Synthesis and spectroscopic properties of reconstituted zinc-myoglobin appending a DNA-binding platinum(II) complex

A reconstituted zinc-myoglobin (ZnMb) dyad, ZnMb-[Pt(bpy)(en)]2+, has been prepared by incorporating chemically-modified zinc-porphyrin, being capable of DNA-binding of the Pt complex, [Pt(bpy)(en)]2+, where bpy and en are 2,2'-bipyridine and ethylenediamine, respectively. The steady-state fluorescence of the cofactor, [Pt(bpy)(mu-enPP)Zn]Cl2, in MeOH indicates that the excited singlet state of zinc--porphyrin was almost quenched, probably because of the strong hydrophobic and pi-pi stacking interactions between the [Pt(bpy)(mu-enPP)Zn]2+ ions. In the reconstituted ZnMb-[Pt(bpy)(en)]2+, the quenching reaction of 1(ZnMb)* with the [Pt(bpy)(en)]2+ moiety does not occur, indicating apo-Mb matrix is essential. On the other hand, when the [Pt(bpy)(en)]2+ moiety was excited, the enhancement of the fluorescence from ZnMb unit was observed. It is suggested that the energy transfer from (1)([Pt(bpy)(en)]2+)* to ZnMb occurs. The spectroscopic changes of ZnMb-[Pt(bpy)(en)]2+ in the presence of calf-thymus DNA were also provided. Soret band at 428 nm gradually decreased, and isosbestic points at 321, 414, and 432 nm were observed with increasing the DNA concentration. When the Pt(II) moiety was excited at lambda(ex) 321 nm, the fluorescence signal around 600 nm similarly decreased. The synthetic manipulation of ZnMb by using a DNA-binding Pt(II) complex demonstrates sensitive fluorescent signal for DNA and valuable information to study photoinduced electron transfer within a Mb-DNA complex.     

Stereoselective and driving-force-dependent photoinduced electron-transfer reactions of zinc myoglobin with optically active N,N′-dimethylcinchoninium and N,N′-dimethylcinchonidinium ions

In order to understand the detailed mechanism of the stereoselective photoinduced electron-transfer (ET) reactions of zinc-substituted myoglobin (ZnMb) with optically active molecules by flash photolysis, we designed and prepared new optically active agents, such as N,N′-dimethylcinchoninium diiodide ([MCN]I₂) and N,N′-dimethylcinchonidinium diiodide ([MCD]I₂). The photoexcited triplet state of ZnMb, ³(ZnMb)*, was successfully quenched by [MCN]²⁺ and [MCD]²⁺ ions to form the radical pair of ZnMb cation (ZnMb^·+) and reduced [MCN]^·+ and [MCD]^·+, followed by a thermal back ET reaction to the ground state. The rate constants (k _q) for the ET quenching at 25 °C were obtained as k _q(MCN)=(1.9±0.1)×10⁶ M⁻¹ s⁻¹ and k _q(MCD)=(3.0±0.2)×10⁶ M⁻¹ s⁻¹, respectively. The ratio of k _q(MCD)/k _q(MCN)=1.6 indicates that the [MCD]²⁺ preferentially quenches ³(ZnMb)*. The second-order rate constants (k _b) for the thermal back ET reaction from [MCN]^·+ and [MCD]^·+ to ZnMb^·+ at 25 °C were k _b(MCN)=(0.79±0.04)×10⁸ M⁻¹ s⁻¹ and k _b(MCD)=(1.0±0.1)×10⁸ M⁻¹ s⁻¹, respectively, and the selectivity was k _q(MCD)/k _q(MCN)=1.3. Both quenching and thermal back ET reactions are controlled by the ET step. In the quenching reaction, the energy differences of ΔΔH ^≠(MCD–MCN) and ΔΔS ^≠(MCD–MCN) at 25 °C were obtained as −1.1 and 0 kJ mol⁻¹, respectively. On the other hand, ΔΔH ^≠(MCD–MCN)=11±2 kJ mol⁻¹ and TΔΔS ^≠(MCD–MCN)=−10±2 kJ mol⁻¹ were given in the thermal back ET reaction. The highest stereoselectivity of 1.7 for [MCD]^·+ found at low temperature (10 °C) was due to the ΔΔS ^≠ value obtained in the thermal back ET reaction. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The charge-transfer complex between protochlorophyllide and NADPH: an intermediate in protochlorophyllide photoreduction

