Polyamines and terminal deoxynucleotidyl transferase expression In KM 3 pre-B cell line during phorbol ester induced differentiation.

The aliphatic polyamines, putrescine, spermine and spermidine belong to a category of molecules implicated in DNA replication. Their synthesis is strongly activated during the G₁ period and they have been implicated in the regulation of cell proliferation and differentiation. Terminal transferase is a DNA polymerase present in pre-T and pre-B cells and its expression can be modulated by phorbol ester treatment. In this study we have monitored the relationship of intracellular polyamine levels with terminal deoxynucleotidyl transferase down-regulation induced by 12-0-tetradecanoyl phorbol myristate 13-acetate treatment in the human pre-B KM-3 cell line. Phorbol myristate acetate can cause an increase, at 4 and 8 hours of differentiation, of intracellular levels of putrescine as well as a decrease in terminal deoxynucleotidyl transferase synthesis showing the probable involvement that polyamines have in the differentiation process.     

Dependence of protective functions of <Emphasis Type="Italic">Escherichia coli</Emphasis> polyamines on strength of stress caused by superoxide radicals

Mechanisms of antioxidant effect of polyamines were studied in dependence on the strength of superoxide stress. Under conditions of weak stress, polyamines from Escherichia coli cultures were shown to function mainly as a scavenger of free superoxide radicals, whereas under conditions of strong stress they mainly acted as positive modulators of antioxidant genes. Spectrofluorimetry was used to show that both polyamine-dependent mutants and wild type cells treated with inhibitors of polyamine synthesis contained an elevated amount of free oxygen radicals, which could be decreased to the normal level by addition of exogenous polyamines. Under conditions of strong stress, polyamines positively influenced expression of the soxRS regulon genes of antioxidant defense, which was accompanied by an increase in the quantity (activity) of their gene products, such as glucose-6-phosphate dehydrogenase (Zwf) and fumarase (FumC). These effects led to an increase in the number of live cells in the cultures subjected to superoxide stress.     

Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme (AZ). The synthesis of mammalian AZ was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. We identified a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian AZ. We show that synthesis of yeast AZ (Oaz1) involves polyamine-regulated frameshifting as well. Degradation of yeast ODC by the proteasome depends on Oaz1. Using this novel model system for polyamine regulation, we discovered another level of its control. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is inhibited by polyamines. We propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation.     

In vitro studies on the entry of polyamines into normal red blood cells

Polyamines are mainly transported in the blood by erythrocytes: Putrescine, spermidine and spermine can be taken up in vitro by red blood cells (RBC); their entry is greater in the presence of serum than in the presence of plasma, and spermine entry is lower than that observed for the two other polyamines. In the presence of serum, the affinity of RBC for spermidine is 30 fold greater than that for putrescine. The majority of RBC polyamines are present in the hemolysate and are not complexed to high molecular weight material. At + 4 degrees C the polyamine uptake is considerably reduced and for putrescine and spermine practically non existent, but it seems that it is internalization rather than binding which constitutes the dependent step. Though intracellular spermidine and spermine levels reflect differences in uptake rather than in outward flux across the cell membrane, the values of putrescine appear to be the resultant of influx and efflux. The presence of specific receptor sites for polyamines visualized by SEM on the surface of RBC using latex-putrescine spheres, confirms the results obtained with labelled polyamines. Therefore, only the understanding of the polyamine repartition inside the blood compartments would permit the clinical use of those molecules as non statistical tumor markers.     

Polyamines, storage substances and abscisic acid-like inhibitors during dormancy and very early activation of Helianthus tuberosus tuber tissues

Abstract Dormancy and break of dormancy of tubers of Helianthus tuberosus L. (Jerusalem artichoke) have been investigated in regard to the possibility that polyamines can control these processes. Polyamines were detected by the method of direct dansylation and abscisic acid was bioassayed using wheat coleoptile growth test. Arginine and glutamine, which are the main store nitrogenous organic compounds of Helianthus tuberosus tuber, decrease during the last phase of dormancy as well as abscisic acid; moreover the corresponding increase in polyamines (putrescine, spermidine and spermine) seems to be strictly related to the break of dormancy of tuber. The artificial break of dormancy induced by 2,4-dichlorophenoxyacetic acid stimulates a great increase in polyamines, just evident within 15 min after activation, and a corresponding decrease in arginine and glutamine. The levels of polyamines, at 1 h of activation are sufficient to stimulate protein synthesis in vitro.     

