5,10,15,20-Tetra-(N-methyl-3-pyridyl)porphyrin destabilizes the anti-parallel telomeric quadruplex d(TTAGGG)4

We studied the parameters of binding of 5,10,15,20-tetra-(N-methyl-3-pyridyl)porphyrin (TMPyP3) to the anti-parallel human telomeric G-quadruplex d(TTAGGG)4, the oligonucleotide dTTAGGGTTAGAG(TTAGGG)2 that does not form a quadruplex structure, as well as to the double stranded d(AC)8 x d(GT) and single stranded d(AC)8 and d(GT)8 DNAs. The analysis of absorption revealed that the binding constants and the number of DNA binding sites for TMPyP3 were d(AC)8 < d(GT)8 < d(AC)8 x d(GT)8 = d(TTAGGG)4 < dTTAGGGTTAGAG(TTAGGG)2. We demonstrated for the first time that the binding constant of TMPyP3 with the non-quadruplex chain dTTAGGGTTAGAG(TTAGGG)2 (1.3 x 10(7) M(-1)) is approximately 3 times bigger than the binding constant with the quadruplex d(TTAGGG)4 (4.6 x 10(6) M(-1)). Binding of two TMPyP3 molecules to d(TTAGGG)4 led to a decrease of thermostability of the G-quadruplex (deltaT(m) = -8 degrees C). Circular dichroism spectra of TMPyP3:d(TTAGGG)4 complexes revealed a shift of DNA structure from the G-quadruplex to an irregular chain. We hypothesize that partial destabilization of the telomeric G-quadruplex by TMPyP3 might be a reason for relatively low potency of this ligand as a telomerase inhibitor, as well as its marginal cytotoxicity for cultured tumor cells.     

Bcl-2 Promoter Sequence G-Quadruplex Interactions with Three Planar and Non-Planar Cationic Porphyrins: TMPyP4, TMPyP3, and TMPyP2

The interactions of three related cationic porphyrins, TMPyP4, TMPyP3 and TMPyP2, with a WT 39-mer Bcl-2 promoter sequence G-quadruplex were studied using Circular Dichroism, ESI mass spectrometry, Isothermal Titration Calorimetry, and Fluorescence spectroscopy. The planar cationic porphyrin TMPyP4 (5, 10, 15, 20-meso-tetra (N-methyl-4-pyridyl) porphine) is shown to bind to a WT Bcl-2 G-quadruplex via two different binding modes, an end binding mode and a weaker mode attributed to intercalation. The related non-planar ligands, TMPyP3 and TMPyP2, are shown to bind to the Bcl-2 G-quadruplex by a single mode. ESI mass spectrometry experiments confirmed that the saturation stoichiometry is 4:1 for the TMPyP4 complex and 2:1 for the TMPyP2 and TMPyP3 complexes. ITC experiments determined that the equilibrium constant for formation of the (TMPyP4)₁/DNA complex (K₁ = 3.7 × 10⁶) is approximately two orders of magnitude greater than the equilibrium constant for the formation of the (TMPyP2)₁/DNA complex, (K₁ = 7.0 × 10⁴). Porphyrin fluorescence is consistent with intercalation in the case of the (TMPyP4)₃/DNA and (TMPyP4)₄/DNA complexes. The non-planar shape of the TMPyP2 and TMPyP3 molecules results in both a reduced affinity for the end binding interaction and the elimination of the intercalation binding mode.     

Interaction Between Nanoparticulate Anatase TiO<Subscript>2</Subscript> and Lactate Dehydrogenase

