Absence Of Spermine Research Articles

Thermospermine Is an Evolutionarily Ancestral Phytohormone Required for Organ Development and Stress Responses in Marchantia Polymorpha.

Thermospermine suppresses auxin-inducible xylem differentiation, whereas its structural isomer, spermine, is involved in stress responses in angiosperms. The thermospermine synthase, ACAULIS5 (ACL5), is conserved from algae to land plants, but its physiological functions remain elusive in non-vascular plants. Here, we focused on MpACL5, a gene in the liverwort Marchantia polymorpha, that rescued the dwarf phenotype of the acl5 mutant in Arabidopsis. In the Mpacl5 mutants generated by genome editing, severe growth retardation was observed in the vegetative organ, thallus, and the sexual reproductive organ, gametangiophore. The mutant gametangiophores exhibited remarkable morphological defects such as short stalks, fasciation and indeterminate growth. Two gametangiophores fused together, and new gametangiophores were often initiated from the old ones. Furthermore, Mpacl5 showed altered responses to heat and salt stresses. Given the absence of spermine in bryophytes, these results suggest that thermospermine has a dual primordial function in organ development and stress responses in M. polymorpha. The stress response function may have eventually been assigned to spermine during land plant evolution.

Structural and Kinetic Characterization of the SpeG Spermidine/Spermine N-acetyltransferase from Methicillin-Resistant Staphylococcus aureus USA300.

Polyamines are simple yet critical molecules with diverse roles in numerous pathogenic and non-pathogenic organisms. Regulating polyamine concentrations affects the transcription and translation of genes and proteins important for cell growth, stress, and toxicity. One way polyamine concentrations are maintained within the cell is via spermidine/spermine N-acetyltransferases (SSATs) that acetylate intracellular polyamines so they can be exported. The bacterial SpeG enzyme is an SSAT that exhibits a unique dodecameric structure and allosteric site compared to other SSATs that have been previously characterized. While its overall 3D structure is conserved, its presence and role in different bacterial pathogens are inconsistent. For example, not all bacteria have speG encoded in their genomes; in some bacteria, the speG gene is present but has become silenced, and in other bacteria, it has been acquired on mobile genetic elements. The latter is the case for methicillin-resistant Staphylococcus aureus (MRSA) USA300, where it appears to aid pathogenesis. To gain a greater understanding of the structure/function relationship of SpeG from the MRSA USA300 strain (SaSpeG), we determined its X-ray crystal structure in the presence and absence of spermine. Additionally, we showed the oligomeric state of SaSpeG is dynamic, and its homogeneity is affected by polyamines and AcCoA. Enzyme kinetic assays showed that pre-incubation with polyamines significantly affected the positive cooperativity toward spermine and spermidine and the catalytic efficiency of the enzyme. Furthermore, we showed bacterial SpeG enzymes do not have equivalent capabilities to acetylate aminopropyl versus aminbutyl ends of spermidine. Overall, this study provides new insight that will assist in understanding the SpeG enzyme and its role in pathogenic and non-pathogenic bacteria at a molecular level.

Colorimetric chemosensor for spermine based on pyrocatechol violet and anionic phenylboronic acid in aqueous solution

A new sensing method for colorimetric detection of spermine has been developed using a simple mixture of anionic pyrocatechol violet (PV) and 3-carboxyphenylboronic acid (CPB) in a neutral aqueous solution. In the absence of spermine, the boronate ester between PV and CPB formed to a small extent, resulting in slight spectral and insignificant color changes. The addition of spermine to the mixture caused solution color change from yellow to reddish-brown with a bathochromic shift in absorption spectra by the boronate ester formation between PV and CPB against the electrostatic repulsive interactions, while basic amino acids and other amines induced no or minimal color and spectral changes. The detection limit was determined to be 6.24 μM for spermine, which was within the critical concentration for cancer diagnosis. The mixture was demonstrated as a novel colorimetric chemosensor for spermine with high sensitivity and sensitivity. This present study can contribute to the facile method for colorimetric detection of cationic analytes by mixing anionic catechol dyes and similarly charged boronic derivatives.