A hypothesis describing the mechanism of photoactive protochlorophyllide (P) photoreduction in vivo, relating mainly to the molecular nature of the intermediates, is proposed. The hypothesis is compatible with currently published experimental data. After illumination of etiolated barley leaves at 143 to 153 K, the absorption of P remains essentially unchanged, but a new absorption band at 690 nm is observed. Appearance of this new intermediate enables to distinguish between light and dark stages of the photoconversion reaction. When returned to the higher temperature in the dark, the treated leaves begin accumulating chlorophyllide (Chlide), concomitant with the disappearance of the 690-nm band. The decay time of the excited P (P^*) is estimated at 300 ps, which approximates the time constant of photoinduced electron transfer (ET). It is suggested that the charge-transfer complex (CTC) in its ground state (GS) (ground state of CTC formed by the partial (δ) electron transfer), i.e. (P^δ−•••H–D^δ+), between P and NADPH – the electron and proton donor (H–D) – accumulates in the following sequence: P^* + H–D → (P^*•••H–D)→[(P^*•••H–D)←(P⁻•••H–D⁺)] → ¹(P⁻•••H–D⁺)] → ³(P⁻•••H–D⁺) → (P^δ−•••H–D ^δ+), where an equilibrium state (ES) – [(P^*•••H–D)←(P⁻•••H–D⁺)] – with a lifetime of about 1 to 2 ns, exists between the local excited (LE) and ET states. The existence of a triplet ET state – ³(P⁻•••H–D⁺) – is proposed because the time interval between recording of the ES and appearance of the CTC GS (35–250 ns) does not fit the lifetime of the singlet excited complex (exciplex). It is feasible that apart from NADPH, other intermediate proton carriers are contemporaneously involved in the dark reaction (P^δ−•••H–D^δ+) → Chlide, because proton binding to the C₇–C₈ bond in vivo takes place in the trans-configuration. The hydride ion may approach the C₇–C₈ bond from one side by heterolytic fission and an additional proton, donated by the protein group, may be simultaneously added to this bond from the opposite side of the porphyrin nucleus surface. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modulation of DNA intercalation by resveratrol and genistein

Time correlated Single Photon Counting study (TCSPC) was performed for the first time to evaluate the effect of resveratrol (RES) and genistein (GEN) at 10–100 μM and 10–150 μM respectively, in modulating the DNA conformation and the variation induced due to intercalation by the dyes, ethidium bromide (EtBr) and acridine orange (AO). It is demonstrated using UV-absorption and fluorescence spectroscopy that RES and GEN, at 50 μM and 100 μM respectively can bind to DNA resulting in significant de-intercalation of the dyes, preventing their further intercalation within DNA. Hyperchromicity with red/blue shifts in DNA when bound to dyes was reduced upon addition of RES and GEN. DNA-dependent fluorescence of EtBr and AO was quenched in the presence of RES by 87.97% and 79.13% respectively, while similar quenching effect was observed for these when interacted with GEN (85.52% and 83.85%). It is found from TCSPC analysis that the higher lifetime component or constituent of intercalated dyes (τ₂, A ₂) decreased with the subsequent increase in smaller component or constituent of free dye (τ₁, A ₁) after the interaction of drugs with the intercalated DNA. Thus these findings signify that RES and GEN can play an important role in modulating DNA intercalation, leading to the reduction in DNA-directed toxicity.     

