The effect of spermine binding on the reactivity of DNA towards carcinogenic alkylating agents

The effect of spermine binding on the electrostatic potential of DNA is evaluated. The calculations are performed for the essential reactive sites, atoms N7 and O6 of guanine, N3 and N7 of adenine, of the nucleic acid and for its surface envelope. An important weakening of the potential is found affecting all the important reactive sites in both grooves and spreading moreover along the polynucleotide chain far away from the site of binding of the ligand. These results are discussed in connection with the experimentally observed inhibitory effect of spermine binding on DNA methylation by carcinogenic agents.     

Different Binding Modes of Spermine to A-T and G-C Base Pairs Modulate the Bending and Stiffening of the DNA Double Helix

Abstract

The influence of base composition (and sequence) on the process of interaction between synthetic polynucleotides and spermine, has been investigated using ultraviolet (including second derivative) spectroscopy, and electric dichroism.

Different binding modes of spermine to poly(dG-dC) as compared to A-T containing polynucleotides, were evidenced. An interaction with the N7 and 06 of guanine is probably partially involved in the former case while simple electrostatic interaction with the phosphate groups would dominate in the latter.

In the intermediate binding range (spermine over DNA phosphate molar ratios Sp/P of the order of 0.1 to 0.2), the complexes with poly(dA) · poly(dT) and those with poly(dA-dT) displayed an important contribution of a permanent dipole moment to the orientation mechanism, as detected by the application of bipolar pulses in electric dichroism experiments. Just prior to precipitation (at Sp/P slightly larger than 0.3), these polynucleotides show electric dichroism and relaxation times characteristics corresponding to toroidal particles formation resulting from a bending of their chains. This implies asymmetric binding to phosphate sites on A-T containing polynucleotides. At low Sp/P ratios, spermine induced a stiffening of poly (dG-dC). No influence of spermine on the orientation mechanism of this polynucleotide was detected for Sp/P values ranging from zero to 0.35. The spermine-induced bending of A-T rich regions thus appears to be essential for DNA condensation into toroidal particles.     

Insight into the molecular recognition of spermine by DNA quadruplexes from an NMR study of the association of spermine with the thrombin‐binding aptamer

The preferred residence sites and the conformation of DNA‐bound polyamines are central to understanding the regulatory roles of polyamines. To this end, we have used a series of selective ¹³C‐edited and selective total correlation spectroscopy‐edited one‐dimensional (1D) nuclear Overhauser effect spectroscopy NMR experiments to determine a number of intramolecular ¹H nuclear Overhauser effect (NOE) connectivities in ¹³C‐labelled spermine bound to the thrombin‐binding aptamer. The results provide evidence that the aptamer‐bound spermine adopts a conformation that optimizes electrostatic and hydrogen bond contacts with the aptamer backbone. The distance between the nitrogen atoms of the central aminobutyl is reduced by an increase in the population of gauche conformers at the C6–C7 bonds, which results in either a curved or S‐shaped spermine conformation. Molecular modelling contributes insight toward the mode of spermine binding of these spermine structures within the narrow grooves of DNA quadruplexes. In each case, the N5 ammonium group makes hydrogen bonds with two nearby phosphates across the narrow groove. Our results have implications for the understanding of chromatin structure and the rational design of quadruplex‐binding drugs. Copyright © 2013 John Wiley & Sons, Ltd.     

A theoretical evaluation of the effect of netropsin binding on the reactivity of DNA towards alkylating agents.

The effect of netropsin binding on the electrostatic potential of DNA reactive sites is presented. Calculations are performed for atoms N7 and O6 of guanine, N3 and N7 of adenine of model, 25 base pair long, DNA-netropsin complexes. An important weakening of the potential is found spreading along all the oligonucleotide chain studied. The results are discussed in connection with the inhibitory effect of a related ligand, distamycin A, on DNA methylation.     

Use of Chemically Modified Nucleotides to Determine a 62-Nucleotide RNA Crystal Structure: A Survey of Phosphorothioates,Br, Pt and Hg

Abstract

Two important challenges confronting RNA crystallographers are producing crystals and finding isomorphous heavy-atom derivatives. Non-isomorphism can be addressed by determining the phases using the multiwavelength anomalous dispersion (MAD) method. These phases can be greatly improved by combining phases from MAD experiments done on different heavy-atom derivatives. Heavy-atom derivatives can be created by chemically modifying the RNA through covalent attachment of bromine or mercury to C5 of pyrimidines or [Pt(NH₃)₃]²⁺ to N7 of guanine. While phosphorothioates can provide mercury binding sites, disorder can reduce their value for phase determination. The location of these chemical modifications is critical since crystallization of these derivatized RNAs is sensitive to heavy atom induced conformational alterations and crystal packing.     