Can Changes in Temperature or Ionic Conditions Modify the DNA Organization in the Full Bacteriophage Capsid?

We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.

Na+,K+-ATPase Activity in the Posterior Gills of the Blue Crab, Callinectes ornatus (Decapoda, Brachyura): Modulation of ATP Hydrolysis by the Biogenic Amines Spermidine and Spermine

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K⁺, Na⁺, NH₄⁺ and Mg²⁺ and on inhibition by ouabain of posterior gill microsomal Na⁺,K⁺-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na⁺,K⁺-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na⁺,K⁺-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis-Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K⁺, Na⁺, NH₄⁺ and Mg²⁺ varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na⁺,K⁺-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.

DNA Morphologic Changes Induced by Spermine on a Gold Surface under DNA Crowding Conditions

Abstract DNA immobilized on a gold surface was globule in the absence of spermine. Upon addition of spermine, the DNA underwent a structural transition that was dependent on DNA length from a globular structure to a random coil. This effect was opposite the DNA morphologic changes observed in dilute solutions.

Voltage-dependent block by internal spermine of the murine inwardly rectifying K+channel, Kir2.1, with asymmetrical K+concentrations

Effects of internal spermine on outward single-channel currents through a strongly inwardly rectifying K(+) channel (Kir2.1) were studied at asymmetrical K(+) concentrations (30 mm external and 150 mm internal K(+)). The current-voltage (I-V) relation for the single channel was almost linear and reversed at -37 ± 3 mV (V(R); n = 19). The channel conductance was 26.3 ± 1.3 pS (n = 24). The open-time and closed-time histograms were fitted with a single exponential function. Internal spermine at a concentration of 1-100 nm reduced the open time of the outward currents in a concentration-dependent manner and produced a blocked state. The steady-state open probability of the outward current decreased with larger depolarizations in both the absence and presence of internal spermine. The steady-state open probability with asymmetrical K(+) and symmetrical (150 mm external and internal K(+)) concentrations plotted against driving force (V - V(R)) coincided with smaller depolarizations in the absence of spermine and larger depolarizations and higher spermine concentrations in the presence of spermine. The blocking rate constants and unblock rates with 30 mm and 150 mm external K(+) were similar at the same driving force. The dissociation constant-membrane potential relation for 30 mm external K(+) was shifted in the negative direction from that for 150 mm external K(+) by 36 mV. These results suggested that the blocking kinetics depends on driving force to produce driving force-dependent inward rectification when the equilibrium potential for K(+) is altered by changing external K(+) and that the energy barriers and wells for blocking ions from passing or lodging are not stable but affected by external K(+) ions.

A study on the activity and thermal stability of adenosine deaminase in the presence of spermine

Adenosine Deaminase is an aminohydrolase (EC 3.5.4.4) which participates in the purine metabolism where it degrades either adenosine or 2'-deoxyadenosine producing inosine or 2'deoxyinosine, respectively. The enzyme contains a parallel alpha/beta -barrel motif with eight central beta strands and eight peripheral alpha helices. ADA is located both in the cytosol and on the cell membrane. Since spermine, a natural metabolite, exists in all cells and tissues and its effect on the cell proliferation and enzyme regulation have been reported, thermal inactivation of the ADA and spermine regulatory effect on the ADA activity have been investigated in this study. Percentage of ADA activity in the presence and absence of spermine (1000 µM) in Tris buffer 50 mM, pH 7.5 at physiologic and pathologic temperatures have been reported in the present study. Thermal inactivation curves for ADA in the absence and presence of spermine (1000 µM) in different temperatures ranging from 55 o C to 70 o C have been drawn. Our data showed that spermine activates the enzyme in the low concentrations of adenosine at 37 o C. However, it inhibits ADA activity at 42 o C in the same concentrations of substrate. It is concluded that spermine regulatory effect depends on combined influence of temperature and adenosine concentration. Furthermore, thermal stability of the enzyme also depends on temperature in presence of spermine. Binding site of spermine on the enzyme has been identified by docking analysis.