Quenching of DNA-ethidium fluorescence by amsacrine and other antitumor agents: a possible electron-transfer effect

The antitumor agent amsacrine, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), when bound to double-stranded DNA, particularly poly(deoxyadenylicthymidylic acid), reduced the fluorescence of bound ethidium without physically displacing it from DNA. Fluorescence lifetime measurements showed that the reduction of fluorescence was not due to reduction of the lifetime of the excited state of ethidium. Rather, a proportion of the DNA-bound ethidium changed to a state where the fluorescence was highly quenched. Several other 9-anilinoacridine derivatives, and also 9-hydroxyellipticine, caused quenching of ethidium-DNA fluorescence, whereas 9-aminoacridine, proflavin, and ellipticine had no effect. Resonance energy transfer (F?rster transfer) is not responsible for the effect since there is no spectral overlap between the absorption spectrum of any of the agents and the fluorescence emission spectrum of ethidium. It is suggested that quenching may occur as a result of reversible formation of electron-transfer complexes between the intercalating drug and the excited state of ethidium.     

Charge separation in photosynthesis via a spin exchange coupling mechanism

A new mechanism for the primary photoinduced charge separation in photosynthesis is proposed. It involves as a real intermediate between the excited special pair state P^* and the primary charge separated state P⁺ H_L ^– a trip-trip-singlet B^T B_L ^T, which consists of a triplet on the dimer P and a further triplet on the monomer B_L. Both combine to a singlet. The electron transfer is caused by spin exchange couplings. The transient spectrum of the short lived intermediate, formerly taken as evidence for the charge transfer state P⁺ B_L ^–, is reinterpreted as a transient excitation of this trip-trip singlet. Received: 2 June 1997 / Accepted: 18 July 1997     

Presteady-State Currents of the Rabbit Na+/Glucose Cotransporter (SGLT1)

The rabbit Na⁺/glucose cotransporter (SGLT1) exhibits a presteady-state current after step changes in membrane voltage in the absence of sugar. These currents reflect voltage-dependent processes involved in cotransport, and provide insight on the partial reactions of the transport cycle. SGLT1 presteady-state currents were studied as a function of external Na⁺, membrane voltage V _m , phlorizin and temperature. Step changes in membrane voltage—from the holding V _h to test values, elicited transient currents that rose rapidly to a peak (at 3–4 msec), before decaying to the steady state, with time constants τ≈4–20 msec, and were blocked by phlorizin (K _i ≈30 μm). The total charge Q was equal for the application of the voltage pulse and the subsequent removal, and was a function of V _m . The Q-V curves obeyed the Boltzmann relation: the maximal charge Q _max was 4–120 nC; V _0.5, the voltage for 50% Q _max was −5 to +30 mV; and z, the apparent valence of the moveable charge, was 1. Q _max and z were independent of V _h (between 0 and −100 mV) and temperature (20–30°C), while increasing temperature shifted V _0.5 towards more negative values. Decreasing [Na⁺] _o decreased Q _max, and shifted V _0.5 to more negative voltages 9by −100 mV per 10-fold decrease in [Na⁺] _o ). The time constant τ was voltage dependent: the τ-V relations were bell-shaped, with maximal τ_max 8–20 msec. Decreasing [Na⁺] _o decreased τ_max, and shifted the τ-V curves towards more negative voltages. Increasing temperature also shifted the τ-V curves, but did not affect τ_max. The maximum temperature coefficient Q ₁₀ for τ was 3–4, and corresponds to an activation energy of 25 kcal/mole. Simulations of a 6-state ordered kinetic model for rabbit Na⁺/glucose cotransport indicate that charge-movements are due to Na⁺-binding/dissociation and a conformational change of the empty transporter. The model predicts that (i) transient currents rise to a peak before decay to steady-state; (ii) the τ-V relations are bell-shaped, and shift towards more negative voltages as [Na⁺] _o is reduced; (iii) τ_max is decreased with decreasing [Na⁺] _o ; and (iv) the Q-V relations are shifted towards negative voltages as [Na⁺] _o is reduced. In general, the kinetic properties of the presteady-state currents are qualitatively predicted by the model. Received: 12 August 1996/Revised: 30 September 1996     

Copper (II) complex of 1,10-phenanthroline and <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine with DNA oxidative cleavage activity in the gallic acid

