Detection of the Gua/Cyt-to-Cyt/Gua mutation in a Gua/Cyt-lined sequence using Zn2+-cyclen polyacrylamide gel electrophoresis

We previously reported a method for the detection of single-nucleotide polymorphisms by polyacrylamide gel electrophoresis (PAGE) with an additive Zn²⁺–cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecane), called Zn²⁺–cyclen–PAGE. The method is based on the difference in mobility of mutant DNA (in the same length) in PAGE that is due to Zn²⁺–cyclen binding to thymine bases accompanying a total charge decrease and a local conformation change of target DNA. In combination with a heteroduplexing technique, the method is more accurate, as shown by clear gel-shifting bands. However, the question remains as to whether the Gua/Cyt-to-Cyt/Gua mutation, which is far apart from the Thy/Ade base (i.e., in a Gua/Cyt-lined sequence), can be detected by this Thy-dependent method. In this study, we determined the potency of Zn²⁺–cyclen–PAGE for the detection of the Gua/Cyt-to-Cyt/Gua single substitutions in some artificial Gua/Cyt-lined sequences derived from a human cardiac sodium channel gene, SCN5A. All Gua/Cyt-to-Cyt/Gua substitutions in the 28-set samples tested, which are 1 to 10 bases away from the nearest Thy/Ade, were successfully detected by designing DNA fragments of the appropriate length.     

Conformational Changes of DNA by Photoirradiation of DNA-Bis(ZnII-Cyclen)-Azobenzene Complex

Bis(Zn^II-cyclen)-azobenzene derivative, which has two Zn^II-macrocyclic tetraamine complexes connected through azobenzene spacer, has been synthesized as a cross-linking agent for double stranded DNA in aqueous solution. The Zn^II-cyclen derivative selectively binds to A-T base pairs producing complexes between the Zn^II-cyclen moiety and the imide-deprotonated thymine with breaking A-T base pairs. The azobenzene spacer undergoes cis/trans photoisomerization in the complex between the Zn^II-cyclen derivative and the DNA duplex. The conformation of the DNA remarkably changed by photoisomerization of the azobenzene linker, when the Zn^II-cyclen derivative binds to the DNA duplex with an interstrand cross-linking manner.     

Associate Hydrations and Energies of Nucleotide Bases as Revealed by Low-Temperature Field Ionization Mass-Spectrometric Data

Abstract

The formation of water clusters, polyhydrates of nucleotide bases and their associates during simultaneous condensation of water and base molecules in vacuo onto a surface of a needle emitter cooled to 170 K was studied by field ionization mass spectrometry. It was found that different emitter temperatures are characterized by a specific distribution of intensities of cluster currents, depending on the number of water molecules in clusters. These distributions correlate with structural peculiarities and the relative energetics of formation of water clusters, polyhydrates of nucleotide bases and their associates at low temperature. The features observed in mass spectra for clusters m⁹Ade (H₂O)₅, m¹Ura (H₂O)₄ and m⁹Ade m¹Ura (H₂O)₂ are treated as a result of formation of energetically favorable structures stabilized by H-bonded bridges of water molecules.

The relative association constants and formation enthalpies of the noncomplementary pairs Ade Cyt, Gua Ura and the associates which model the aminoacid-base complexes m¹Ura Gin and m₂ ^1,3Thy Gin were determined from the temperature dependencies of the intensities of mass spectra peaks in the range 290–320 K.     

DNA Staining in Agarose Gels with ZN2+-Cyclen-Pyrene

A pyrene-labeled Zn²⁺-cyclen complex for the staining of DNA in agarose gels is reported. The metal chelate coordinates reversibly to the DNA phosphate backbone, which induces the formation of pyrene excimers. The typical pyrene excimer emission is used for the detection of the DNA. Staining is limited to agarose gels and is less sensitive than ethidium bromide, but DNA amounts as low as 10 ng and short DNA strands (～300 b.p.) are detectable. Gel extraction as a standard technique in molecular biology was successfully performed after staining with Zn²⁺-cyclen-pyrene. Cytotoxicity tests on HeLa and V-79 cells reveal that the zinc-cyclen pyrene probe is significant less toxic compared to ethidium bromide.     