In order to study the mechanisms underlying the effects of TiO₂ nanoparticles on lactate dehydrogenase (LDH, EC1.1.1.27), Institute of Cancer Research region mice were injected with nanoparticulate anatase TiO₂ (5 nm) of various doses into the abdominal cavity daily for 14 days. We then examined LDH activity in vivo and in vitro and direct evident for interaction between nanoparticulate anatase TiO₂ and LDH using spectral methods. The results showed that nanoparticulate anatase TiO₂ could significantly activate LDH in vivo and in vitro; the kinetics constant (Km) and Vmax were 0.006 μM and 1,149 unit mg⁻¹ protein min⁻¹, respectively, at a low concentration of nanoparticulate anatase TiO₂, and 3.45 and 0.031 μM and 221 unit mg⁻¹ protein min⁻¹, respectively, at a high concentration of nanoparticulate anatase TiO₂. By fluorescence spectral assays, the nanoparticulate anatase TiO₂ was determined to be directly bound to LDH, and the binding constants of the binding site were 1.77 × 10⁸ L mol⁻¹ and 2.15 × 10⁷ L mol⁻¹, respectively, and the binding distance between nanoparticulate anatase TiO₂ and the Trp residue of LDH was 4.18 nm, and nanoparticulate anatase TiO₂ induced the protein unfolding. It was concluded that the binding of nanoparticulate anatase TiO₂ altered LDH structure and function.     

β-Adrenoreceptor agonists and antagonists modulate the activity of α<Subscript>2</Subscript>-adrenoreceptors in rat cortex cerebral membranes

The influence of isoprenaline- and propranolole-induced activation and inhibition of β-adrenoreceptors on the specific nonselective α₂-antagonist [³H]RX821002 binding was studied on rat cerebral cortex subcellular membrane fractions. It was shown that the ligand-receptor interaction for α₂-adrenoreceptors corresponded to the model that assumed the presence of one receptor pool and binding of two ligand molecules to a receptor dimer. The following parameters were determined for [³H]RX821002 binding to α₂-adrenoreceptors: K _d1 = 1.57 ± 0.27 nM, B _max = 7.24 ± 1.63 fmol/mg of protein, n = 2. In the case of isoprenaline-induced activation of β-adrenoreceptors the binding of radiolabeled ligand to α₂-adrenoreceptors was described by the same model. The affinity of α₂-adrenoreceptors for [³H]RX821002 decreased more than twofold (K _d = 3.55 ± 0.02 nM) and the quantity of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg of protein). Propranolole changed the model of ligand binding, and two pools of receptors were detected with the following parameters: K _d1 = 0.61 ± 0.02 nM, K _d2 = 3.41 ± 0.13 nM, B _ml = 1.88 ± 0.028 fmol/mg of protein, B _m2 = 9.27 ± 0.08 fmol/mg of protein, n = 2. The data suggest that α₂-adrenoreceptors in subcellular membrane fractions from rat cerebral cortex exist in dimeric form. Isoprenaline and propranolole exhibit modulating effect on the specific antagonist binding to α₂-adrenoreceptors, which results in the inhibition and alteration of [³H]RX821002 binding parameters.     

Peculiarities of cyanide binding to the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript>-type cytochrome oxidase from the thermophilic bacterium <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Cytochrome c oxidase of the ba ₃-type from Thermus thermophilus does not interact with cyanide in the oxidized state and acquires the ability to bind heme iron ligands only upon reduction. Cyanide complexes of the reduced heme a ₃ in cytochrome ba ₃ and in mitochondrial aa ₃-type cytochrome oxidase are similar spectroscopically, but the a ₃²⁺-CN complex of cytochrome ba ₃ is strikingly tight. Experiments have shown that the K _d value of the cytochrome ba ₃ complex with cyanide in the presence of reductants of the enzyme binuclear center does not exceed 10⁻⁸ M, which is four to five orders of magnitude less than the K _d of the cyanide complex of the reduced heme a ₃ of mitochondrial cytochrome oxidase. The tightness of the cytochrome ba ₃ complex with cyanide is mainly associated with an extremely slow rate of the ligand dissociation (k _off ≤ 10⁻⁷ sec⁻¹), while the rate of binding (k _on ∼ 10² M⁻¹·sec⁻¹) is similar to the rate observed for the mitochondrial cytochrome oxidase. It is proposed that cyanide dissociation from the cytochrome ba ₃ binuclear center might be hindered sterically by the presence of the second ligand molecule in the coordination sphere of Cu_B²⁺. The rate of cyanide binding with the reduced heme a ₃ does not depend on pH in the neutral area, but it approaches linear dependence on H⁺ activity in the alkaline region. Cyanide binding appears to be controlled by protonation of an enzyme group with pK _a = 8.75.     