Copper (II) complex of formulation [Cu–Phen–Tyr](H₂O)](ClO₄) (Phen = 1,10-phenanthroline, l-Tyr = l-tyrosine), has been prepared, and their induced DNA oxidative cleavage activity studied. The complex binds to DNA by intercalation, as deduced from the absorption and fluorescence spectral data. Scatchard plots constructed from the absorption titration data gave binding constant 2.44 × 10⁴ M⁻¹ of base pairs. Extensive hypochromism, broadening, and red shifts in the absorption spectra were observed. Upon binding to DNA, the fluorescence from the DNA–ethidium bromide system was efficiently quenched by the copper (II) complex. Stern–Volmer quenching constant 0.61 × 10³ M⁻¹ obtained from the linear quenching plots. [Cu–Phen–Tyr] complex efficiently cleave the supercoiled DNA to its nicked circular form with gallic acid as biological reductant at appropriate complex concentration. The gallic acid as reductant could observably improve copper (II) complex to DNA damage. The pseudo-Michaelis–Menten kinetic parameters (k _cat, K _M) were calculated to be 1.32 h⁻¹ and 5.46 × 10⁻⁵ M for [Cu–Phen–Tyr] complex. Mechanistic studies reveal the involvement of superoxide anions and hydroxyl radical (HO^·) as the reactive species under an aerobic medium.     

2-(3,5-Dibromo-4-hydroxyphenyl)imidazo[4,5-<Emphasis Type="Italic">f</Emphasis>][1,10]phenanthrolinoruthenium(II) complexes: synthesis,characterization, cytotoxicity,apoptosis, DNA-binding and antioxidant activity

A new ligand DBHIP and its two ruthenium(II) complexes [Ru(dmb)₂(DBHIP)](ClO₄)₂ (1) and [Ru(dmp)₂(DBHIP)](ClO₄)₂ (2) have been synthesized and characterized. The cytotoxicity of DBHIP and complexes 1 and 2 has been assessed by MTT assay. The apoptosis studies were carried out with acridine orange/ethidium bromide (AO/EB) staining methods. The binding behaviors of these complexes to calf thymus DNA (CT-DNA) were studied by absorption titration, viscosity measurements, thermal denaturation and photoactivated cleavage. The DNA-binding constants of complexes 1 and 2 were determined to be 8.64 ± 0.16 × 10⁴ (s = 1.34) and 2.79 ± 0.21 × 10⁴ (s = 2.17) M⁻¹. The results suggest that these complexes interact with DNA through intercalative mode. The studies on the mechanism of photocleavage demonstrate that superoxide anion radical (O₂ ^•–) and singlet oxygen (¹O₂) may play an important role in the DNA cleavage. The experiments on antioxidant activity show that these compounds also exhibit good antioxidant activity against hydroxyl radical (OH^•).     

Efficient cycloreversion of cis,syn-thymine photodimer by a Zn2+–1,4,7,10-tetraazacyclododecane complex bearing a lumiflavin and tryptophan by chemical reduction and photoreduction of a lumiflavin unit

DNA photolyases (EC 4.1.99.3) are enzymes that catalyze photoreversion of cis,syn-thymine photodimer (T[c,s]T), which is one of major photolesion products in DNA, by utilizing UV light. In this work, we have designed and synthesized Zn²⁺–1,4,7,10-tetraazacyclododecane complexes bearing a lumiflavin and l-tryptophan (ZnL³) or l-phenylalanine (ZnL⁴) as artificial DNA photolyases. We have found that (ZnL³)_red, whose flavin unit was reduced in situ by Na₂S₂O₄, accelerates the photoreversion of T[c,s]T utilizing near-UV light in aqueous solution at pH 7.6 and 11. Interestingly, more efficient photoreversion of T[c,s]T was achieved by UV irradiation of an oxidized form of ZnL³ [(ZnL³)_ox] in the presence of an excess amount of Et₃N at pH 11. UV–vis and fluorescence measurements and action spectra showed that an oxidized form of flavin of (ZnL³)_ox was photoreduced by Et₃N into its reduced form (ZnL³)_red, which promoted the photoreduction of T[c,s]T. Comparison of the photochemical properties of ZnL³ with those of ZnL⁴ suggested that a tryptophan unit in ZnL³ contributed to the stabilities of the flavin through intramolecular photoinduced electron transfer.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Benzothiazole bipyridine complexes of ruthenium(II) with cytotoxic activity