Base modifications in plasmid DNA caused by potassium permanganate

KMnO4 is a powerful oxidizing agent which has been used to modify DNA bases. In previous studies, mild KMnO4 treatment has been shown to preferentially modify Thy; Cyt and Gua are modified only under harsher conditions to as yet unidentified products. In the present study, denatured plasmid pCMV beta gal DNA was exposed to 0.015-1.5 mM KMnO4, pH 8.6, at 4 degrees C for 5 min, after which the DNA was hydrolyzed in formic acid, trimethylsilylated, and analyzed for modified base content by gas chromatography-mass spectrometry/selected ion monitoring. KMnO4 treatment, even at concentrations as low as 0.015 mM, caused a concentration-dependent increase in the Thy products Thy glycol and 5-hydroxy-5-methylhydantoin, the Cyt products Cyt glycol, 5,6-dihydroxycytosine, and 5-hydroxyhydantoin, the Ade product 8-hydroxyadenine, and the Gua product 8-hydroxyguanine. The Ade product 4,6-diamino-5-formamidopyrimidine and the Gua product 2,6-diamino-4-hydroxy-5-formamidopyrimidine were minimally (less than or equal to 2-fold) increased by treatment with greater than or equal to 0.8 mM KMnO4. These data demonstrate that, in addition to Thy, Cyt, Gua, and Ade bases in plasmid DNA may be modified by treatment with KMnO4, even under mild conditions. They represent the first identification of Cyt, Gua, and Ade products caused by KMnO4 treatment. Furthermore, these data suggest that previous studies which have used treatment with KMnO4 to study the mutagenicity of Thy glycol specifically or as a Thy-specific probe in DNA structure should be interpreted with caution.     

Binding of Ca<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> to factor IX/X-binding protein from venom of <Emphasis Type="Italic">Agkistrodon halys</Emphasis> Pallas: stabilization of the structure during GdnHCl-induced and thermally induced denaturation

Coagulation factor IX/coagulation factor X binding protein from the venom of Agkistrodon halys Pallas (AHP IX/X-bp) is a unique coagulation factor IX/coagulation factor X binding protein (IX/X-bp). Among all IX/X-bps identified, only AHP IX/X-bp is a Ca²⁺- and Zn²⁺-binding protein. The binding properties of Ca²⁺ and Zn²⁺ ions binding to apo-AHP IX/X-bp and their effects on the stability of the protein have been investigated by isothermal titration calorimetry, fluorescence spectroscopy, and differential scanning calorimetry. The results show that AHP IX/X-bp has two metal binding sites, one specific for Ca²⁺ with lower affinity for Zn²⁺ and one specific for Zn²⁺ with lower affinity for Ca²⁺. The bindings of Ca²⁺ and Zn²⁺ in the two sites are entropy- and enthalpy-driven. The binding affinity of AHP IX/X-bp for Zn²⁺ is 1 order of magnitude higher than for Ca²⁺ for either high-affinity binding or low-affinity binding, which accounts for the existence of one Zn²⁺ in the purified AHP IX/X-bp. Guanidine hydrochloride (GdnHCl)-induced and thermally induced denaturations of Ca²⁺–Ca²⁺-AHP IX/X-bp, Zn²⁺–Zn²⁺-AHP IX/X-bp, and Ca²⁺–Zn²⁺-AHP IX/X-bp are all a two-state processes with no detectable intermediate state(s), indicating the Ca²⁺/Zn²⁺-induced tight packing of the protein. Ca²⁺ and Zn²⁺ increase the structural stability of AHP IX/X-bp against GdnHCl or thermal denaturation to a similar extent. Although Ca²⁺ and Zn²⁺ have no obvious effect on the secondary structure of AHP IX/X-bp, they induce different rearrangements in local conformation. The Zn²⁺-stabilized specific conformation of AHP IX/X-bp may be helpful to its recognition of the structure of coagulation factor IX. This work suggests that in vitro, Ca²⁺ plays a structural rather than an active role in the anticoagulation of AHP IX/X-bp, whereas Zn²⁺ plays both structural and active roles in the anticoagulation. In blood, Ca²⁺ binds to AHP IX/X-bp and stabilizes its structure, whereas Zn²⁺ cannot bind to AHP IX/X-bp owing to the low Zn²⁺ concentration. AHP IX/X-bp prolongs the clotting time in vivo through its binding only with coagulation factor X/activated coagulation factor X.     

