the cells. The characteristics of the protoplast and vacuolar uptake system induce us to put forward the hypothesis of a passive influx of polyamines through the plasmalemma and of the presence of a carrier-mediated transport system localized in the tonoplast.     

Human mononuclear leukocyte transglutaminase activity is enhanced by streptococcal erythrogenic toxin and a staphylococcal mitogenic factor associated with toxic shock syndrome

Transglutaminase activity in human peripheral lymphocytes is enhanced after incubation of the cells with concanavalin A. Streptococcal proliferative factor toxin (erythrogenic toxin) from Streptococcus pyogenes and Toxic shock syndrome toxin from Staphylococcus aureus were purified and tested for their ability to enhance transglutaminase activity. Mononuclear leukocyte transglutaminase activity was enhanced 3–5-fold 30 min after incubation with either toxin. Enhancement occurred only when toxin was incubated with intact cells; addition of toxin to cell lysates was without effect. Transglutaminase was not measurable extracellularly. Histamine and dansyl cadaverine, competitive substrates for transglutaminase, inhibited [³H]putrescine incoporation into casein and [³H]thymidine incorporation into DNA. Incubation of lymphocytes with cycloheximide and either toxin or concanavalin A did not inhibit enzyme activity. These bacterial toxins, like phytomitogens, may perturb the cellular membrane and mediate their effect by transglutaminase-mediated cross-linking of membrane proteins.     

Liposome-Mediated transfer of bacterial RNA into carrot protoplasts

The uptake of liposome-encapsulated E. coli [³H]RNA by carrot (Daucus carota L.) protoplasts was examined. [³H]RNA extracted from protoplasts that had been incubated with [³H]RNA-containing, large, unilamellar lipid vesicles (liposomes) obtained by ether infusion, and examined by sucrose gradient centrifugation and formamide-polyacrylamide gel electrophoresis, appeared substantially degraded, with a total elimination of 23S RNA and a partial loss of 16S RNA. In contrast, no breakdown of the [³H]RNA was apparent in the liposomes after sequestration, even in the presence of externally added ribonuclease, or in the unfused liposomes remaining after incubation of protoplasts with liposomes. Thus, the degradation of the [³H]RNA extracted from the protoplasts must have occurred within the protoplasts and represents evidence for liposome-mediated RNA uptake. Naked RNA added to the protoplast culture was found to be totally degraded after incubation with the protoplasts. The uptake of liposome-sequestered RNA by protoplasts was demonstrated to be a function both of the lipid composition of the liposomal membrane and of the temperature of incubation of the liposomeprotoplast mixture. Furthermore, the mode of this uptake (fusion versus endocytosis) could be manipulated by adjusting the cholesterol content of the liposomal membrane. The implications of the ability to insert RNA into protoplasts without degradation by extracellular nucleases are discussed.     

Reversal of the deoxyhypusine synthesis reaction. Generation of spermidine or homospermidine from deoxyhypusine by deoxyhypusine synthase

Deoxyhypusine synthase catalyzes the first step in hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) synthesis in a single cellular protein, eIF5A precursor. The synthesis of deoxyhypusine catalyzed by this enzyme involves transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein to form a deoxyhypusine-containing eIF5A intermediate, eIF5A(Dhp). We recently discovered the efficient reversal of deoxyhypusine synthesis. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD, and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD, and putrescine. Thus it was found that [14C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The Km value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 microM). Using [14C]putrescine as an acceptor, various spermidine analogs were evaluated as donor substrates for human deoxyhypusine synthase. Comparison of spermidine analogs as inhibitors of deoxyhypusine synthesis, as donor substrates for synthesis of deoxyhypusine (or its analog), and for synthesis of homospermidine (or its analog) provides new insights into the intricate specificity of this enzyme and versatility of the deoxyhypusine synthase reaction.     

Localization and biosynthesis of polyamines in insulin-producing cells.

Two recently developed fluorescence cytochemical methods, specific for spermidine and spermine, were used to localize polyamines in the endocrine pancreas. The polyamines were restricted to the insulin-producing beta-cells and were mainly associated with the secretory granules. Chemical polyamine determinations carried out on isolated rat and mouse pancreatic islets revealed large amounts of polyamines. Compared with extracts of whole pancreas, the islets contained very high concentrations of spermine relative to spermidine. Biosynthesis of polyamines from [3H]ornithine or from [3H]putrescine in isolated islets was significantly stimulated at high glucose concentrations. Moreover, significant incorporation of label from [3H]putrescine was also detected in gamma-aminobutyric acid. This incorporation, however, was not stimulated by high glucose. Possible roles for polyamines associated with the secretory granules in insulin-producing cells are discussed.     

Chemically defined media for growing anaerobic bacteria of the genus Veillonella

A chemically defined medium for Veillonella parvula and V. alcalescens is described. Some nutritional aspects of the two strains used were examined: the optimum concentration of reducing agents, the requirement for amino acids, diamines, vitamins and other growth factors, and the conditions needed for well balanced nutrition.No specific requirements for single amino acids were observed. A combination of l-cysteine, dl-aspartic acid, l-glutamic acid, l-serine and l-tyrosine, promoted growth. In V. alcalescens, serine could substitute both arginine and tryptophan (or histidine). No growth was obtained with ammonium salts as the sole N source.Decarboxylation of l-ornithine, l-lysine and l-arginine was not demonstrated in the Veillonella parvula strain, which required putrescine or cadaverine for growth. Spermine, spermidine, l-lysine, l-ornithine and l-arginine, could not substitute putrescine in Veillonella parvula. Veillonella alcalescens, which does not require putrescine in the medium, was able to decarboxylate l-ornithine while forming putrescine.     

Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunts label from 3,4-[C]methionine into spermidine in aged orange peel discs

The flux of radioactivity from 3,4-[(14)C]methionine into S-adenosyl-l-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC), spermine, and spermidine while inhibiting conversion of ACC to ethylene by 100 millimolar phosphate and 2 millimolar Co(2+) was studied in aged peel discs of orange (Citrus sinensis L. Osbeck) fruit. Inhibition up to 80% of ethylene production by phosphate and cobalt was accompanied by a 3.3 times increase of label in ACC while the radioactivity in SAM was only slightly reduced. Aminoethoxyvinylglycine (AVG) increased the label in SAM by 61% and reduced it in ACC by 47%. Different combinations of standard solution, in which putrescine or spermidine were administered alone or with AVG, demonstrated clearly that inhibition of ethylene biosynthesis-at the conversion of SAM to ACC-by AVG, exogenous putrescine or exogenous spermidine, stimulated the incorporation of 3,4-[(14)C]methionine into spermidine.