The Mechanism of CeCl<Subscript>3</Subscript> on the Activiation of Alanine Aminotransferase from Mice

The activity of alanine aminotransferase (ALT; E.C. 2.6.1.2) is often changed upon inflammatory responses in animals. Rare earths was shown to provoke various inflammatory responses both in rats and mice; however, the molecular mechanism by which rare earths exert its toxicity has not been completely understood, especially, we know little about the mechanism of the interaction between CeCl₃ and ALT. In this report, we investigated the mechanisms of CeCl₃ on ALT activity in vivo and in vitro. Our results showed that Ce³⁺ could significantly activate ALT in vivo and in vitro; the kinetics constant (Km) and Vmax were 0.018 μM and 1,380 unit mg⁻¹ protein min⁻¹, respectively, at a low concentration of Ce³⁺, and 0.027 μM and 624 unit mg⁻¹ protein min⁻¹, respectively, at a high concentration of Ce³⁺. By UV absorption and fluorescence spectroscopy assays, the Ce³⁺ was determined to be directly bound to ALT; the binding site of Ce³⁺ to ALT was 1.72, and the binding constants of the binding site were 4.82 × 10⁸ and 9.05 × 10⁷ L mol⁻¹. Based on the analysis of the circular dichroism spectra, it was concluded that the binding of Ce³⁺ altered the secondary structure of ALT, suggesting that the observed enhancement of ALT activity was caused by a subtle structural change in the active site through the formation of the complex with Ce³⁺.     

Ab initio studies on the decomposition kinetics of CF<Subscript>3</Subscript>OCF<Subscript>2</Subscript>O radical

The present study deals with the decomposition of CF₃OCF₂O radical formed from a hydrofluoroether, CF₃OCHF₂ (HFE-125), in the atmosphere. The study is performed using ab initio quantum mechanical methods. Two plausible pathways of decomposition of the titled species have been considered, one involving C-O bond scission and the other occurring via F atom elimination. The geometries of the reactant, products and transition states involved in the decomposition pathways are optimized and characterized at DFT (B3LYP) level of theory using 6-311G(d,p) basis set. Single point energy calculations have been performed at G2M(CC,MP2) level of theory. Out of the two prominent decomposition channels considered, the C-O bond scission is found to be dominant involving a barrier height of 15.3 kcal mol⁻¹ whereas the F-elimination path proceeds with a barrier of 26.1 kcal mol⁻¹. The thermal rate constants for the above two decomposition pathways are evaluated using canonical transition state theory (CTST) and these are found to be 1.78 × 10⁶ s⁻¹ and 2.83 × 10⁻⁷ s⁻¹ for C-O bond scission and F-elimination respectively at 298 K and 1 atm pressure. Transition states are searched on the potential energy surfaces involved during the decomposition channels and each of the transition states is characterized. The existence of transition states on the corresponding potential energy surface is ascertained by performing intrinsic reaction coordinate (IRC) calculation.     

Disordering of human telomeric G-quadruplex with novel antiproliferative anthrathiophenedione

Linear heteroareneanthracenediones have been shown to interfere with DNA functions, thereby causing death of human tumor cells and their drug resistant counterparts. Here we report the interaction of our novel antiproliferative agent 4,11-bis[(2-{[acetimido]amino}ethyl)amino]anthra[2,3-b]thiophene-5,10-dione with telomeric DNA structures studied by isothermal titration calorimetry, circular dichroism and UV absorption spectroscopy. New compound demonstrated a high affinity (K_ass∼10⁶ M⁻¹) for human telomeric antiparallel quadruplex d(TTAGGG)₄ and duplex d(TTAGGG)₄∶d(CCCTAA)₄. Importantly, a ∼100-fold higher affinity was determined for the ligand binding to an unordered oligonucleotide d(TTAGGG TTAGAG TTAGGG TTAGGG unable to form quadruplex structures. Moreover, in the presence of Na⁺ the compound caused dramatic conformational perturbation of the telomeric G-quadruplex, namely, almost complete disordering of G-quartets. Disorganization of a portion of G-quartets in the presence of K⁺ was also detected. Molecular dynamics simulations were performed to illustrate how the binding of one molecule of the ligand might disrupt the G-quartet adjacent to the diagonal loop of telomeric G-quadruplex. Our results provide evidence for a non-trivial mode of alteration of G-quadruplex structure by tentative antiproliferative drugs.     