The effect of spermine on the initiation of mitochondrial protein synthesis

Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine is a quaternary aliphatic amine that is cationic under physiological conditions. In this paper, we demonstrate that spermine stimulates fMet-tRNA binding to mammalian mitochondrial 55S ribosomes. The stimulatory effect of spermine is independent of the identity of the mRNA. The degree of stimulation of spermine is the same at all concentrations of mitochondrial initiation factor 2 (IF2mt) and mitochondrial initiation factor 3 (IF3mt). This observation indicates that IF2mt and IF3mt, while essential for initiation, are not the primary components of the translation initiation system affected by spermine. IF3mt dissociates 55S ribosomes detectably in the absence of spermine, but this effect is strongly inhibited in the presence of spermine. This observation indicates that the positive effect of spermine on initiation is not due to an increase in the availability of the small subunits for initiation. Spermine also promotes fMet-tRNA binding to small subunits of the mitochondrial ribosome in the presence of IF2mt. The major effect of spermine in promoting initiation complex formation thus appears to be on the interaction of fMet-tRNA with the ribosome.

The impact of spermine competition on the efficacy of DNA-binding Fe(II), Co(II), and Cu(II) complexes of dimeric chromomycin A3

Chromomycin (Chro) forms a 2:1 drug/metal complex through the chelation with Fe(II), Co(II), or Cu(II) ion. The effects of spermine on the interaction of Fe(II), Co(II), and Cu(II) complexes of dimeric Chro with DNA were studied. Circular dichroism (CD) measurements revealed that spermine strongly competed for the Fe(II) and Cu(II) cations in dimeric Chro-DNA complexes, and disrupted the structures of these complexes. However, the DNA-Co(II)(Chro)(2) complex showed extreme resistance to spermine-mediated competition for the Co(II) cation. According to surface plasmon resonance (SPR) experiments, a 6mM concentration of spermine completely abolished the DNA-binding activity of Fe(II)(Chro)(2) and Cu(II)(Chro)(2) and interfered with the associative binding of Co(II)(Chro)(2) complexes to DNA duplexes, but only slightly affected dissociation. In DNA integrity assays, lower concentrations of spermine (1 and 2mM) promoted DNA strand cleavage by Cu(II)(Chro)(2), whereas various concentrations of spermine protected plasmid DNA from damage caused by either Co(II)(Chro)(2) or Fe(II)(Chro)(2). Additionally, DNA condensation was observed in the reactions of DNA, spermine, and Fe(II)(Chro)(2). Despite the fact that Cu(II)(Chro)(2) and Fe(II)(Chro)(2) demonstrated lower DNA-binding activity than Co(II)(Chro)(2) in the absence of spermine, while Cu(II)(Chro)(2) and Fe(II)(Chro)(2) exhibited greater cytoxicity against HepG2 cells than Co(II)(Chro)(2), possibly due to competition of spermine for Fe(II) or Cu(II) in the dimeric Chro complex in the nucleus of the cancer cells. Our results should have significant relevance to future developments in metalloantibiotics for cancer therapy.

Spermine Metabolism and Anticancer Therapy

The natural polyamines (PA), putrescine (PUT), spermidine (SPD) and spermine (SPM) are ubiquitous constituents of eukaryotic cells. The increase of PA in malignant and proliferating cells attracted the interest of scientists during last decades, addressing PA depletion as a new strategy to inhibit cell growth. Selective enzyme inhibitors were developed for decreasing PA metabolism and to act as chemotherapeutic anticancer agents. Indeed, the complexity of the PA homoeostasis overcomes the PA perturbation by a single enzyme to take effect therapeutically. Recently, an increasing interest has been posed on spermine-oxidase (SMO), the only catabolic enzyme able to specifically oxidise SPM. Interestingly, the absence of SPM is compatible with life, but its accumulation and degradation is lethal. Augmented SMO activity provokes an oxidative stress rendering cells prone to die, and appears to be important in the cell differentiation pathway. Extra-cellular SPM is cytotoxic, but its analogues are capable of inhibiting cell growth at low concentrations, most likely by intracellular SPM depletion. These pivotal roles seem to evoke the biological processes of stress response, wherein balance is mandatory to live or to die. Thus, altering SPM metabolism could allow a multi-tasking therapeutic strategy, addressed not only to inhibit PA metabolism. Several tetramines are presently in early phases (I and II) of clinical trials, and it will be a matter of a few more years to understand whether SPM-related therapeutic approaches would be of benefit for composite treatment protocols of cancer.