A series of benzothiazole-substituted trisbipyridine ruthenium(II) analogues {[Ru(bpy)₂(4,5′-bbtb)]²⁺, [Ru(bpy)₂(5,5′-bbtb)]²⁺ and [Ru(bpy)₂(5-mbtb)]²⁺ [bpy is 2,2′-bipyridine, bbtb is bis(benzothiazol-2-yl)-2,2′-bipyridine, 5-mbtb is 5-(benzothiazol-2-yl),5′-methyl-2,2′-bipyridine]} have been prepared and compared with the complex [Ru(bpy)₂(4,4′-bbtb)]²⁺ reported previously. From the UV–vis spectral studies, substitution at the 5-position of the bpy causes the ligand-centred transitions to occur at considerably lower energy than for those with the functionality at the 4-position, while at the same time causing the emission to be effectively quenched. However, substitution at the 4-position causes the metal-to-ligand charge transfer to occur at lower energies. Fluorescent intercalator displacement studies indicate that the doubly substituted complexes displace ethidium bromide from a range of oligonucleotides, with the greater preference shown for bulge and hairpin sequences by the Λ enantiomer. Since the complexes only show small variation in the UV–vis spectra on the introduction of calf thymus DNA and a small increase in fluorescence they do not appear to be intercalators, but appear to associate within one of the grooves. All of the reported bisbenzothiazole complexes show reasonable cytotoxicity against a range of human cancer cell lines. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A differential molecualr topography of the Pr and Pfr forms of native oat phytochrome as probed by fluoresence quenching

Tryptophan (Trp) surface topography of the red- and far-red-absorbing forms of phytochrome (Pr, Pfr) ofAvena sativa L. has been investigated by analyzing quenching of the two components of Trp fluorescence decay, in order to understand the differences in the two forms at the molecular level. Stern-Volmer kinetic analysis of the quenching data for two cationic surface quenchers, Cs⁺ and Tl⁺, showed strong quenching of the short component of the Pr fluorescence (Stern-Volmer constants,K _sv , 27.2 and 21.4 M⁻¹, respectively) relative to that of Pfr fluorescenceK _sv , 10.4 and 12.3 M⁻¹, respectively). The long component of the Trp fluorescence was quenched differentially by Cs⁺ and Tl⁺, withK _sv of 9.0 and 19.8 M⁻¹, respectively, for the Pr fluorescence andK _sv of 13.7 and 8.7 M⁻¹, respectively, for the Pfr fluorescence. The results indicate that the phytochrome Trp residues with short fluorescence lifetime are more accessible to the cationic surface quenchers than those with long fluorescence lifetime. The data, taken together with our earlier study (Singh et al. 1988, Biochim, Biophys. Acta936, 395–405), indicate that most, if not all the ten Trp residues of phytochrome, are fluorescent and exist in distinct groups differing in their topography and microenvironment, and the peptide segment containing Trp-774 and Trp-778 within the 55-kilodalton C-terminal domain of phytochrome also undergoes a subtle alteration in its surface topography during Pr→Pfr phototransformation. This paper is dedicated to Professor Hans Mohr in commemoration of his 60th birthday     

Renaturation of DNA in the presence of ethidium bromide

The rate of renaturation of T2 DNA has been studied as a fuction of ethidium bound per nucleotide of denatured DNA. The Binding constants and number of binding sites for ethidium have been determined by spectral titration for denatured DNA at 55, 65, and 75°C and for native DNA at 65°C in 0.4M Na⁺. The rate of renaturation of T2 DNA was found to be independentof ethidium binding up to 0.03 moles per mole of nucleotide. Above 0.03 moles, the rate drops off precipitously approaching zero at 0.08 and 0.06 moles bound ethidium per nucleotide at 65°C respectively. A study was also made of the use of bound ethidium fluorescence as a probe for monitoring DNA renaturation reactions.     