Plastid‐associated polyamines: their role in differentiation,structure, functioning,stress response and senescence

Polyamines are low‐molecular weight biogenic amines. They are a specific group of cell growth and development regulators. In the past decade biochemical, molecular and genetic studies have contributed much to a better understanding of the biological role of polyamines in the plant cell. Substantial evidence has also been added to our understanding of the role of polyamines in plastid development. In developing chloroplasts, polyamines serve as a nitrogen source for protein and chlorophyll synthesis. In chloroplast structure, thylakoid proteins linked to polyamines belong mainly to antenna proteins of light‐harvesting chlorophyll a/b–protein complexes. The fact that LHCII oligomeric forms are much more intensely labelled by polyamines, in comparison to monomeric forms, suggests that polyamines participate in oligomer stabilisation. In plastid metabolism, polyamines modulate effectiveness of photosynthesis. The role of polyamines in mature chloroplasts is also related to the photo‐adaptation of the photosynthetic apparatus to low and high light intensity and its response to environmental stress. The occurrence of polyamines and enzymes participating in their metabolism at every stage of plastid development indicates that polyamines play a role in plastid differentiation, structure, functioning and senescence.     

Ornithine decarboxylase transgene in tobacco affects polyamines,in vitro-morphogenesis and response to salt stress

Transgenic tobacco plants expressing the putrescine synthesis gene ornithine decarboxylase from mouse were raised to study the effects of up-regulation of a metabolic pathway as critical as the polyamine biosynthesis on the plant growth and development, in vitro-morphogenesis and their response to salt stress. Further, the response of the alternate pathway (arginine decarboxylase) for putrescine synthesis to the modulation of the ornithine decarboxylase pathway has also been investigated. The over-expression of the odc gene and increased levels of putrescine in tobacco led to a delay in plant regeneration on selection medium which could be overcome by the exogenous application of polyamine biosynthesis inhibitors and spermidine. Further, the lines generated had a variable in vitro morphogenic potential, which could be correlated to the shifts in their polyamine metabolism. These studies have brought forward the critical role played by polyamines in the normal development of plants and also their role in plant regeneration. Since polyamines are known to accumulate in cells under abiotic stress conditions, the tolerance of the transgenics to salt stress was also investigated and the transgenics with their polyamine metabolism up-graded showed increased tolerance to salt stress.     

Physiological polyamines: simple primordial stress molecules

Physiological polyamines are ubiquitous polycations with pleiotropic biochemical activities, including regulation of gene expression, cell proliferation and modulation of cell signalling. Reports that the polyamines with cytoprotective activities were induced by diverse stresses raised the hypothesis that physiological polyamines may play a role in inducing stress response. In a wide range of organisms, physiological polyamines were not only induced by diverse stresses, such as reactive oxygen species (ROS), heat, ultraviolet (UV) and psychiatric stress but were able to confer beneficial effects for survival. Recent biochemical and genetic evidences show that polyamines can function as an ROS scavenger, acid tolerance factor and chemical chaperone, and positive regulators for expression of stress response genes which may explain their protective functions against diverse stresses. Taken together, these data suggest that physiological polyamines can function as primordial stress molecules in bacteria, plants and mammals, and may play an essential role in regulation of pathogen-host interactions.     

Generation of large insert yeast artificial chromosome libraries

The development of YAC cloning technology has directly enhanced the relationship among genetic, physical, and functional mapping of genomes. Because of their large size, YACs have enabled the rapid construction of physical maps by ordered clone mapping and contig building, and they complement other molecular approaches for mapping complex genomes. Large insert libraries are constructted by size fractionating large DNA embedded in agarose and protecting DNA from degradation with polyamines.     

Amelioration in secretion of hyperthermostable and Ca2+ -independent alpha-amylase of Geobacillus thermoleovorans by some polyamines and their biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone

AIM: Effect of polyamines and their biosynthesis inhibitors on the production of hyperthermostable and Ca2+ -independent alpha-amylase by Geobacillus thermoleovorans MTCC 4220. METHODS AND RESULTS: The alpha-amylase was produced in starch-yeast extract-tryptone (SYT) broth with different polyamines (PA) and polyamine biosynthesis inhibitors, methylglyoxal-bis-guanylhydrazone (MGBG) and cyclohexylammonium sulphate (CHA) at 70 degrees C. The bacterial pellets were obtained after growing G. thermoleovorans at different temperatures, and used in determining total PA. The cell-free culture filtrates were used in alpha-amylase assays. During growth, total polyamines in biomass increased till 2 h, and thereafter, decreased gradually. The total polyamine content was very high in the biomass cultivated at 55 degrees C when compared with that of higher temperatures. Enzyme titre enhanced up to 70 degrees C, and thereafter declined. Extracellular enzyme and protein levels declined in the presence of exogenously added PA. The intracellular enzyme titres, however, were higher in putrescine (put) and spermidine (spd) than in spermine (spm). Polyamine biosynthesis inhibitor, MGBG enhanced secretion of alpha-amylase in a laboratory fermentor as well as shake flasks, although CHA did not affect it. CONCLUSIONS: The intracellular accumulation of put in the presence of MGBG appeared to enhance synthesis and secretion of alpha-amylase. Extracellular enzyme and protein levels were low in the presence of exogenously added PA, but their intracellular levels, however, were higher in put and spd than in spm. SIGNIFICANCE AND IMPACT OF THE STUDY: A substantial increase in the synthesis and secretion of alpha-amylase was attained in G. thermoleovorans in the presence of polyamine biosynthesis inhibitor MGBG.     

Hormonal control of proliferation in meristematic agglomerates of Eucalyptus camaldulensis Dehn

Summary Eucalyptus camaldulensis can be micropropagated through so-called meristematic agglomerates (MAs). MAs (4–6 mm diameter) are dense shoot clusters initiated by the outgrowth of numerous successive buds. Their reddish nature is associated with an increase in their endogeneous cytokinin level during the exponential phase of growth. A simultaneous decrease in the auxin level favors a high cytokinin/auxin ratio. A low level of polyamines occurs at the time of the lowest level of auxins. Slow hormone release by activated charcoal plays a role in this very prolific organogenesis.     

The role of calcium, polyamines and centrosomes in the formation and organization of cleavage furrows in amphibian eggs.

Cleavage furrows of amphibian eggs exhibit characteristic morphological features: the presence of finger-like microvilli (MV) along their outer edges, the formation of furrow walls from new plasma membrane lacking MV, and the subsequent retrieval of this membrane during the infolding of the furrow. A similar structure can be induced, specifically, by certain cytoplasmic components such as centrosomes, polyamines and calcium. Their respective roles in the events associated with the furrowing process have been investigated by injecting these agents into nucleated and enucleated Pleurodeles eggs and evaluating their effects using cytochemical labelling of the egg surface with a biotin-streptavidin system. The injection of polyamines (spermine or spermidine) and in some cases, calcium into enucleated eggs provoked MV elongation and the appearance of newly formed, smooth plasma membrane. In these eggs, this membrane was not incorporated into the furrows, and as a consequence, the blastomeres did not actually separate. In contrast, the injection of centrosomes into enucleated eggs induced both the incorporation and internalization of new membrane, resulting in the formation of furrows and a true cellularization of the eggs, identical to the cleavage process observed in fertilized eggs. The present results provide further evidence that the establishment of the furrow depends on two complementary interacting systems: the contractile elements of the egg cortex which regulate the insertion of new membrane and the mitotic center which is essential for the invagination of the furrow.     

植物多胺代谢途径研究进展

多胺是一类小分子生物活性物质,广泛存在于生物体内,与植物的生长发育、衰老及抗逆性都有着密切的联系。目前,在植物中的多胺合成途径已经基本揭示,其生理作用在分子水平上逐步得到阐明。对多胺合成突变体和各种转基因植物的研究也使得人们更深入地了解了多胺以及其合成代谢相关酶在植物生长发育等生理过程中的重要作用。以下概述了植物多胺代谢途径,重点综述了代谢途径中各基因的功能及遗传操作的最新进展,并对将来的研究方向尤其是相关基因在植物抗逆境 (包括生物和非生物逆境) 基因工程方面的应用作了讨论。     

NaCl处理下大麦根系质膜微囊结合多胺与Na+/H+逆向运输的关系

NaCl10 0mmol/L处理结合外施Spd和Put以及多胺代谢抑制剂邻二氮杂菲和MGBG ,以改变大麦根系质膜结合多胺种类和数量 ,研究了大麦根系质膜上两种形态多胺与质子泵和Na /H 逆向运输活性的关系。结果发现 ,NaCl处理后大麦根系质膜微囊上存在Na /H 逆向运输活性。质膜H ATPase活性与膜上非共价键结合多胺数量间呈显著正相关 ,其中 ,Spd对H ATPase的激活程度大于Put。膜蛋白上共价键结合多胺数量与Na /H 逆向运输活性间呈极显著正相关关系 ,说明大麦根系质膜Na /H 逆向运输的盐诱导似乎与Na /H 逆向运输蛋白的从头合成有关。此外 ,质膜Na /H 逆向运输活性仅与膜蛋白上共价键结合多胺数量有关 ,而与多胺种类关系不大。     

Interactions of polyamines and nitrogen nutrition in plants

Biogenic amines occupy an important position among the many nitrogenous plant compounds. Polyamines are part of the overall metabolism of nitrogenous compounds, yet they do not seem to function in the 'normal' nitrogen nutrition. Rather, these widespread polycations (e. g. putrescine, spermidine and spermine) are involved in the regulation of growth and stress, probably by binding to negatively charged macromolecules. In addition, some diamines and polyamines are metabolized to yield 'secondary 'metabolites such as nicotine and other alkaloids. Previous studies have indicated that the ratio of nitrate to ammonium nutrition affects polyamine biosynthesis and content in intact plants. Thus, an increase in putrescine accumulation was found under conditions of excess ammonium ions, relative to nitrate. Modifications of nitrogen sources in the culture medium of tobacco cell suspensions (depletion of ammonium nitrate, or potassium nitrate, or both) resulted in marked changes in the content of cellular free polyamines. Considerable changes in the content of specific polyamines were also found with exposure to specific inhibitors of polyamine biosynthesis (difluoromethyl ornithine, difluoromethyl arginine, cyclohexylamine, methylglyoxal-bis-guanylhydrazone). However, a combination of nitrogen depletion of the medium and some inhibitors resulted in a very marked over-production of spermidine and spermine. The significance of these findings is discussed in relation to the assumption that polyamines act as a metabolic buffer, and maintain cellular pH under conditions where ammonium assimilation produces an excess of protons.     

Association of polyamines to different parts of various plant species

The variation in polyamine content in different plant species and in different parts within a plant can be considerable. To get general information about levels of polyamines in plants and about the association of polyamines to different types of tissue, 30 plants from 13 plant families were examined for their polyamine content before and after germination using high pressure liquid chromatography (HPLC) analysis. A marked increase in polyamine content occurs in the cotyledons or endosperms in the seeds on germination, i.e. in the nutrient storing and exporting part of the plant. In the radicle, hypocotyl or coleoptile, i.e. growing parts of the plant, an increase in polyamine content is rarely observed. Additionally, polyamine levels can be very low [below 1 nmol (g fresh weight)^-1] in different parts of various species. Obviously, levels in the pmol (g fresh weight)^-1 range satisfy the needs of many growing plant parts. The high levels of polyamines found especially in cotyledons cannot be explained by their postulated association with increased cell division rates.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by polyamines

The influence of polyamines (putrescine, spermidine, and spermine) on the activity of human platelet soluble guanylate cyclase and the stimulation of the enzyme by sodium nitroprusside (SNP), YC-1 and their combination was investigated. All these polyamines stimulated the guanylate cyclase activity and potentiated its activation by sodium nitroprusside. The stimulatory effects of sodium nitroprusside and putrescine (or spermine) were addidive; spermidine produced a synergistic activation and increased the additive effect. All the polyamines inhibited the enzyme activation by YC-1 and decreased the synergistic activation of SNP-stimulated guanylate cyclase activity by YC-1 with nearly the same potency. The ability of the investigated polyamines to potentiate and to increase synergistically (similar to to YC-1, but less effective) NO-dependent activation of soluble guanylate cyclase represents a new biochemical effect of these compounds; this effect should be taken into consideration, especially due to the endogenous nature of polyamines. The data obtained suggest, that specific biological functions of polyamines in the processes of growth and differentiation of cells may be also related to the ability of compounds to activate soluble guanylate cyclase and to increase intracellular cGMP level.     

Polyamine-sensitive protein kinase from chick intestinal mucosa

Summary A cyclic nucleotide-independent protein kinase which phoshorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [-³²P]ATP.When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.     

Circular Dichroism and NMR Studies of Metabolically Stableα-Methylpolyamines: Spectral Comparison with Naturally Occurring Polyamines

Three synthetic polyamine analogs, α-methylspermine, and α,α′-dimethylspermine, were compared with their naturally occurring counterparts, spermidine and spermine, by two different spectral techniques. The interaction of polyamines with oligodeoxynucleotides was measured by circular dichroism in order to monitor the polyamine-induced conversion of right-handed B-DNA to the left-handed Z-form. The methylated analogs were shown to be equally effective as the natural polyamines in inducing the B → Z transition. The pH dependence of the chemical shift of all carbon atoms in each of the five polyamines was measured by ¹³C-NMR spectroscopy. With the exception of expected changes in chemical shift due to the presence of the α-methyl substituents, the chemical shifts and pH dependence of all carbon atoms in the three α-methyl polyamines were similar to the corresponding naturally occurring polyamines. The combined data indicate that α-methyl polyamines have physical properties that are very similar to their natural counterparts. The two metabolically stable polyamine analogs, α-methylspermidine and α,α′-dimethylspermine, are therefore useful surrogates for spermidine and spermine in the study of numerous polyamine-mediated effects in mammalian cell cultures and can be used in such studies without the requirement for coadministration of amine oxidase inhibitors.