Identification of Stimulators and Inhibitors of Cdc7 Kinase in Vitro

Cdc7 is a serine-threonine kinase that regulates initiation and progression of DNA replication. The activity of purified Cdc7 kinase is significantly stimulated by polyamines such as spermine or spermidine. Positively charged polymers of lysine or arginine also stimulate its kinase activity, whereas the negatively charged substances such as polyglutamate or nucleic acids significantly inhibit the kinase activity. Spermine affects both the K(m) and V(max) of Cdc7 kinase for a minichromosome maintenance (MCM) substrate. We also found that histones, lysine- and arginine-rich basic proteins, can stimulate Cdc7 kinase activity, and a MCM complex in association with histone is a more efficient substrate of Cdc7 than the free MCM complex. These results identify potential cellular inhibitors and stimulators of Cdc7 kinase and suggest that Cdc7 may be another target of cellular polyamines and that histones may stimulate Cdc7-mediated phosphorylation of chromatin-bound substrates. Ectopic expression of an antizyme, known to reduce the cellular polyamine levels, resulted in reduction of Cdc7-mediated phosphorylation of MCM4 protein, suggesting physiological roles of polyamines in regulation of Cdc7 kinase activity in the cells.

Improvement of intestinal absorption of water-soluble macromolecules by various polyamines: Intestinal mucosal toxicity and absorption-enhancing mechanism of spermine

The absorption-enhancing effects of three different polyamines, spermine (SPM), spermidine (SPD) and putrescine (PUT) on the intestinal absorption of water-soluble macromolecules were examined in rats. Fluorescein isothiocyanate-labeled dextrans (FDs) with different average molecular weights were chosen as models of water-soluble macromolecules and intestinal absorption of FDs with or without these polyamines was examined by an in situ closed loop method. The intestinal absorption of fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4400 (FD4) was relatively low in the absence of these polyamines. However, its absorption was improved in the presence of 5–10 mM SPM and 10 mM SPD in the jejunum and 10 mM SPM in the colon, while 10 mM PUT had almost no absorption-enhancing effect on the intestinal absorption of FD4. Overall, the enhancing effects of these polyamines were greater in the jejunal membranes than in the colonic membranes. The absorption-enhancing effect of SPM decreased as the molecular weights of FDs increased. The intestinal membrane toxicity of 10 mM SPM was evaluated by measuring the amount of protein and activity of lactate dehydrogenase (LDH) released from the intestinal epithelial cells. We also observed the morphological changes of intestinal mucosa in the presence or absence of SPM. The results indicated that the amount of protein and LDH was not changed in the presence of 10 mM SPM, although we observed a significant increase in these biological markers in the presence of 3% Triton X-100, as a positive control. Furthermore, we found no significant change in the intestinal membrane with 10 mM SPM by the morphological observation. These findings suggested that 10 mM SPM did not cause any significant membrane damage to the intestinal epithelium. To investigate the absorption-enhancing mechanism of SPM, the transepithelial electrical resistance (TEER) of the rat jejunal membranes was studied by using a diffusion chamber method. SPM decreased the TEER values in a concentration dependent manner and 10 mM SPM had almost the same effect to decrease the TEER value compared with 10 mM EDTA as a positive control. These findings suggest that SPM may loosen the tight junction of the epithelium, thereby increasing the intestinal absorption of drugs via a paracellular route. In summary, polyamines, especially SPM would be one of the suitable absorption enhancers with high effectiveness and low intestinal membrane toxicity.

Modulation of Arginase Pathway by Chloroquine and Its Congener May Underlie the Enhanced Cell Commitment towards Erythroid Differentiation.