Theoretical study of N<Subscript>4</Subscript>X (X = O,S, Se) systems

A series of N₄X (X = O, S, Se) compounds have been examined with ab initio and density functional theory (DFT) methods. To our knowledge, these compounds, except for the C_2v ring and the C_3v towerlike isomers of N₄O, are first reported here. The ring structures are the most energetically favored for N₄X (X = O and S) systems. For N₄Se, the cagelike structure is the most energetically favored. Several decomposition and isomerization pathways for the N₄X species have been investigated. The dissociation of C_2v ring N₄O and N₄S structures via ring breaking and the barrier height are only 1.1 and −0.2 kcal mol⁻¹ at the CCSD(T)/6-311+G*//MP2/6-311+G* level of theory. The dissociation of the cagelike N₄X species is at a cost of 12.1–16.2 kcal mol⁻¹. As for the towerlike and triangle bipyramidal isomers, their decomposition or isomerization barrier heights are all lower than 10.0 kcal mol⁻¹. Although the C_S cagelike N₄S isomer has a moderate isomerization barrier (18.3–29.1 kcal mol⁻¹), the low dissociation barrier (−1.0 kcal mol⁻¹) indicates that it will disappear when going to the higher CCSD(T) level. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of shelf life of <Emphasis Type="Italic">Spirulina platensis</Emphasis> (MI<Subscript>2</Subscript>) grown in the Philippines

Gamma linolenic acid (GLA) degradation in Spirulina followed first-order reaction kinetics. At an accelerated temperature range of 45 to 55°C, the degradation rate constants (k _r) of GLA obtained were 4.0 × 10⁻² to 8.8 × 10⁻² day⁻¹. The energy of activation (E _a) was 16.53 kcal mol⁻¹, and the Q₁₀ was 2.22. Based on 20% GLA degradation, the shelf life of sun-dried Spirulina at 30°C is 263 days or 8.6 months using the Arrhenius plot, and 258 days or 8.5 months using the Q ₁₀ approach. Presented at the 6th Meeting of the Asia Pacific Society of Applied Phycology, Manila, Philippines.     

Kinetics of CO conversion into H<Subscript>2</Subscript> by <Emphasis Type="Italic">Carboxydothermus hydrogenoformans</Emphasis>

The objective of this study was to improve the biological water–gas shift reaction for producing hydrogen (H₂) by conversion of carbon monoxide (CO) using an anaerobic thermophilic pure strain, Carboxydothermus hydrogenoformans. Specific hydrogen production rates and yields were investigated at initial biomass densities varying from 5 to 20 mg volatile suspended solid (VSS) L⁻¹. Results showed that the gas–liquid mass transfer limits the CO conversion rate at high biomass concentrations. At 100-rpm agitation and at CO partial pressure of 1 atm, the optimal substrate/biomass ratio must exceed 5 mol CO g⁻¹ biomass VSS in order to avoid gas–liquid substrate transfer limitation. An average H₂ yield of 94 ± 3% and a specific hydrogen production rate of ca. 3 mol g⁻¹ VSS day⁻¹ were obtained at initial biomass densities between 5 and 8 mg VSS⁻¹. In addition, CO bioconversion kinetics was assessed at CO partial pressure from 0.16 to 2 atm, corresponding to a dissolved CO concentration at 70°C from 0.09 to 1.1 mM. Specific bioactivity was maximal at 3.5 mol CO g⁻¹ VSS day⁻¹ for a dissolved CO concentration of 0.55 mM in the culture. This optimal concentration is higher than with most other hydrogenogenic carboxydotrophic species.     

