Polyamine stress at high pH in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

Background  

Polyamines such as spermine and spermidine are required for growth ofEscherichia coli; they interact with nucleic acids, and they bind to ribosomes. Polyamines block porins and decrease membrane permeability, activities that may protect cells in acid. At high concentrations, however, polyamines impair growth. They impair growth more severely at high pH, probably due to their increased uptake as membrane-permeant weak bases. The role of pH is critical in understanding polyamine stress.     

My favourite molecule: Polyamines,chromatin structure and transcription

Nucleosomes are the basic elements of chromatin structure. Polyamines, such as spermine and spermidine, are small ubiquitous molecules absolutely required for cell growth. Photoaffinity polyamines bind to specific locations in nucleosomes and can change the helical twist of DNA in nucleosomes. Acetylation of polyamines reduces their affinity for DNA and nucleosomes, thus the helical twist of DNA in nucleosomes could be regulated by cells through acetylation. I suggest that histone and polyamine acetylation act synergistically to modulate chromatin structure. On naked DNA, the photoaffinity spermine bound preferentially to a specific ‘TATA’ sequence element, suggesting that polyamines may be involved in the unusual chromatin structure in this region. Further work is needed to test whether the specificities shown by photoaffinity polyamines are also shown by cellular polyamines; such experiments are now feasible.     

Modulation of Arabidopsis CYCB1 expression patterns by polyamines and salt stress

Polyamines are new plant growth regulators that participate in various physiological processes modulating cell division and differentiation, stimulating secondary metabolite production and in stress responsiveness. In the present study, we evaluated the effect of polyamine application on CYCB1-GUS reporter line in Arabidopsis, in order to monitor changes in cell division. We observed that polyamines modulate the expression of CYCB1-GUS, most likely in an amine-specific manner. In particular, spermidine and spermine induced significant increases in CYCB1-GUS expression in shoot apex and root meristems. According of this view, mainly the higher polyamines stimulate the lateral root formation in Arabidopsis. Furthermore, the application of d-arginine and methylglyoxal bis-(guanylhydrazone) polyamine inhibitors drastically reduced Arabidopsis CYCB1-GUS root growth and plant fresh weight, as well as CYCB1-GUS expression. Another key point on this study was to analyze the effect of polyamines on CYCB1-GUS expression under salt stress. Salt stress treatments repressed CYCB1-GUS expression in a concentration dependent manner; this negative effect was ameliorated by polyamine application, in particular by spermidine and spermine, even at 125 mM NaCl, allowing the maintenance of CYCB1-GUS levels under salt stress. This work is one more contribution on the role of polyamines in cell cycle modulation and abiotic stress protection.     

Endogenous polyamine profiles in different tissues of <Emphasis Type="Italic">Coffea</Emphasis> sp., and their levels during the ontogeny of fruits

Polyamines are essential compounds for growth and development in plants. An attempt has been made to find out the endogenous polyamine profiles in various parts and during the ontogeny of fruit formation of two commercially important Coffea species viz., arabica and canephora. Putrescine (Put), spermine (Spm) and spermidine (Spd) are the predominant polyamines during the ontogeny of fruit and their level increased with the advancement of fruit development. However, in the initial stages of flower and fruit development Spm levels were found to be decreased. Elevated levels of major polyamines Put, Spd, and Spm were observed in zygotic embryos than in somatic embryos. Along with this cadavarine (Cad) and other biogenic amines viz., tyramine (Tyr) and tryptamine (Try) were also found during the ontogeny of fruit in C. canephora. In this study the enodogenous polyamine profiles in coffee tissues and beans have been addressed.     