Arginase is a nitric oxide synthase-alternative pathway enzyme for L-arginine breakdown leading to biosynthesis of urea and L-ornithine. Evidence suggests that reduction of arginine bioavailability for NO production secondary to increased plasma arginase levels in sickle cell disease, may contribute to severe pathophysiological derangements and increase mortality. Recently, we showed that Chloroquine (CQ), an anti-malarial and anti-rheumatoid drug, displays a linear competitive mode of inhibition on sickle erythrocyte arginase (Iyamu et al., Brit J. Haematol, 2007: in press). Utilizing the K562 cell line as a model system in our present study, we obtained evidence of the anti-proliferative and differentiation effects of CQ and its analog, Hydroxychloroquine (OHCQ) at pharmacologically attainable concentrations (5–20μM). Specifically, CQ (IC50= 8.0 ±1.2 μM) and OHCQ (IC50 =17.0 ± 2.4 μM) inhibited K562 cell proliferation in a dose-dependent manner. This inhibitory effect was accompanied by enhanced commitment towards erythroid maturation as assessed by hemoglobinization (Iyamu et al., Exp Hematol, 2003); this was linked to a dose-dependent inhibition of arginase activity. To assess the role of the arginine-polyamine pathway in the induction of differentiation of K562 cells by CQ or OHCQ, cells were treated with 10 μM CQ or 20 μM OHCQ in the presence or absence of spermine (5μM) or spermidine (5 μM). Our results indicate that spermine and spermidine (intermediate products of the arginine polyamine pathway) partially reverted the effects of CQ and OHCQ on arginase activity. But this reversal did not affect the commitment of K562 cells towards erythroid maturation. We also show that CQ and OHCQ enhanced fetal hemoglobin (Hb F) synthesis by 3.4 and 3.2-fold (vs control), and maximally stimulated intracellular cGMP levels (as determined by immunosorbent assay) by 6.6- and 3.0-fold at 6hrs and 3hrs respectively. The induction of these macromolecules by CQ or OHCQ correlated with arginase inhibition. Again, the partial reversal of arginase activity by spermine or spermidine could not reduce the Hb F induction by CQ or OHCQ. This observation suggests that the commitment of cells towards erythroid maturation precedes the effects of spermine or spermidine on arginase activity. We further investigated the effects of 8-Bromo-cAMP (cAMP analog) on arginase activity in lipopolysaccharide (LPS)-stimulated K562 cells. The inhibitory effect of CQ on arginase was reversed in LPS-stimulated cells but this reversal of arginase activity was not observed in Bromo-cAMP-LPS- treated cells. Moreover, the moderate increase of arginase activity in LPS-treated cells in the presence of CQ failed to reduce the CQ-dependent enhancement of total hemoglobin and Hb F production. Indeed, the combination of 8-Bromo-cAMP with CQ in LPS-treated cells resulted in a significant inhibition (>35%) of arginase activity. This inhibitory effect was associated with a 3.9-fold increase of Hb F synthesis (vs CQ alone) as well as enhancement of total Hb production. This surprising observation demonstrates the possible involvement of the arginase pathway as well as additional, but yet unidentified mechanism(s). In conclusion, these observations that CQ and OHCQ may inhibit the initial step of the arginine-polyamine pathway, resulting in the enhancement of erythroid differentiation may provide a paradigm for targeted therapy of hemoglobinopathies.

The polyamine spermine protects against high salt stress in Arabidopsis thaliana

It is well known that changes in abiotic conditions such as the concentration of ions, temperature and humidity lead to modulation of polyamine contents in plants. However, little is known about the relevant parts these polyamines play in abiotic stress responses. Here we addressed a specific role of spermine during high salt stress using an Arabidopsis double knockout-mutant plant (acl5/spms) which cannot produce spermine. The mutant showed higher sensitivity to high salt than wild type plants. This phenotype was cured by exogenous spermine but not by the other polyamines putrescine and spermidine, suggesting a strong link between spermine-deficiency and NaCl-hypersensitivity. The mutant was also hypersensitive to high levels of KCl but not to MgCl2 or to high osmoticum. NaCl-hypersensitivity of the mutant was compromised by treatment with Ca2+ channel blockers. Moreover, the mutant showed poor growth on Ca2+-depleted Murashige–Skoog agar media. The data suggest that the absence of spermine causes an imbalance in Ca2+ homeostasis in the mutant plant. Based on the data obtained, we propose a model for a role of spermine in high salt stress responses.