High cytotoxicity and membrane permeability of Et3Pb+ in mammalian and plant cells

Cells of mammalian origin as well as those of higher plants appear to be very sensitive to triethyllead ion (Et₃Pb⁺). Neuroblastoma cells kept in the presence of 1 μM Et₃Pb⁺ lost their viability within 6 h. Growth of suspension culture cells of soybean (G. max(L.)Merr.) was inhibited by 1 μM Et₃Pb⁺, and finally the cells died. Morphologically, Et₃Pb⁺ caused the complete breakdown of microtubular structures in neuroblastoma cells; thus microtubules appeared to be the main target for the toxin. While in a previous study the effect of Et₃Pb⁺ on microtubules has been well documented at concentrations of 50–200 μM ¹, the present study demonstrates that the formation of microtubules from pig brain tubulin is disturbed at concentrations of Et₃Pb⁺ as low as 0.5 to 1 μM . We conclude from these data that Et₃Pb⁺ freely permeates the plasma membranes of mammalian as well as plant cells.     

Nonionic but water soluble, [Glycine-Pd-Alanine] and [Glycine-Pd-Valine] complexes. Their synthesis,characterization, antitumor activities and rich DNA/HSA interaction studies

Two novel, neutral and water soluble Pd(II) complexes of formula [Pd(Gly)(Ala)] (1) and [Pd(Gly)(Val)] (2) (Gly, Ala, and Val are anionic forms of glycine, alanine, and valine amino acids, respectively) have been synthesized and characterized by FT-IR, UV–Vis, ¹H-NMR, elemental analysis, and molar conductivity measurement. The data revealed that each amino acid binds to Pd(II) through the nitrogen of –NH₂ and the oxygen of –COO^– groups and acts as a bidentate chelate. These complexes have been assayed against leukemia cells (K₅₆₂) using MTT method. The results indicated that both of the complexes display more cytotoxicity than the well-known anticancer drug, cisplatin. The interaction of the compounds with calf thymus DNA (CT-DNA) and human serum albumin (HSA) were assayed by a series of experimental techniques including electronic absorption, fluorescence, viscometry, gel electrophoresis, and FT-IR. The results indicated that the two complexes have interesting binding propensities toward CT-DNA as well as HSA and the binding affinity of (1) is more than (2). The fluorescence data indicated that both complexes strongly quench the fluorescence of ethidium bromide–DNA system as well as the intrinsic fluorescence of HSA via static quenching procedures. The thermodynamic parameters (ΔH°, ΔS°, and ΔG°) calculated from the fluorescence studies showed that hydrogen bonds and van der Waals interactions play a major role in the binding of the complexes to DNA and HSA. We suggest that both of the Pd(II) complexes exhibit the groove binding mode with CT-DNA and interact with the main binding pocket of HSA.

Communicated by Ramaswamy H. Sarma     

II. Voltage-activated Proton Currents in Human THP-1 Monocytes

Depolarization-activated H⁺-selective currents were studied using whole-cell and excised-patch voltage clamp methods in human monocytic leukemia THP-1 cells, before and after being induced by phorbol ester to differentiate into macrophage-like cells. The H⁺ conductance, g _H, activated slowly during depolarizing pulses, with a sigmoidal time course. Fitted by a single exponential following a delay, the activation time constant, τ_act was roughly 10 sec at threshold potentials, decreasing at more positive potentials. Tail currents upon repolarization decayed mono-exponentially at all potentials. The tail current time constant, τ_tail, was voltage dependent, decreasing with hyperpolarization from 2–3 sec at 0 mV to ∼200 msec at −100 mV. Surprisingly, although τ_act depended strongly on pH _o , τ_tail was completely independent of pH _o . H⁺ currents were inhibited by Zn²⁺. Increasing pH _o or decreasing pH _i shifted the voltage-activation relationship to more negative potentials, tending to activate the g _H at any given voltage. Studied in excised, inside-out membrane patches, H⁺ currents were larger and activated much more rapidly at lower bath pH (i.e., pH _i ). In THP-1 cells differentiated into macrophages, the H⁺ current density was reduced by one-half, and τ_act was slower by about twofold. The properties of H⁺ channels in THP-1 cells and in other macrophage-related cells are compared. Received: 19 September 1995/Revised: 14 March 1996     