In vitro DNA binding activity and molecular docking reveals pierisin-5 as an anti-proliferative agent against gastric cancer

Abstract

Pierisin-5 is a DNA dependent ADP ribosyltransferase (ADRT) protein from the larvae of Indian cabbage white butterfly, Pieris canidia. Interestingly, Pierisin-5 ADP-ribosylates the DNA as a substrate, but not the protein and subsequently persuades apoptotic cell death in human cancer cells. This has led to the investigation on the DNA binding activity of Pierisin-5 using in vitro and in silico approaches in the present study. However, both the structure and the mechanism of ADP-ribosylation of pierisin-5 are unknown. In silico modeled structure of the N-terminal ADRT catalytic domain interacted with the minor groove of B-DNA for ribosylation with the help of β-NAD⁺ which lead to a structural modification in DNA (DNA adduct). The possible interaction between calf thymus DNA (CT-DNA) and purified pierisin-5 protein was studied through spectral–spatial studies and the blue shift and hyperchromism in the UV–Visible spectra was observed. The DNA adduct property of pierisin-5 protein was validated by in vitro cytotoxic assay on human gastric (AGS) cancer cell lines. Our study is the first report of the mechanism of DNA binding property of pierisin-5 protein which leads to the induction of cytotoxicity and apoptotic cell death against cancer cell lines.

Communicated by Ramaswamy H. Sarma     

Inhibition of acetylpolyamine and spermine oxidases by the polyamine analogue chlorhexidine

Acetylpolyamine and spermine oxidases are involved in the catabolism of polyamines. The discovery of selective inhibitors of these enzymes represents an important tool for the development of novel anti-neoplastic drugs. Here, a comparative study on acetylpolyamine and spermine oxidases inhibition by the polyamine analogue chlorhexidine is reported. Chlorhexidine is an antiseptic diamide, commonly used as a bactericidal and bacteriostatic agent. Docking simulations indicate that chlorhexidine binding to these enzymes is compatible with the stereochemical properties of both acetylpolyamine oxidase and spermine oxidase active sites. In fact, chlorhexidine is predicted to establish several polar and hydrophobic interactions with the active site residues of both enzymes, with binding energy values ranging from ?7.6 to ?10.6 kcal/mol. In agreement with this hypothesis, inhibition studies indicate that chlorhexidine behaves as a strong competitive inhibitor of both enzymes, values of K_i being 0.10 μM and 0.55 μM for acetylpolyamine oxidase and spermine oxidase, respectively.     

3种水稻土中7株固氮蓝细菌的分离与特征

【背景】蓝细菌是水生和陆地生态系统中生物固氮的主要贡献者。【目的】增加对稻田土壤固氮蓝细菌的了解,获得用于进一步研究的可培养固氮蓝细菌菌株。【方法】选择3种具有不同固氮能力的水稻土,采用BG11-N培养基分离培养固氮蓝细菌菌株,对新分离菌株进行形态特征观察,通过基因组DNA的nifH基因扩增明确其固氮潜力,进一步采用乙炔还原法和~(15)N_2示踪法定量测定其固氮能力,通过基因组DNA的16SrRNA基因序列比对进行鉴定。【结果】在光照培养条件下,采用BG11-N培养基共分离纯化得到自养菌株7株,细胞呈圆形或椭圆形、单列、无分枝、丝状和念珠状,在固体培养基上形成团垫状菌落。新分离菌株在BG11-N培养基中生长状况良好,以基因组DNA为模板可扩增出nifH基因,乙炔还原法和~(15)N_2示踪法测定结果显示具有较高固氮能力,同时具有铁载体生成能力。结合16S rRNA基因序列比对和形态特征,7株菌被初步鉴定隶属于念珠藻科(Nostocaceae)。【结论】从水稻土中分离到在稻田生物固氮中发挥重要作用的蓝细菌(念珠藻科)菌株,可培养固氮蓝细菌菌株固氮能力较高,兼具铁载体生成能力,可作为进一步深入研究的微生物资源,具有潜在的研究应用价值。     

Synthesis and DNA threading properties of quaternary ammonium [Ru(phen)2(dppz)] derivatives