Spermine Causes Loss of Innate Immune Response to Helicobacter pylori by Inhibition of Inducible Nitric-oxide Synthase Translation

Helicobacter pylori infection of the stomach elicits a vigorous but ineffective host immune and inflammatory response, resulting in persistence of the bacterium for the life of the host. We have reported that in macrophages, H. pylori up-regulates inducible NO synthase (iNOS) and antimicrobial NO production, but in parallel there is induction of arginase II, generating ornithine, and of ornithine decarboxylase (ODC), generating polyamines. Spermine, in particular, has been shown to restrain immune response in activated macrophages by inhibiting proinflammatory gene expression. We hypothesized that spermine could prevent the antimicrobial effects of NO by inhibiting iNOS in macrophages activated by H. pylori. Spermine did not affect the up-regulation of iNOS mRNA levels but in a concentration-dependent manner significantly attenuated iNOS protein levels and NO production. Reduction in iNOS protein was due to inhibition of iNOS translation and not due to iNOS degradation. ODC knockdown with small interfering (si) RNA resulted in increased H. pylori-stimulated iNOS protein expression and NO production without altering iNOS mRNA levels. When macrophages were cocultured with H. pylori, killing of bacteria was enhanced by transfection of ODC siRNA and prevented by addition of spermine. These results identify a mechanism of immune dysregulation induced by H. pylori in which stimulated spermine synthesis by the arginase-ODC pathway inhibits iNOS translation and NO production, leading to persistence of the bacterium and risk for peptic ulcer disease and gastric cancer.

Polyamines Modulate AMPA Receptor–Dependent Synaptic Responses in Immature Layer V Pyramidal Neurons

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) mediate the majority of fast excitation in the CNS. Receptors lacking GluR2 exhibit inward rectification and paired-pulse facilitation (PPF) due to polyamine (PA)-dependent block and unblock, respectively. In this study, we tested whether rectification and PPF in immature, but not mature, pyramidal neurons depend not only on the absence of functional GluR2 but also on the level of endogenous PAs. Whole cell recordings were obtained from layer V pyramidal neurons of P12-P14 or P16-P20 rats in the presence or absence of spermine in the pipette (50 microM). Isolated minimal excitatory synaptic responses were obtained, and paired (20 Hz) stimuli were used to investigate the rectification index (RI) and paired-pulse ratio (PPR). Spermine and its synthetic enzyme, ornithine decarboxylase (ODC), expression was examined using immunostaining and Western blot, respectively. At the immature stage (<P15) inclusion of intracellular spermine increased rectification and PPF for evoked excitatory postsynaptic currents (EPSCs) but had little or no effect on either measure in more mature (P16-P20) pyramidal neurons. Depletion of PAs reduced rectification suggesting that endogenous PAs play a critical role in functional regulation of AMPARs. Spermine immunoreactivity and ODC expression in immature rat neocortex (<P15) were greater than more mature tissue by approximately 20 and 60%, respectively. These results provide further support for the idea that excitatory synapses on immature neocortical pyramidal neurons ubiquitously contain AMPA receptors lacking the GluR2 subunit and that the level of endogenous PAs plays an important role in modulating AMPAR-dependent neurotransmission.