Analysis of Plasma Membrane Integrity by Fluorescent Detection of Tl+ Uptake

The exclusion of polar dyes by healthy cells is widely employed as a simple and reliable test for cell membrane integrity. However, commonly used dyes (propidium, Yo-Pro-1, trypan blue) cannot detect membrane defects which are smaller than the dye molecule itself, such as nanopores that form by exposure to ultrashort electric pulses (USEPs). Instead, here we demonstrate that opening of nanopores can be efficiently detected and studied by fluorescent measurement of Tl⁺ uptake. Various mammalian cells (CHO, GH3, NG108), loaded with a Tl⁺-sensitive fluorophore FluxOR™ and subjected to USEPs in a Tl⁺-containing bath buffer, displayed an immediate (within <100 ms), dose-dependent surge of fluorescence. In all tested cell lines, the threshold for membrane permeabilization to Tl⁺ by 600-ns USEP was at 1–2 kV/cm, and the rate of Tl⁺ uptake increased linearly with increasing the electric field. The lack of concurrent entry of larger dye molecules suggested that the size of nanopores is less than 1–1.5 nm. Tested ion channel inhibitors as well as removal of the extracellular Ca²⁺ did not block the USEP effect. Addition of a Tl⁺-containing buffer within less than 10 min after USEP also caused a fluorescence surge, which confirms the minutes-long lifetime of nanopores. Overall, the technique of fluorescent detection of Tl⁺ uptake proved highly effective, noninvasive and sensitive for visualization and analysis of membrane defects which are too small for conventional dye uptake detection methods.     

Mixed-ligand complexes of ruthenium(II) containing new photoactive or electroactive ligands: synthesis, spectral characterization and DNA interactions

Mixed-ligand ruthenium(II) complexes of three photoactive ligands, viz., (E)-1-[2-(4-methyl-2-pyridyl)-4-pyridyl]-2-(1-naphthyl)-1-ethene (mppne), (E)-1-(9-anthryl)-2-[2-(4-methyl-2-pyridyl)-4-pyridyl]-1-ethene (mppae) and (E)-1-[2-(4-methyl-2-pyridyl)-4-pyridyl]-2-(1-pyrenyl)-1-ethene (mpppe), in which a 2,2′-bipyridyl unit is linked via an ethylinic linkage to either a naphthalene, an anthracene or a pyrene chromophore and three electroactive ligands, viz., 4-(4-pyridyl)-1,2-benzenediol (catpy), 5,6-dihydroxy-1,10-phenanthroline (catphen) and 1,2-benzenediol (cat), were synthesized in good to moderate yields. Complexes [Ru(bpy)₂(mppne)]²⁺ (bpy is 2, 2′–bipyridyl), [Ru(bpy)₂(mppae)]²⁺, [Ru(bpy)₂(mpppe)]²⁺, [Ru(bpy)₂(sq-py)]⁺, [Ru(bpy)₂(sq-phen)]⁺ and [Ru(phen)₂(bsq)]⁺ (phen is 1,10-phenanthroline) were fully characterized by elemental analysis, IR, ¹H NMR, fast-atom bombardment or electron-impact mass, UV–vis and cyclic voltammetric methods. In the latter three complexes, the ligands catpy, catphen and cat are actually bound to the metal center as the corresponding semiquinone species, viz., 4-(4-pyridyl)-1,2-benzenedioleto(+I) (sq-py), 1,10-phenanthroline-5,6-dioleto(+I) (sq-phen) and 1,2-benzenedioleto(+I) (bsq), thus making the overall charge of the complexes formally equal to + 1 in each case. These three complexes are electron paramagnetic resonance active and exhibit an intense absorption band between 941 and 958 nm owing to metal-to-ligand charge transfer (MLCT, d _Ru→π*_sq) transitions. The other three ruthenium(II) complexes containing three photoactive ligands, mppne, mppae and mpppe, exhibit MLCT (d _Ru→π*_bpy ) bands in the 454–461-nm region and are diamagnetic. These can be characterized by the ¹H NMR method. [Ru(bpy)₂(mppne)]²⁺, [Ru(bpy)₂(mppae)]²⁺ and [Ru(bpy)₂(mpppe)]²⁺ exhibit redox waves corresponding to the Ru^III/Ru^II couple along with the expected ligand (bpy and substituted bpy) based ones in their cyclic and differential pulse voltammograms (CH₃CN, 0.1 M tetrabutylammonium hexafluorophosphate)—corresponding voltammograms of [Ru(bpy)₂(sq-py)]⁺, [Ru(bpy)₂(sq-phen)]⁺ and [Ru(phen)₂(bsq)]⁺ are mainly characterized by waves corresponding to the quinone/semiquinone (q/sq) and semiquinone/1,2-diol (sq/cat) redox processes. The results of absorption and fluorescence titration as well as thermal denaturation studies reveal that [Ru(bpy)₂(mppne)]²⁺ and [Ru(bpy)₂(mppae)]²⁺ are moderate-to-strong binders of calf thymus DNA with binding constants ranging from 10⁵ to 10⁶ M⁻¹. Under the identical conditions of drug and light dose, the DNA (supercoiled pBR 322) photocleavage activities of these two complexes follow the order:[Ru(bpy)₂(mppne)]²⁺>[Ru(bpy)₂(mppae)]²⁺, although the emission quantum yields follow the reverse order. The other ruthenium(II) complexes containing the semiquinone-based ligands are found to be nonluminescent and inefficient photocleavage agents of DNA. However, experiments shows that [Ru(bpy)₂(sq)]⁺-based complexes oxidize the sugar unit and could be used as mild oxidants for the sugar moiety of DNA. Possible explanations for these observations are presented.Electronic Supplementary Material Supplementary material is available for this article at .     