Effects of elevated CO<Subscript>2</Subscript> and/or O<Subscript>3</Subscript> on hormone IAA in needles of Chinese pine

This study examined the effects of season-long exposure of Chinese pine (Pinus tabulaeformis) to elevated carbon dioxide (CO₂) and/or ozone (O₃) on indole-3-acetic acid (IAA) content, activities of IAA oxidase (IAAO) and peroxidase (POD) in needles. Trees grown in open-top chambers (OTC) were exposed to control (ambient O₃, 55 nmol mol⁻¹ + ambient CO₂, 350 μmol mol⁻¹, CK), elevated CO₂ (ambient O₃ + high CO₂, 700 μmol mol⁻¹, EC) and elevated O₃ (high O₃, 80 ± 8 nmol mol⁻¹ + ambient CO₂, EO) OTCs from 1 June to 30 September. Plants grown in elevated CO₂ OTC had a growth increase of axial shoot and needle length, compared to control, by 20% and 10% respectively, while the growth in elevated O₃ OTC was 43% and 7% less respectively, than control. An increase in IAA content and POD activity and decrease in IAAO activity were observed in trees exposed to elevated CO₂ concentration compared with control. Elevated O₃ decreased IAA content and had no significant effect on IAAO activity, but significantly increased POD activity. When trees pre-exposed to elevated CO₂ were transferred to elevated O₃ (EC–EO) or trees pre-exposed to elevated O₃ were transferred to elevated CO₂ (EO–EC), IAA content was lower while IAAO activity was higher than that transferred to CK (EC–CK or EO–CK), the change in IAA content was also related to IAAO activity. The results indicated that IAAO and POD activities in Chinese pine needles may be affected by the changes in the atmospheric environment, resulting in the change of IAA metabolism which in turn may cause changes in Chinese pine’s growth. An erratum to this article can be found at     

CO<Subscript>2</Subscript>-enriched microenvironment affects sucrose and macronutrients absorption and promotes autotrophy in the <Emphasis Type="Italic">in vitro</Emphasis> culture of kiwi (<Emphasis Type="Italic">Actinidia deliciosa</Emphasis> Chev. Liang and Ferguson)

In traditional in vitro culture, the low CO₂ concentration inside the vessels restricts photosynthesis and necessitates the addition of sucrose to the culture medium as the main energy source, thus bringing about changes in the absorption of mineral elements from the culture medium. In this study, we investigated macronutrient absorption and sugar consumption in Actinidia deliciosa Chevalier Liang and Ferguson cv. Hayward (kiwi), cultured on medium supplemented with varying amounts of sucrose (0, 10, and 20 g l⁻¹) under both heterotrophy and autotrophy, flushed with different concentrations of CO₂ (non-ventilation, 300, 600, and 2,000 μl l⁻¹). In ventilated systems with 20 g l⁻¹ of sucrose, sucrose absorption was less than under non-ventilation. The lowest rate of sucrose absorption was recorded when the explants were cultured on medium supplemented with 20 g l⁻¹ of sucrose and flushed with 600 μl l⁻¹ CO₂. Absorption of NO₃ ⁻, PO₄ ³⁻, and Mg²⁺ were high (maximum) at the end of the culture period (40 d) in explants flushed with 600 μl l⁻¹ CO₂ that have been cultured 20 d in the presence of sucrose and then transferred to a sucrose-free medium. These autotrophic conditions promoted maximum plant growth in terms of both fresh and dry mass as well as the length and number of shoots and leaves. The study shows that to maintain an optimum regime of mineral nutrition for prolonged culture of kiwi in vitro, an increased amount of these three ions should be supplemented in Murashige and Skoog’s medium.     

Complexes of antiparallel telomeric G-quadruplex d(TTAGGG)4 with carboxymethyl tetracationic porphyrins

Porphyrins are a chemical class that is widely used in drug design. Cationic porphyrins may bind to DNA guanine quadruplexes. We report the parameters of the binding of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium) porphyrin (P1) and 5,10,15,20-tetrakis(N-etoxycarbonylmethyl-4-pyridinium) porphyrin (P2) to antiparallel telomeric G-quadruplex formed by d(TTAGGG)₄ sequence (TelQ). The binding constants (K _i ) and the number of binding sites (N _j ) were determined from absorption isotherms generated from the absorption spectra of complexes of P1 and P2 with TelQ. Compound P1 demonstrated a high affinity to TelQ (K _i = (40 ± 6) × 10⁶ M^?1, N ₁ = 1; K ₂ = (5.4 ± 0.4) × 10⁶ M^?1, N ₂ = 2). In contrast, the binding constants of P2-TelQ complexes (K ₁ = (3.1 ± 0.2) × 10⁶ M^?1, N ₁ = 1; K ₂ = (1.2 ± 0.2) × 10⁶ M^?1, N ₂ = 2) were one order of magnitude smaller than the corresponding values for P2-TelQ complexes. Measurements of the quantum yield and fluorescence lifetime of the drug’s TelQ complexes revealed two types of binding sites for P1 and P2 on the quadruplex oligonucleotide. We concluded that strong complexes can result from the interaction of the porphyrins with TTA loops whereas the weaker complexes are formed with G-quartets. The altered TelQ conformation detected by the circular dichroism spectra of P1-TelQ complexes can be explained by the disruption of the G-quartet. We conclude that peripheral carboxy groups contribute to the high affinity of P1 for the antiparallel telomeric G-quadruplex.     