Influence of Spermine on DNA Conformation in a Molecular Dynamics Trajectory of d(CGCGAATTCGCG)2: Major Groove Binding by One Spermine Molecule Delays the A→B Transition

The effect of spermine on the A-DNA to B-DNA transition in d(CGCGAATTCGCG)2 has been investigated by five A-start molecular dynamics simulations, using the Cornell et al. potential. In the absence of spermine an A→B transition is initiated immediately and the DNA becomes equidistant from the A- and B-forms at 200ps. In three DNA-spermine simulations, when a spermine is located across the major groove of A-DNA in one of three different initial locations, the time taken to reach equidistance from the A- and B-forms is delayed until 800, 950 or 1000ps. In each case the A-form appears to be temporarily stabilized by spermine's electrostatic interactions with phosphates on both sides of the major groove. The onset of the A→B transition can be correlated with the spermine losing contact with phosphates on one side of the groove and with A-like → B-like sugar pucker transitions in the vicinity of the spermine bridge. However in the fifth trajectory, in which the spermine initially threads from the major groove via the backbone into the minor groove, the B→A transition occurs rapidly once again and the DNA is equidistant between the A- and B-forms within 300ps. This indicates that the mere presence of spermine is insufficient to delay the transition and that major groove binding stabilizes A-DNA.

Effect of spermine synthase on the sensitivity of cells to anti-tumour agents.

The role of spermine in the sensitivity of cells to various established and experimental anti-tumour agents was examined, using paired cell lines that possess or lack spermine synthase. All spermine-synthase-deficient cells had no detectable spermine, and elevated spermidine, content. Spermine content did not alter the cell growth rate. There was little or no difference in sensitivity of immortalized mouse embryonic fibroblasts to doxorubicin, etoposide, cisplatin, methylglyoxal bis(guanylhydrazone) or H(2)O(2) and only a slight increase in sensitivity to vinblastine and nocodazole. However, the absence of spermine clearly increased the sensitivity to 1,3-bis(2-chloroethyl)- N -nitrosourea, suggesting that depletion of spermine may be a useful way to increase the anti-neoplastic effects of anti-tumour agents that form chloroethyl-mediated interstrand DNA cross-links. The effects of spermine on the response to polyamine analogues (which have been proposed to be useful anti-neoplastic agents) were complex, and depended on the compound examined and on the cells tested. Sensitivity to CHENSpm ( N (1)-ethyl- N (11)-[(cycloheptyl)methyl]-4,8-diazaundecane) was substantially greater in immortalized fibroblasts that lack spermine. In contrast, BE-3-4-3 [ N (1), N (12)-bis(ethyl)spermine] and BE-3-3-3 [ N (1), N (11)-bis(ethyl)norspermine] were more active against cells that contained spermine. The presence of spermine correlated with a greater induction of spermidine/spermine- N (1)-acetyltransferase by BE-3-3-3, which is consistent with suggestions that this induction is important for the response to this drug. These findings support the concepts that different polyamine analogues have different sites of action and that CHENSpm has a different site of action from BE-3-3-3.

Voltage-dependent gating and block by internal spermine of the murine inwardly rectifying K+ channel, Kir2.1

The mechanism of inward rectification was investigated by recording single-channel currents through an inwardly rectifying K+ channel (Kir2.1). cDNA encoding a wild-type (WT) channel, a mutant replacing Asp 172 with Asn (D172N), and a tandem tetramer WT-(D172N)2-WT, was transfected into COS-1 cells using the liposome method, and after 48–72 h single-channel currents were recorded in the inside-out configuration at 150 mm internal and external K+. Steady-state open probability of outward currents decreased with larger depolarizations. The activation curve was fitted with a single Boltzmann equation. The voltages of half-activation in the absence of spermine were +35.9 mV (WT), +55.0 mV (WT-(D172N)2-WT) and +76.7 mV (D172N). Open-time and zero-current-time histograms were constructed. The open-time histogram was fitted with a single exponential function. Two exponential functions were necessary to fit the closed-time histogram. In each channel, internal spermine at a concentration of 1–100 nm reduced the open time of the outward currents in a concentration-dependent manner and produced one blocked state without affecting the inward currents, suggesting that spermine acts as an open channel blocker. The normalized steady-state open probability-spermine concentration curve was fitted by saturation kinetics with a Hill coefficient of 1. On the assumption of the linear sequential state model, the unblock and blocking rates were estimated in each channel. Unblock rates depended on the number of D172N mutant subunits, but blocking rates did not. The results suggest that closing gates work independently of the spermine block and D172 is involved in both intrinsic gating and the spermine block.