Ruthenium complexes with one dipyrido[3,2-a:2′-3′-c]phenazine (dppz) ligand, e.g. [Ru(phen)₂(dppz)]²⁺ (phen = phenanthroline), shows strong binding to double helical DNA and are well-known DNA “light-switch” molecules. We have here investigated four new [Ru(phen)₂(dppz)]²⁺ derivatives with different bulky quaternary ammonium substituents on the dppz ligand to find relationships between molecular structure and intercalation kinetics, which is considered to be of importance for antitumor applicability. Linear dichroism spectroscopy shows that the enantiomers of the new complexes exhibit very similar binding geometries (intercalation of dppz moiety between adjacent DNA base pairs) as the enantiomers of the parent [Ru(phen)₂(dppz)]²⁺ complex. Absorption spectra and luminescence properties provide further evidence for a final intercalative binding mode which has to be reached by threading of a bulky moiety between the strands of the DNA. Δ-enantiomers of all the new complexes show much slower association and dissociation kinetics than that of a reference complex without a cationic substituent. Kinetics were not very different whether the bulky quaternary group was derived from hexamethylene tetramine or 1,4-diazabicyclo-(2,2,2)octane (DABCO) or whether it had one or two positive charges. However, a complex in which the hexamethylene tetramine substituent is attached via a phenyl group showed a lowered association rate, in addition to an improved quantum yield of luminescence. A second positive charge on the DABCO substituent resulted in a much slower dissociation rate, suggesting that the distance from the Ru-centre and the amount of charge are both important for threading intercalation kinetics.     

Analogues of tentoxin: Tools for mechanistic investigations

Summary Tentoxin [cyclo-(MeAla¹-Leu²-MeΔPhe³-Gly⁴] is a metabolite isolated from a phytopathogenic fungusAlternaria alternata, which induces chlorosis of many plants. This potential natural herbicide binds specifically to the soluble part CF₁ of the chloroplastic coupling factor, which is a proton ATP-synthase. The effect of the toxin is concentration dependent: at low concentration it is a powerful inhibitor, while at higher concentration, it stimulates the enzyme. We synthesized tentoxin and we designed new analogues in order to vary the hydrophobicity on the side chain and to study the structure activity relationships. Comparative activities suggest that it is possible to separate inhibitory and activating effects using tentoxin analogues, showing some evidence for the existence of two binding sites with different affinity constant.     

Identification and characterization of improved nitrogen efficiency in interspecific hybridized new-type Brassica napus

Background and Aims

Oilseed rape (Brassica napus) is an important oil crop worldwide. The aim of this study was to identify the variation in nitrogen (N) efficiency of new-type B. napus (genome A^rA^rC^cC^c) genotypes, and to characterize some critical physiological and molecular mechanisms in response to N limitation.

Methods

Two genotypes with contrasting N efficiency (D4-15 and D1-1) were identified from 150 new-type B. napus lines, and hydroponic and pot experiments were conducted. Root morphology, plant biomass, N uptake parameters and seed yield of D4-15 and D1-1 were investigated. Two traditional B. napus (genome AⁿAⁿCⁿCⁿ) genotypes, QY10 and NY7, were also cultivated. Introgression of exotic genomic components in D4-15 and D1-1 was evaluated with molecular markers.

Key Results

Large genetic variation existed among traits contributing to the N efficiency of new-type B. napus. Under low N levels at the seedling stage, the N-efficient new-type D4-15 showed higher values than the N-inefficient D1-1 line and the traditional B. napus QY10 and NY7 genotypes with respect to several traits, including root and shoot biomass, root morphology, N accumulation, N utilization efficiency (NutE), N uptake efficiency (NupE), activities of nitrate reductase (NR) and glutamine synthetase (GS), and expression levels of N transporter genes and genes that are involved in N assimilation. Higher yield was produced by the N-efficient D4-15 line compared with the N-inefficient D1-1 at maturity. More exotic genome components were introgressed into the genome of D4-15 (64·97 %) compared with D1-1 (32·23 %).

Conclusions

The N-efficient new-type B. napus identified in this research had higher N efficiency (and tolerance to low-N stress) than traditional B. napus cultivars, and thus could have important potential for use in breeding N-efficient B. napus cultivars in the field.     

Synthesis and Physiological Activity of Methyl 6′6′-Didemethyl Abscisate and New Chiral Analogs

Methyl 6′, 6′-didemethyl abscisate (5) was synthesized and assayed to elucidate the physiological activity of methyl substituents on the cyclohexene ring of abscisic acid (ABA). During this study two new chiral stereoisomeric analogs 6 and 7 were synthesized from l-and d-carvone. The rice seedling assay and germination assay of garden radish showed that 6′-methyl groups of ABA were not important in biological activity and that 5′-isopropenyl analogs 6 and 7 were inactive.     

Binding Sites of the Polyamines Putrescine,Cadaverine, Spermidine and Spermine on A- and B-DNA Located by Simulated Annealing

Abstract

Molecular dynamics simulations with simulated annealing are performed on polyamine-DNA systems in order to determine the binding sites of putrescine, cadaverine, spermidine and spermine on A- and B-DNA. The simulations either contain no additional counterions or sufficient Na⁺ ions, together with the charge on the polyamine, to provide 73% neutralisation of the charges on the DNA phosphates. The stabilisation energies of the complexes indicate that all four polyamines should stabilise A-DNA in preference to B-DNA, which is in agreement with experiment in the case of spermine and spermidine, but not in the case of putrescine or cadaverine. The major groove is the preferred binding site on A-DNA of all the polyamines. Putrescine and cadaverine tend to bind to the sugar-phosphate backbone of B-DNA, whereas spermidine and spermine occupy more varied sites, including binding along the backbone and bridging both the major and minor grooves.     

Inhibitory role of TGIF in the As<Subscript>2</Subscript>O<Subscript>3</Subscript>-regulated p21<Superscript><Emphasis Type="Italic">WAF1/CIP1</Emphasis></Superscript> expression

Although arsenic is an infamous carcinogen, it has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we had demonstrated that opposing effects of ERK1/2 and JNK on p21 expression in response to arsenic trioxide (As₂O₃) are mediated through the Sp1 responsive elements of the p21 promoter in A431 cells. Presently, we demonstrate that Sp1, and c-Jun functionally cooperate to activate p21 promoter expression through Sp1 binding sites (−84/−64) by using DNA affinity binding, chromatin immunoprecipitation, and promoter assays. Surprisingly, As₂O₃-induced c-Jun(Ser63/73) phosphorylation can recruit TGIF/HDAC1 to the Sp1 binding sites and then suppress p21 promoter activation. We suggest that, after As₂O₃ treatment, the N-terminal domain of c-Jun phosphorylation by JNK recruits TGIF/HDAC1 to the Sp1 sites and then represses p21 expression. That is, TGIF is involved in As₂O₃-inhibited p21 expression, and then blocks the cell cycle arrest.     

Regulation of the Ca2+-ATPase by cholesterol: A specific or non-specific effect?

Abstract

Like other integral membrane proteins, the activity of the Sarco/Endoplasmic Reticulum Ca²⁺-ATPase (SERCA) is regulated by the membrane environment. Cholesterol is present in the endoplasmic reticulum membrane at low levels, and it has the potential to affect SERCA activity both through direct, specific interaction with the protein or through indirect interaction through changes of the overall membrane properties. There are experimental data arguing for both modes of action for a cholesterol-mediated regulation of SERCA. In the current study, coarse-grained molecular dynamics simulations are used to address how a mixed lipid-cholesterol membrane interacts with SERCA. Candidates for direct regulatory sites with specific cholesterol binding modes are extracted from the simulations. The binding pocket for thapsigargin, a nanomolar inhibitor of SERCA, has been suggested as a cholesterol binding site. However, the thapsigargin binding pocket displayed very little cholesterol occupation in the simulations. Neither did atomistic simulations of cholesterol in the thapsigargin binding pocket support any specific interaction. The current study points to a non-specific effect of cholesterol on SERCA activity, and offers an alternative interpretation of the experimental results used to argue for a specific effect.     

Structural Changes of Yeast tRNATyr Caused by the Binding of Divalent Ions in the Presence of Spermine