Thermal tolerance role of novel polyamine,caldopentamine, identified in fifth instar Bombyx mori

Silkworms have limited ability to regulate their body temperature; therefore, environmental changes, such as global warming, can adversely affect their viability. Polyamines have shown protection to various organisms against heat stress. This study evaluated the qualitative and quantitative changes in heat-stressed Bombyx mori larvae polyamines. Fifth instar Bombyx mori larvae were divided into two groups; control group, reared at room temperature, i.e., 28?±?2 °C, and the heat shock group, exposed to 40 °C. Dansylation of the whole worm polyamines and subsequent thin-layer chromatography revealed the presence of components with the same Rf value as dansyl–putrescine, spermidine, and spermine. The dansyl–putrescine, spermidine, and spermine polyamines were identified by mass spectrometric analyses. After heat shock, the thin-layer chromatography of the whole-larvae tissue extracts showed qualitative and quantitative changes in dansylated polyamines. A new polyamine, caldopentamine, was identified, which showed elevated levels in heat-stressed larvae. This polyamine could play a role in helping the larvae tolerate various stress, including thermal stress. No significant changes in silk fiber’s economic and mechanical properties were observed in our study. This study indicated that PA, caldopentamine, supplementation could improve heat-stress tolerance in Bombyx mori.

     

Probing tRNA interaction with biogenic polyamines

Biogenic polyamines are found to modulate protein synthesis at different levels. This effect may be explained by the ability of polyamines to bind and influence the secondary structure of tRNA, mRNA, and rRNA. We report the interaction between tRNA and the three biogenic polyamines putrescine, spermidine, spermine, and cobalt(III)hexamine at physiological conditions, using FTIR spectroscopy, capillary electrophoresis, and molecular modeling. The results indicated that tRNA was stabilized at low biogenic polyamine concentration, as a consequence of polyamine interaction with the backbone phosphate group. The main tRNA reactive sites for biogenic polyamine at low concentration were guanine-N7/O6, uracil-O2/O4, adenine-N3, and 2′OH of the ribose. At high polyamine concentration, the interaction involves guanine-N7/O6, adenine-N7, uracil-O2 reactive sites, and the backbone phosphate group. The participation of the polycation primary amino group, in the interaction and the presence of the hydrophobic contact, are also shown. The binding affinity of biogenic polyamine to tRNA molecule was in the order of spermine > spermidine > putrescine with K_Spm = 8.7 × 10⁵ M⁻¹, K_Spd = 6.1 × 10⁵ M⁻¹, and K_Put = 1.0 × 10⁵ M⁻¹, which correlates with their positively charged amino group content. Hill analysis showed positive cooperativity for the biogenic polyamines and negative cooperativity for cobalt-hexamine. Cobalt(III)hexamine contains high- and low-affinity sites in tRNA with K₁ = 3.2 × 10⁵ M⁻¹ and K₂ = 1.7 × 10⁵ M⁻¹, that have been attributed to the interactions with guanine-N7 sites and the backbone PO₂ group, respectively. This mechanism of tRNA binding could explain the condensation phenomenon observed at high Co(III) content, as previously shown in the Co(III)–DNA complexes.     

Selective regulation of polyamine metabolism with methylated polyamine analogues

Polyamine metabolism is intimately linked to the physiological state of the cell. Low polyamines levels promote growth cessation, while increased concentrations are often associated with rapid proliferation or cancer. Delicately balanced biosynthesis, catabolism, uptake and excretion are very important for maintaining the intracellular polyamine homeostasis, and deregulated polyamine metabolism is associated with imbalanced metabolic red/ox state. Although many cellular targets of polyamines have been described, the precise molecular mechanisms in these interactions are largely unknown. Polyamines are readily interconvertible which complicate studies on the functions of the individual polyamines. Thus, non-metabolizable polyamine analogues, like carbon-methylated analogues, are needed to circumvent that problem. This review focuses on methylated putrescine, spermidine and spermine analogues in which at least one hydrogen atom attached to polyamine carbon backbone has been replaced by a methyl group. These analogues allow the regulation of both metabolic and catabolic fates of the parent molecule. Substituting the natural polyamines with methylated analogue(s) offers means to study either the functions of an individual polyamine or the effects of altered polyamine metabolism on cell physiology. In general, gem-dimethylated analogues are considered to be non-metabolizable by polyamine catabolizing enzymes spermidine/spermine-N ¹-acetyltransferase and acetylpolyamine oxidase and they support short-term cellular proliferation in many experimental models. Monomethylation renders the analogues chiral, offering some advantage over gem-dimethylated analogues in the specific regulation of polyamine metabolism. Thus, methylated polyamine analogues are practical tools to meet existing biological challenges in solving the physiological functions of polyamines.     