The platinum-olefin binding energy in series of (PH<Subscript>3</Subscript>)<Subscript>2</Subscript>Pt(olefin) complexes - a theoretical study

Theoretical investigation of Pt(0)-olefin organometallic complexes containing tertiary phosphine ligands was focused on the strength of platinum-olefin electronic interaction. DFT theoretical study of electronic effects in a substantial number of ethylene derivatives was evaluated in terms of the Pt-olefin binding energy using MP2 correlation theory. Organometallics bearing coordinated olefins with general formula (R¹R²C = CR³R⁴)Pt(PH₃)₂ [R = various substituents] had been selected, including olefins containing both electron-donor substituents as well as electron-withdrawing groups. The stability of the corresponding complexes increases with a strengthening electron-withdrawal ability of the olefin substituents. Figure Representation of (CH₂ = CHR)Pt(PPh₃)₂ and the stability chart     

Spectroscopic study on the binding of a cationic porphyrin to DNA G-quadruplex under different K+ concentrations

We have performed systematic spectroscopic titrations to characterize the binding reaction of cationic meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) with the G-quadruplex (G4) of human telomeric single-strand oligonucleotide d[TAGGG(TTAGGG)3T] (S24), for which special effort was made to examine the TMPyP4-G4 binding stoichiometry, the binding modes, and the conformational conversion of the G4 structure under different potassium ion (K+) concentration. It is found that, in the presence of 0, 10 mM, and 100 mM K+, TMPyP4 forms a complex with the anti-parallel G4 in a TMPyP4-to-G4 molar ratio of 5, 5 and 3, respectively, and the increase of K+ concentration would reduce the binding affinity of TMPyP4 to G4. For the TMPyP4-G4 complex, the end-stacking mode and groove binding mode were presumed mainly by the results of time-resolved fluorescence spectroscopy in the three cases. Most importantly, it is found that TMPyP4 can directly induce the formation of the anti-parallel G4 structure from the single-strand oligonucleotide S24 in the absence of K+, and that it can preferentially induce the conformational conversion of the G4 structure from the hybrid-type to the anti-parallel one in the presence of K+.     

Clorgyline and other propargylamine derivatives as inhibitors of succinate-dependent H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at NADH:UBIQUINONE oxidoreductase (Complex I) in brain mitochondria

Complex I is the main O₂ ⁻ producer of the mitochondrial respiratory chain. O₂ ⁻ release is low with NAD-linked substrates and increases strongly during succinate oxidation, which increases the QH₂/Q ratio and is rotenone sensitive. We show that the succinate dependent O₂ ⁻ production (measured as H₂O₂ release) is inhibited by propargylamine containing compounds (clorgyline, CGP 3466B, rasagiline and TVP-1012). The inhibition does not affect membrane potential and is unaffected by ΔpH modifications. Mitochondrial respiration is similarly unaffected. The propargylamines inhibition of O₂ ⁻/H₂O₂ production is monitored also in the presence of the Parkinson's disease toxin dopaminochrome which stimulates O₂ ⁻ release. Propargylamine-containing compounds are the first pharmacological inhibitors described for O₂ ⁻ release at Complex I.     