Abstract

The existence of specific sites in tRNA for the binding of divalent cations has been seriously questioned by electrostatic considerations [Leroy & Guéron (1979) Biopolymers, 16, 2429–2446], However, our earlier studies of the binding of Mg²⁺ and Mn²⁺ to yeast tRNA^Tyr have indicated that spermine creates new binding sites for divalent cations [Weygand-Durasevi? et al. (1977) Biochim. Biophys. Acta, 479, 332–344; Nöthig-Laslo et al. (1981) Eur. J. Biochem. 117, 263–267]. We have now used yeast tRNA^Tyr, spin labeled at the hypermodified purine (i⁶A-37) in the anticodon loop, to study the effect of spermine on the binding of manganese ions. The presence of eight spermine molecules per tRNA^Tyr at high ionic strength (0.2 M NaCl, 0.05 M triethanolamine-HCl) and at low temperature (7°C) enhances the binding of manganese to tRNA^Tyr. This effect could not be explained by electrostatic binding. The initial binding of manganese to tRNA^Tyr affects the motional properties of the spin label indicating a change of the conformation of the anticodon loop. From the absence of the paramagnetic effect of manganese on the ESR spectra of the spin label one can conclude that the first binding site for manganese is at a distance from i⁶A-37, influencing the spin label motion through a long-range effect. The enhancement of the binding of manganese to tRNA^Tyr by spermine is lost upon destruction of its specific macromolecular structure and it does not occur in single stranded or in double-stranded polynucleotides. The observed effect can be explained by the binding of Mn²⁺ to new sites, created by the binding of spermine, which are specific for the macromolecular structure of tRNA.     

Structures and stability of N<Subscript>13</Subscript><Superscript>+</Superscript> and N<Subscript>13</Subscript><Superscript>−</Superscript> clusters

The structures and stabilities of eleven N₁₃ ⁺ and N₁₃ ^– isomers have been investigated with second-order Møller–Plesset (MP2) and density functional theory (DFT) methods. Five N₁₃ ⁺ isomers and six N₁₃ ^– isomers are all reasonable local minima on their potential energy hypersurfaces. The most stable N₁₃ ⁺ cation is structure C-2 with C_2v symmetry, which contains a pentazole ring and two N₄ open chains. It is different from those of the N₇ ⁺ and N₉ ⁺ clusters, but similar to the N₁₁ ⁺ cluster. Meanwhile, the most stable N₁₃ ^– structure A-2 is composed of a pentazole ring and a six-membered ring connected by two nitrogen atoms. It is not only different from those of the N₇ ^– and N₉ ^– clusters, but also from the N₁₁ ^– cluster. The decomposition pathways of structures C-2 and A-2 were investigated at the B3LYP/(aug)-cc-pVDZ level. From the barrier heights of the structures C-2 and A-2 decomposition processes, it is suggested that C-2 is difficult to observe experimentally and A-2 may be observed as a short-lived species. Figure Optimized geometrical parameters of N₁₃ ⁺ isomer C-2      

Application of 15N natural abundance technique for evaluating biological nitrogen fixation in oil palm ecotypes at nursery stage in pot experiments and at mature plantation sites

A range of different species of diazotrophic bacteria has been found in tissues and the rhizosphere of oil palm plants, suggesting a potential to benefit from biological nitrogen fixation (BNF). A few studies have confirmed that plantlets at nursery stage can benefit significantly from BNF after inoculation with Azospirillum spp. but no data are available regarding the benefit from naturally-occurring diazotrophic bacteria in oil palm. The results described here were derived from two pot trials laid out under controlled conditions with plantlets from two important regions for palm oil production in Brazil, as well as from different field sites of mature oil palm plantations. The ¹⁵N natural abundance technique was employed to estimate plant dependence on BNF (%Ndfa) by the different ecotypes grown in soil and previously characterized as hosting diazotrophic bacteria. From both pot trials it was possible to identify some ecotypes of high potential for N₂-fixation that reached in some cases approximately 50%Ndfa. However, the accuracy of measurement still needs to be improved using more suitable reference plants for pot experiments. Values of δ ¹⁵N signals from oil palm and reference plants in the field were inconclusive concerning any benefit from BNF to oil palm, owing to apparently high temporal and spatial variability of δ ¹⁵N of the plant-available N in the heterogeneous soil matrix for the different palm and reference plant tested.     

Demonstration and Characterization of two Classes of Cardiac Glycoside Binding Sites to Rat Heart Membrane Preparations Using Quantitative Computer Modeling

Abstract

Cardiac glycoside binding to rat heart membrane preparations was measured by rapid filtration technique. The binding data were analyzed using quantitative computer analysis. The experimental results using [³H]-ouabain as the labeled ligand were consistent with a model in which cardiac glycoside specific binding occurs at two independent classes of sites. The high affinity sites were characterized by a dissociation constants of 40 nM, 50 nM, and 61 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 1.3 pmoles/mg protein. The lower affinity sites for ouabain were characterized by dissociation constants of 2.3 µM, 67 nM and 71 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 3 pmoles/mg protein. Potassium ions inhibit [³H]-ouabain binding in a dose dependent manner with an IC₅₀ of 500 µM. Quantitative computer modelling indicated that potassium inhibits ouabain binding at both binding sites.