Role of polyamines in the cytokinin-dependent physiological processes. I. Effect of benzyladenine on polyamine levels during chloroplast differentiation in the tissue culture of Dianthus caryophyllus

Tissue culture of Dianthus caryophyllus L. (cv. William Sim.) obligatory requiring N⁶-benzyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry. Green callus growing on benzyladenine — containing medium showed decrease in the contents of free, conjugated and bound putrescine and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence of cytokinin. Spermidine was dominant polyamine bound to chromatin isolated from control callus. Chromatin isolated from green callus was characterized by a lower level of each polyamine in comparison to chlorophyll-less callus. Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine) in comparison to chlorophyll-less callus (− benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an inhibitor of S-adenosylmethionine decarboxylase depressed the greening process. Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic apparatus is discussed.     

Polyamine‐independent growth and biofilm formation,and functional spermidine/spermine N‐acetyltransferases in Staphylococcus aureus and Enterococcus faecalis

Polyamines such as spermidine and spermine are primordial polycations that are ubiquitously present in the three domains of life. We have found that Gram‐positive bacteria Staphylococcus aureus and Enterococcus faecalis have lost either all or most polyamine biosynthetic genes, respectively, and are devoid of any polyamine when grown in polyamine‐free media. In contrast to bacteria such as Pseudomonas aeruginosa, Campylobacter jejuni and Agrobacterium tumefaciens, which absolutely require polyamines for growth, S. aureus and E. faecalis grow normally over multiple subcultures in the absence of polyamines. Furthermore, S. aureus and E. faecalis form biofilms normally without polyamines, and exogenous polyamines do not stimulate growth or biofilm formation. High levels of external polyamines, including norspermidine, eventually inhibit biofilm formation through inhibition of planktonic growth. We show that spermidine/spermine N‐acetyltransferase (SSAT) homologues encoded by S. aureus USA300 and E. faecalis acetylate spermidine, spermine and norspermidine, that spermine is the more preferred substrate, and that E. faecalis SSAT is almost as efficient as human SSAT with spermine as substrate. The polyamine auxotrophy, polyamine‐independent growth and biofilm formation, and presence of functional polyamine N‐acetyltransferases in S. aureus and E. faecalis represent a new paradigm for bacterial polyamine biology.     

Polyamine inhibition of lipoperoxidation. The influence of polyamines on iron oxidation in the presence of compounds mimicking phospholipid polar heads.

Polyamines appear to inhibit peroxidation of vesicles containing acidic phospholipids. A correlation exists between polyamine binding to phospholipid vesicles and its protective effect. However, phosphatidylinositol-containing vesicles which bind spermine are not protected by the polyamine [Tadolini, Cabrini, Landi, Varani & Pasquali (1985) Biogenic Amines 3, 97-106]. In the present paper I tested the hypothesis that polyamines, in particular spermine, by forming a ternary complex with iron and the phospholipid polar head may change the susceptibility of Fe2+ to autoxidation and thus its ability to generate free oxygen radicals. Different compounds mimicking phospholipid polar heads were studied, namely AMP, mimicking phosphatidic acid, CDP-choline, mimicking phosphatidylcholine, and glycerophosphoinositol, mimicking phosphatidylinositol. The results support the proposed hypothesis. In the presence of CDP-choline or of glycerophosphoinositol, spermine poorly affects Fe2+ autoxidation, whereas a considerable inhibition is observed in the presence of AMP. The ability of other phosphorus-containing compounds (ATP, ADP, cyclic AMP, sodium phosphate) to affect Fe2+ autoxidation in the presence of polyamines was also evaluated to understand the molecular mechanism of this phenomenon. It is proposed that polyamines may be part of the passive cellular defence mechanism against the oxidative damage caused by Fe2+.     