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase

Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a k_cat/K_M of 3.2 × 10⁷ and 3.0 × 10⁷ s^{? 1}M^{? 1}, respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site‐directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10‐fold decrease in k_cat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828–840. © 2016 Wiley Periodicals, Inc.     

Formation of ternary complexes involving zinc or copper ions, polynucleotides, and polypeptides containing glutamic acid and tyrosine residues

Metal ions such as Zn²⁺ and Cu²⁺ can mediate interactions between copolypeptides (Glu_x, Tyr_y)_n and polynucleotides. CD data show that these ternary complexes are characterized by an unstacking of nucleic acid bases, while the polypeptide adopts an α-helical conformation as observed in the two binary complexes polynucleotide–cation and polypeptide–cation. Fluorescence studies demonstrate that tyrosyl side chains interact with nucleic acid bases in the ternary complexes, leading to a quenching of tyrosine fluorescence.     

Physiology of genotypic differences in zinc and copper uptake in rice and tomato

Summary Excised roots of rice (Oryzae sativa L.) cv IR26 absorbed both Zn²⁺ and Cu²⁺ from 0.01 mM to 0.50 mM external solutions at rates twice those of cv M101 over a 30-min period. However, the latter have a two-fold greater affinity (1/Km) for Zn²⁺ and Cu²⁺ than do those of the former. Zinc²⁺ and Cu²⁺ mutually and competitively inhibited uptake of each other, indicating that both micronutrient cations are absorbed through the same uptake mechanism or carrier sites. Further, these differences in uptake rates are restricted to roots but they cannot be explained by variations in root surface areas. Excised roots of tomato (Lycopersicon esculentum L.) cv Kewalo absorbed Zn²⁺ and Cu²⁺ much more rapidly than did cv Sel 7625-2. Uptake of each cation was competitively and reciprocally inhibited by the other, so Zn²⁺ and Cu²⁺ are seemingly accumulated through the same uptake system in tomato also. Tomato cultivars Kewalo and Sel 7625-2 did not differ with regard to affinities of the root apices for Zn²⁺ and Cu²⁺; however. Vmax values for Zn²⁺ and Cu²⁺ uptake by roots of cv Kewalo were three-fold greater than those for cv Sel 7625-2. Journal Series 2991 of the Hawaii Institute of Tropical Agriculture and Human Resources. Supported by USDA/CSRS Grants Program in Tropical and Subtropical Agriculture (83-CSRS-2-2245).     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.     

Protein Folding: Grand Challenge of Nature

Abstract

Conformational preferences of the hypermodified nucleic acid bases N6-(Δ² -cis-hydroxyisopentenyl)adenine, cis-io⁶Ade also known as cis-zeatin, and N⁶-(Δ² -trans-hydroxyisopentenyl)adenine, trans-io⁶ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms²io⁶Ade or cis-ms²zeatin, and trans-ms²io⁶Ade or trans-ms²zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AMI and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io⁶Ade, trans-io⁶Ade, cis-ms²io⁶Ade and trans-ms²io⁶Ade are such that in each of these molecules the isopentenyl substituent spreads away (has “dista” conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io⁶Ade as well as cis- ms²io⁶Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io⁶Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms²io⁶Ade, possible O-H…S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms²io⁶Ade as well as trans-ms²io⁶Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this nonobstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i⁶Ade and ms²i⁶Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms²-zeatin (ms²io⁶Ade) modification.     