DNA interaction studies of sesamol (3,4-methylenedioxyphenol) food additive

The interaction of native calf thymus DNA (CT-DNA) with sesamol (3,4-methylenedioxyphenol) in Tris–HCl buffer at neutral pH 7.4 was monitored by absorption spectrophotometry, viscometry and spectrofluorometry. It is found that sesamol molecules could interact with DNA outside and/or groove binding modes, as are evidenced by: hyperchromism in UV absorption band, very slow decrease in specific viscosity of DNA, and small increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of sesamol, which indicates that it is able to partially release the bound MB. Furthermore, the enthalpy and entropy of the reaction between sesamol and CT-DNA showed that the reaction is enthalpy-favored and entropy-disfavored (ΔH = ?174.08 kJ mol^?1; ΔS = ?532.92 J mol^?1 K^?1). The binding constant was determined using absorption measurement and found to be 2.7 × 10⁴ M^?1; its magnitude suggests that sesamol interacts to DNA with a high affinity.     

The role of the HCR system in the repair of lethal lesions ofBacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents

The role of the HCR system in the repair of prelethal lesions induced by UV-light, γ-rays and alkylating agents was studied in theBacillus subtilis SPP1 phage, its thermosensitive mutants (N3, N73 endts ₁) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher inhcr ⁺ cells than inhcr cells, the differences being more striking in intact phages than in their transfecting DNA’s. Repair inhibitors reduce the survival inhcr ⁺ cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growinghcr ⁺ cells but has no effect on its survival in competenthcr ⁺ cells; acriflavin and ethidium bromide decrease the survival of UV-irradiated SPP1 phage in both exponentially growing and competenthcr ⁺ cells to the level of survival observed inhcr cells; moreover, ethidium bromide lowers the number of infective centres inhcr ⁺ cells of UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of UV-irradiated phages or their DNA inhcr cells. The repair mechanism under study repairs effectively also lesions induced by polyfunctional alkylating agents in transfecting DNA’s ofB. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in phages and their DNA by ethyl methanesulphonate or γ-rays.