Rapid self-assembly of alpha-synuclein observed by in situ atomic force microscopy

Self-assembly of alpha-synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson's disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy to continuously monitor the self-assembly of wild-type alpha-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant alpha-synuclein(1-108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(+/-0.2)nm in height and presumably representing individual beta-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite substrates, suggesting that negatively charged surfaces promote alpha-synuclein self-assembly. In the presence of spermidine or spermine 5.9(+/-1.0)nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. alpha-Synuclein spheroid formation depended critically on polyamine binding to the C terminus, revealing a promoting effect of the C terminus on alpha-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of alpha-synuclein self-assembly and identifies key factors modulating the aggregation process.     

Mucosal polyamine measurements and colorectal cancer risk

Polyamines are short-chain aliphatic amines required for normal cellular growth that are ubiquitously found in all living tissues. Polyamine content has been shown to correlate with cellular proliferation. Quantitation of polyamines may thus provide a biochemical measure of proliferation in the colorectal mucosa where dysregulated epithelial proliferation is associated with colorectal cancer risk. A case-control study was conducted to validate the hypothesized association between mucosal polyamine measurements and colorectal cancer risk. Polyamines were measured in 4–6 multiple rectal mucosal biopsies from 11 normal control subjects and seven case patients with colon cancer. Compared with the controls, mean polyamine measurements, after adjustment for age and sex, were significantly increased for spermidine (P < 0.003) and spermine (P < 0.017). Subsequent analyses indicated that in controls 1–4 biopsies appeared adequate to characterize an individual. However, mucosal polyamines in the cases exhibited more sampling variability, requiring 4–8 biopsies to achieve an acceptable level of reliability. After adjustment for age and sex, the odds ratios for spermidine and spermine levels, compared to the controls, were 4.8 (95% confidence interval: 1.6–33.7) and 2.3 (1.2–6.3), respectively. The results of this study indicate that increases of mucosal polyamine measurements, after taking the sampling and methodological variability into account, are significantly associated with colorectal cancer risk, and suggest that polyamine measurements in rectal mucosa may play an important role as biomarkers for identifying high-risk individuals and/or for using as intermediate endpoints in prevention trials. © 1996 Wiley-Liss, Inc.     

Spermidine and abscisic acid-mediated phosphorylation of a cytoplasmic protein from rice root in response to salinity stress

Importance of higher polyamines, spermidine, and spermine, in relation to the mechanism and adaptation to combat abiotic stress has been well established in cereals. Owing to their polycationic nature at physiological pH, polyamines bind strongly to negative charges in cellular components such as nucleic acids, various proteins, and phospholipids. To study the physiological role of polyamine during salinity stress, phosphorylation study was carried out in cytosolic soluble protein fraction isolated from the roots of salt tolerant (Nonabokra) and salt sensitive (M-1-48) rice cultivars treated with none (control), NaCl (150 mM, 16 h), spermidine (1 mM, 16 h) or with abscisic acid (100 μM, 16 h). A calcium independent auto regulatory 42 kDa protein kinase was found to phosphorylate myelin basic protein and casein but not histone. Interestingly, this was the only protein to be phosphorylated in root cytosolic fraction during NaCl/abscisic acid/spermidine treatment indicating its importance in salinity mediated signal transduction. This is the first report of polyamine as well as abscisic acid induced protein kinase activity in rice root in response to salinity stress.     

Novel anti-apoptotic effect of the retinoblastoma protein: implications for polyamine analogue toxicity

The retinoblastoma protein (pRb) pathway is frequently altered in breast cancer cells. pRb is involved in the regulation of cell proliferation and cell death. The breast cancer cell line L56Br-C1 does not express pRb and is extremely sensitive to treatment with the polyamine analogue N ¹,N ¹¹-diethylnorspermine (DENSPM) which causes apoptosis. Polyamines are essential for the regulation of cell proliferation, cell differentiation and cell death. DENSPM depletes cells of polyamines, e.g., by inducing the activity of the polyamine catabolic enzyme spermidine/spermine N ¹-acetyltransferase (SSAT). In this study, L56Br-C1 cells were transfected with human pRb–cDNA. Overexpression of pRb inhibited DENSPM-induced cell death and DENSPM-induced SSAT activity. This suggests that the pRb protein level is a promising marker for polyamine depletion sensitivity and that there is a connection between pRb and the regulation of SSAT activity. We also show that SSAT protein levels and SSAT activity do not always correlate, suggesting that there is an unknown regulation of SSAT.     