Interaction of pyrrolobenzodiazepine (PBD) ligands with parallel intermolecular G-quadruplex complex using spectroscopy and ESI-MS

Studies on ligand interaction with quadruplex DNA, and their role in stabilizing the complex at concentration prevailing under physiological condition, has attained high interest. Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the interaction of PBD and TMPyP4 ligands, stoichiometry and selectivity to G-quadruplex DNA. Two synthetic ligands from PBD family, namely pyrene-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrid (PBD1), mixed imine-amide pyrrolobenzodiazepine dimer (PBD2) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) were studied. G-rich single-stranded oligonucleotide d(5'GGGGTTGGGG3') designated as d(T(2)G(8)), from the telomeric region of Tetrahymena Glaucoma, was considered for the interaction with ligands. ESI-MS and spectroscopic methods viz., circular dichroism (CD), UV-Visible, and fluorescence were employed to investigate the G-quadruplex structures formed by d(T(2)G(8)) sequence and its interaction with PBD and TMPyP4 ligands. From ESI-MS spectra, it is evident that the majority of quadruplexes exist as d(T(2)G(8))(2) and d(T(2)G(8))(4) forms possessing two to ten cations in the centre, thereby stabilizing the complex. CD band of PBD1 and PBD2 showed hypo and hyperchromicity, on interaction with quadruplex DNA, indicating unfolding and stabilization of quadruplex DNA complex, respectively. UV-Visible and fluorescence experiments suggest that PBD1 bind externally where as PBD2 intercalate moderately and bind externally to G-quadruplex DNA. Further, melting experiments using SYBR Green indicate that PBD1 unfolds and PBD2 stabilizes the G-quadruplex complex. ITC experiments using d(T(2)G(8)) quadruplex with PBD ligands reveal that PBD1 and PBD2 prefer external/loop binding and external/intercalative binding to quadruplex DNA, respectively. From experimental results it is clear that the interaction of PBD2 and TMPyP4 impart higher stability to the quadruplex complex.     

Characterization of a putative pheromone biosynthesis-activating neuropeptide (PBAN) receptor from the pheromone gland of Heliothis peltigera

The binding of [³H]tyrosyl-PBAN28-33NH₂ to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO₃ ⁻ ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a K_d of 5.73 ± 1.05 × 10⁻⁶ M and a Bmax of 1.85 ± 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH₂ and PBAN28-33ΝΗ₂ with a K_i of 4.3 ± 1.1 × 10⁻⁶ M and 4.9 ± 2.6 × 10⁻⁶ M, respectively. Accepted: 4 February 1999     

Conformational changes induced in the human protein translin and in the single-stranded oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5) upon binding of these oligodeoxynucleotides by translin

Translin is a human single-stranded DNA and RNA binding protein that has been highly conserved in eukaryotic evolution. It consists of eight subunits having a highly helical secondary structure that assemble into a ring. The DNA and the RNA are bound inside the ring. Recently, some of us demonstrated that the human translin specifically binds the single-stranded microsatellite repeats, d(GT)(n), the human telomeric repeats, d(TTAGGG)(n), and the Tetrahymena telomeric repeats, d(GGGGTT)(n). These data suggested that translin might be involved in recombination at d(GT)(n).d(AC)(n) microsatellites and in telomere metabolism. Other data indicated that translin might stimulate binding of telomerase to single-stranded telomeric overhangs by unwinding secondary structures formed by the telomeric repeats. Here we present a circular dichroism (CD) analysis of complexes formed between the human translin and the microsatellite and telomeric oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5). We report that conformational changes occur in both the translin and the oligodeoxynucleotides upon formation of the complexes. In translin octamers bound to the oligodeoxynucleotide d(GT)(12), the fraction of alpha-helices decreases from approximately 67% to approximately 50%, while the fraction of turns and of the unordered structure increases from approximately 11% to approximately 17% and from approximately 19% to approximately 24%, respectively. In the bound oligodeoxynucleotide d(GT)(12), we observed CD shifts which are consistent with a decrease of base stacking and a putative anti-syn switch of some guanines. The oligodeoxynucleotide d(TTAGGG)(5) formed intramolecular quadruplexes under the conditions of our assays and translin was found to unfold the quadruplexes into structures consisting of a single hairpin and three unwound single-stranded d(TTAGGG) repeats. We suggest that such unfolding could account for the stimulation of telomerase activity by translin mentioned above.