Macrocyclic zinc(II) complexes for selective recognition of nucleobases in single- and double-stranded polynucleotides

 As an extension of our earlier discoveries that Zn^II-cyclen complex (1) (cyclen=1,4,7,10-tetraazacyclododecane) and Zn^II-acridine-pendant cyclen complex Zn^II-N-(9-acridin)ylmethyl-cyclen (3) are the first compounds to selectively recognize thymidine and uridine nucleosides in aqueous solution at physiological pH, the interaction of these and a relevant complex, bis(Zn^II-cyclen) (7), has been investigated with a series of polynucleotides, single-stranded poly(U) and poly(G), and double-stranded poly(A)·poly(U), poly(dA)·poly(dT) and poly(dG)·poly(dC). These Zn^II-cyclen complexes interact with the imide-containing nucleobases in the single-stranded poly(U), unperturbed by the presence of the anionic phosphodiester backbone. The affinity constant of 1 for each N(3)-deprotonated uracil base in poly(U) is determined to be log K= 5.1 by a kinetic measurement, which is almost the same as log K=5.2 for the interaction of 1 with uridine. Thus, they disrupt the A-U (or A-T) hydrogen bonds to unzip the duplex of poly(A)·poly(U) or poly(dA)·poly(dT), as demonstrated by lowering of the melting temperatures (T _m) of poly(A)·poly(U) and poly(dA)·poly(dT) in 5 mM Tris-HCl buffer (pH 7.6, 10 mM NaCl) with increase in their concentrations. The order of the denaturing efficiency is well correlated with that of the 1 : 1 affinity constants for each complex with uracil or thymine;7>3>1. The comparison of circular dichroism (CD) spectra for poly(A)·poly(U), poly(A), and poly(U) in the presence of 3 has revealed a structural change from poly(A)·poly(U) to two single strands, poly(A) and poly(U), caused by 3 binding exclusively to uracils in poly(U). On the other hand, the acridine-pendant cyclen complex 3, which earlier was found to associate with guanine by the Zn^II coordinating with guanine N(7), in addition to the π-π stacking, interacts with guanine in the double helix of poly(dG)·poly(dC) from outside and stabilized the double-stranded structure, as indicated by higher T _m. Received: 31 December 1997 / Accepted: 23 February 1998     

Developmental regulation of cytokinin, spore germination inhibitor discadenine and related enzymes in Dictyostelium discoideum

The amounts of discadenine (spore germination inhibitor of Dictyostelium discoideum) and its precursor, N⁶-Δ²-isopentenyladenine (i⁶Ade) in cells of D. discoideum were measured at various stages of differentiation. The activities of the enzymes involved in the biosynthesis of these bases, i.e., discadenine synthetase and 5′-AMP: Δ²-isopentenylpyrophosphate Δ²-isopentenyltransferase (5′-AMP isopentenyltransferase) in the cells were also measured. During differentiation, discadenine appeared in the cells at the stage of culmination before i⁶Ade was detected. The activity of 5′-AMP: Δ²-isopentenylpyrophosphate Δ²-isopentenyltransferase in cell extracts increased after the beginning of culmination, much later than the increase in discadenine synthetase activity. The order of appearance of these bases and enzymes is apparently the reverse of that expected from the biosynthetic route. The implications of these findings are discussed.     

Zinc Induces Expression of the BH3-only Protein PUMA Through p53 and ERK Pathways in SH-SY5Y Neuroblastoma Cells

Accumulating evidence suggests that zinc (Zn²⁺) contributes to neuronal death in pathologic states such as ischemia. p53-upregulated modulator of apoptosis (PUMA), which is a BH3-only protein, is known to promote apoptosis through a tumor suppressor p53-dependent and -independent mechanism. In this study, we examined the effect of Zn²⁺ on the induction of the PUMA gene in human neuroblastoma SH-SY5Y cells. The expression of PUMA was induced by Zn²⁺ in a dose- and time-dependent manner. A reporter assay revealed that Zn²⁺ activated the PUMA promoter. In addition, the mutation of the p53 binding site in the PUMA promoter region reduced promoter activation by Zn²⁺. These findings suggest that p53 participates in Zn²⁺-induced PUMA expression. Furthermore, we also demonstrated here that Zn²⁺ stimulates the phosphorylation of ERK and that the MEK-ERK pathway inhibitor, U0126, suppressed Zn²⁺-induced PUMA expression. Taken together, these results indicate that Zn²⁺ regulates the induction of PUMA through p53 and ERK pathways.     