Rectification of rabbit cardiac ryanodine receptor current by endogenous polyamines.

The actions of three endogenous polyamines (spermine, spermidine, and putrescine) were defined on Ca2+ release channels (ryanodine receptors, RyRs) isolated from rabbit cardiac sarcoplasmic reticulum. The current-voltage relationship of the RyR channel was N-shaped in the presence of polyamine (1-5 mM). Polyamine blocked conduction near 0 mV, but the blockade was relieved at large potentials. Polyamines acted (blocked) from both sides of the channel. Polyamine efficacy was dependent on current direction and was inversely related to the ion selectivity of the RyR pore. This suggests that polyamine interacts with current-carrying ions in the permeation pathway. The apparent half-block concentration of spermine at 0 mV was < 0.1 mM. The features of polyamine blockade suggest that the polyamines are permeable cationic blockers of the RyR channel. Further, the levels of polyamines found in muscle cells are sufficient to block single RyR channels and thus may alter the sarcoplasmic reticulum Ca2+ release process in situ.     

Comparison of soybean cultivars for enhancement of the polyamine contents in the fermented soybean natto using Bacillus subtilis (natto)

Polyamines have beneficial properties to prevent aging-associated diseases. Raw soybean has relatively high polyamine contents; and the fermented soybean natto is a good source of polyamines. However, detailed information of diversity of polyamine content in raw soybean is lacking. The objectives of this study were to evaluate differences of polyamines among raw soybeans and select the high polyamine-containing cultivar for natto production. Polyamine contents were measured chromatographically in 16 samples of soybean, which showed high variation among soybeans as follows: 93–861 nmol/g putrescine, 1055–2306 nmol/g spermidine, and 177–578 nmol/g spermine. We then confirmed the high correlations of polyamine contents between raw soybean and natto (r = 0.96, 0.95, and 0.94 for putrescine, spermidine, and spermine, respectively). Furthermore, comparison of the polyamine contents among 9 Japanese cultivars showed that ‘Nakasen-nari’ has the highest polyamine contents, suggesting its suitability for enhancement of polyamine contents of natto.     

Profile of polyamines during sprouting and growth of saffron (Crocus sativus L.) Corms

The profile of free and conjugated polyamines putrescine, spermidine, and spermine was studied at the onset of sprouting and during various stages of vegetative growth in saffron (Crocus sativus L.) corms. Polyamines were extracted from the shoot meristems and estimated by high performance liquid chromatography. Free putrescine was not detected at the onset of sprouting, whereas free spermidine and spermine levels increased rapidly on sprouting and decreased during further stages of corm development. The levels of conjugated polyamines were several times higher than the free forms indicating their possible role in the developmental processes. A comparison of polyamine levels of vegetative and floral corms showed higher titers of free polyamines in vegetative and conjugated polyamines in floral corms.     

Histamine prevents polyamine accumulation in mouse C57.1 mast cell cultures.

The effects of histamine on polyamine uptake and metabolism was studied in a mouse mast cell line (C57.1), as a cell model in which both biogenic amines are important for maintaining cell function and viability. Results obtained after incubations with exogenous histamine indicated that histamine prevents polyamine accumulation by affecting polyamine uptake. A plasma membrane transport system for polyamines has been also studied in mast cells. It seems to be a Na(+)-dependent uptake with high affinity for both spermine and spermidine and lower affinity for putrescine and agmatine. Polyamine uptake was reduced in both cells treated with exogenous histamine and histamine-preloaded cells. However, ornithine decarboxylase activity and cell proliferation were not affected by histamine. Incubation with histamine enhanced the spermidine/spermine acetyl transferase induction caused by N(1)-ethyl-N(11)-[(cyclopropyl)methyl]-4,8-diazaundecane, suggesting that polyamine acetylation could be another mechanism by which histamine prevents polyamine accumulation in C57.1 mast cells.