A critical role of the transient receptor potential melastatin 2 channel in a positive feedback mechanism for reactive oxygen species-induced delayed cell death

Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn²⁺ homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H₂O₂ induced neuroblastoma SH-SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn²⁺]_i, and therefore used this cell model to investigate the mechanisms underlying ROS-induced TRPM2-mediated delayed cell death. H₂O₂ increased concentration-dependently the [Zn²⁺]_i and caused lysosomal dysfunction and Zn²⁺ loss and, furthermore, mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase-1-dependent TRPM2 channel activation with PJ34 and 3,3′,5,5′-tetra-tert-butyldiphenoquinone, inhibiting the TRPM2 channel with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid, or chelating Zn²⁺ with N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Bafilomycin-induced lysosomal dysfunction also resulted in mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2-APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR-induced Zn²⁺ uptake in isolated mitochondria, which was prevented by TPEN. H₂O₂-induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4-specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H₂O₂-induced ROS generation, lysosomal dysfunction and Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX-mediated ROS generation, lysosomal Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS-induced delayed cell death.     

Calcium-Sensitive Fluorescent Dyes Can Report Increases in Intracellular Free Zinc Concentration in Cultured Forebrain Neurons

Abstract: High concentrations of Zn²⁺ are found in presynaptic terminals of excitatory neurons in the CNS. Zn²⁺ can be released during synaptic activity and modulate postsynaptic receptors, but little is known about the possibility that Zn²⁺ may enter postsynaptic cells and produce dynamic changes in the intracellular Zn²⁺ concentration ([Zn²⁺]_i). We used fura-2 and magfura-2 to detect the consequences of Zn²⁺ influx in cultured neurons under conditions that restrict changes in intracellular Ca²⁺ and Mg²⁺ concentrations. The resulting ratio changes for both dyes were reversed completely by the Zn²⁺ chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, indicating that these dyes are measuring changes in [Zn²⁺]_i. We found that fura-2 was useful in measuring small increases in [Zn²⁺]_i associated with exposure to Zn²⁺ alone that may be mediated by a Na⁺/Ca²⁺ exchanger. Magfura-2, which has a lower affinity for Zn²⁺, was more useful in measuring larger agonist-stimulated increases in [Zn²⁺]_i. The coapplication of 300 µM Zn²⁺ and 100 µM glutamate/10 µM glycine resulted in a [Zn²⁺]_i increase that was ～40–100 nM in magnitude and could be inhibited by the NMDA receptor antagonist, MK-801 (30 µM), or extracellular Na⁺. This suggests that Zn²⁺ influx can occur through at least two different pathways, leading to varying increases in [Zn²⁺]_i. These findings demonstrate the feasibility of measuring changes in [Zn²⁺]_i in neurons.     

Conformational Preferences of Modified Nucleic Acid Bases N6-Methyl-N6-(N-Threonylcarbonyl) Adenine and 2-Methylthio-N6-(N-Threonylcarbonyl) Adenine by the Quantum Chemical PCILO Calculations

Abstract

Conformational preferences of the hypermodified nucleic acid bases N⁶-methyl-N⁶-(N- threonylcarbonyl) Adenine, m⁶ tc⁶ Ade, and 2-methylthio-N⁶-(N-threonylcarbonyl) Adenine, mS² tc⁶ Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m⁶ tc⁶ Ade and mS² tc⁶ Ade alike the threonylcar- bonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N⁶-(N-threonylcarbonyl) Adenine, tc⁶ Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc⁶ Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(l) of the adenine base.

The N⁶-methyl protons, in m⁶ tc⁶ Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS² tc⁶ Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N⁶-(Δ²-isopentenyl) Adenine, mS² i⁶ Ade, where the 2-methylthio group orients trans to the C(2)- N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced.