Real-time detection of Fe.EDTA/H2O2-induced DNA cleavage by linear dichroism

The conditions for the measurement of linear dichroism (LD) can be adjusted so as to solely reflect the length and the flexibility of DNA. The real-time detection of the EDTA.Fe(2+)-induced oxidative cleavage of double-stranded native and synthetic DNAs was performed using LD. The decrease in the magnitude of the LD at 260 nm, which reflects an increase in the flexibility and a decrease in the length of the DNA, can be described by the sum of two or three exponential curves in relation to the EDTA.Fe(2+) concentration. The fast component was assigned to the cleavage of one of the double strands, inducing an increase in the flexibility, while the other slower component was assigned to the cleavage of the double strand, resulting in the shortening of DNA. The decrease in the magnitude of the LD of poly[d(A-T)(2)] was similar to that of poly[d(I-C)(2)], while that of poly[d(G-C)(2)] was found to be the slowest, indicating that the resistance of poly[d(G-C)(2)] against the Fenton-type reagent was the strongest. This observation suggests that the amine group in the minor groove of the double helix may play an important role in slowing the EDTA.Fe(2+)-induced oxidative cleavage.     

DNA binding of a spermine derivative: Spectroscopic study of anthracene-9-carbonyl-n1-spermine with poly[d(G-C)·(d(G-C))] and poly[d(A-T) · d(A-T)]

The binding of polyamines, including spermidine ( 1 ) and spermine ( 2 ), to poly[d(G-C) · d(G-C) ] was probed using spectroscopic studies of anthracene-9-carbonyl-N¹-spermine ( 3 ); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly [(dG-dC) · (dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C) · d(G-C)] has a site size of 6 ± 1 bases, and an equilibrium binding constant of (2.2 ± 1.1) × 10⁷ M^?1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly [d(A-T) · d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 ± 1.1 bases and an equilibrium binding constant of (6.6 ± 3.3) × 10⁵ M^?1. Thus, 3 binds preferentially to poly [d(G-C) · d(G-C)] at these concentrations. © 1994 John Wiley & Sons, Inc.     

Hydration effects of Ni(2+) binding to synthetic polynucleotides with regularly alternating purine-pyrimidine sequences

High precision ultrasonic and densimetric techniques have been used to study the interaction of Ni²⁺ ions with right-handed poly[d(G-C)]·poly[d(G-C)], poly-[d(A-C)]·poly[d(G-T)] and poly[d(A-T)]·poly[d(A-T)] in 5 mM CsCl, 0.2 mM HEPES, pH 7.5 at 20°C. From these measurements the changes in the apparent molar volume and the apparent molar adiabatic compressibility due to the interaction have been obtained. The volume effects of the binding, calculated per mole of Ni²⁺ ions, range from 11.7 to 23.9 cm³ mol^–1 and the compressibility effects range from 19.3 × 10^–4 to 43.1 × 10^–4 cm³ mol^–1 bar^–1. These data are interpreted in terms of dehydration of the polynucleotides and Ni²⁺ ions, i.e. the release of water molecules from the hydration shells of the molecules. An increase in G+C content gives an increase in volume and compressibility effects, indicating a rise in the extent of dehydration. The dehydration effects of Ni²⁺ binding to poly[d(G-C)]·poly[d(G-C)] are approximately twice those of poly[d(A-T)]·poly[d(A-T)]. The volume and compressibility effects of Ni²⁺–EDTA complex formation have also been measured and used as a model system for quantitative estimation. These values revealed that Ni²⁺ ions can coordinate two atomic groups of poly[d(G-C)]·poly[d(G-C)], while in the case of the Ni²⁺–poly[d(A-T)]·poly[d(A-T)] complex volume and compressibility effects correspond to one direct or two indirect (through water) contacts.     

Triple-helix formation in the antiparallel binding motif of oligodeoxynucleotides containing N(9)- and N(7)-2-aminopurine deoxynucleosides

Triplex-forming oligodeoxynucleotide 15mers, designed to bind in the antiparallel triple-helical binding motif, containing single substitutions (Z) of the four isomeric αN⁷-, βN⁷-, αN⁹- and βN⁹-2-aminopurine (ap)-deoxyribonucleosides were prepared. Their association with double-stranded DNA targets containing all four natural base pairs (X-Y) opposite the aminopurine residues was determined by quantitative DNase I footprint titration in the absence of monovalent metal cations. The corresponding association constants were found to be in a rather narrow range between 1.0 × 10⁶ and 1.3 × 10⁸ M^–1. The following relative order in Z × X-Y base-triple stabilities was found: Z = αN⁷ap: T-A > A-T> C-G ~ G-C; Z = βN⁷ap: A-T > C-G > G-C > T-A; Z = αN⁹ap: A-T = G-C > T-A > C-G; and Z = βN⁹ap: G-C > A-T > C-G > T-A.     

DNA structural features responsible for sequence-dependent binding geometries of Hoechst 33258

The complexes of Hoechst 33258 with poly[d(A-T)₂], poly[d(I-C)₂], poly[d(G-C)₂], and poly[d(G-m⁵C)₂] were studied using linear dichroism, CD, and fluorescence spectroscopies. The Hoechst-poly[d(I-C)₂] complex, in which there is no guanine amino group protruding in the minor groove, exhibits spectroscopic properties that are very similar to those of the Hoechst-poly[d(A-T)₂] complex. When bound to both of these polynucleotides, Hoechst exhibits an average orientation angle of near 45° relative to the DNA helix axis for the long-axis polarized low-energy transition, a relatively strong positive induced CD, and a strong increase in fluorescence intensity—leading us to conclude that this molecule also binds in the minor groove of poly[d(I-C)₂]. By contrast, when bound to poly[d(G-C)₂] and poly[d(G-m⁵C)₂], Hoechst shows a distinctively different behavior. The strongly negative reduced linear dichroism in the ligand absorption region is consistent with a model in which part of the Hoechst chromophore is intercalculated between DNA bases. From the low drug:base ratio onset of excitonic effects in the CD and fluorescence emission spectra, it is inferred that another part of the Hoechst molecule may sit in the major groove of poly[d(G-C)₂] and poly[d(G-m⁵C)₂] and preferentially stacks into dimers, though this tendency is strongly reduced for the latter polynucleotide. Based on these results, the importance of the interactions of Hoechst with the exocyclic amino group of guanine and the methyl group of cytosine in determining the binding modes are discussed. © 1996 John Wiley & Sons, Inc.     

Mechanistic Studies of Semicarbazone Triapine Targeting Human Ribonucleotide Reductase in Vitro and in Mammalian Cells: TYROSYL RADICAL QUENCHING NOT INVOLVING REACTIVE OXYGEN SPECIES*

Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the β₂ subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(Fe^III₂-Y·)(Fe^III₂)] cofactor for de novo DNA biosynthesis. Several mechanisms for 3-AP inhibition of β₂ have been proposed; one involves direct iron chelation from β₂, whereas a second involves Y· destruction by reactive oxygen species formed in situ in the presence of O₂ and reductant by Fe(II)-(3-AP). Inactivation of β₂ can thus arise from cofactor destruction by loss of iron or Y·. In vitro kinetic data on the rates of ⁵⁵Fe and Y· loss from [(⁵⁵Fe^III₂-Y·)(⁵⁵Fe^III₂)]-β₂ under aerobic and anaerobic conditions reveal that Y· loss alone is sufficient for rapid β₂ inactivation. Oxyblot^TM and mass spectrometric analyses of trypsin-digested inhibited β₂, and lack of Y· loss from H₂O₂ and O₂^˙̄ treatment together preclude reactive oxygen species involvement in Y· loss. Three mammalian cell lines treated with 5 μm 3-AP reveal Y· loss and β₂ inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo- over [(Fe^III₂-Y·)(Fe^III₂)]-β₂ in lysates, similar iron-content in β₂ immunoprecipitated from 3-AP-treated and untreated [⁵⁵Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited β₂ are most consistent with Y· loss being the predominant mode of inhibition, with β₂ remaining iron-loaded and stable. A model consistent with in vitro and cell-based biochemical studies is presented in which Fe(II)-(3-AP), which can be cycled with reductant, directly reduces Y· of the [(Fe^III₂-Y·)(Fe^III₂)] cofactor of β₂.     

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria

We examined nitrate-dependent Fe²⁺ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe²⁺ and reduced NO₃⁻ to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein⁻¹ min⁻¹, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH₄⁺ mg protein⁻¹ min⁻¹). A ¹⁵N tracer experiment demonstrated that coupling of nitrate-dependent Fe²⁺ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO₃⁻ by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe²⁺ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO₃⁻ ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe²⁺ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe²⁺ oxidation and NO₃⁻ reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein⁻¹ min⁻¹, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe²⁺ oxidation and the anammox reaction decreased the molar ratios of consumed NO₂⁻ to consumed NH₄⁺ (ΔNO₂⁻/ΔNH₄⁺) and produced NO₃⁻ to consumed NH₄⁺ (ΔNO₃⁻/ΔNH₄⁺). These reactions are preferable to the application of anammox processes for wastewater treatment.     

Spectral Analysis of Naturally Occurring Methylxanthines (Theophylline,Theobromine and Caffeine) Binding with DNA

Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR) spectroscopic methods, and especially monitored their binding affinity in the presence of Mg²⁺ and during helix-coil transitions of DNA by temperature (T_m) or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophylline = 3.5×10³ M⁻¹, DNA-theobromine = 1.1×10³ M⁻¹, and DNA-Caffeine = 3.8×10³ M⁻¹. On the other hand T_m/pH melting profiles showed 24–35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C) and phosphate group through hydrogen bond (H-bond) interaction. In the presence of Mg²⁺, methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg²⁺. The spectral analyses indicated the order of binding affinity as “caffeine≥theophylline>theobromine” to the native double helical DNA, and “theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage

The bleomycins (BLMs) are a family of natural glycopeptides used clinically as antitumor agents. In the presence of required cofactors (Fe²⁺ and O₂), BLM causes both single-stranded (ss) and double-stranded (ds) DNA damage with the latter thought to be the major source of cytotoxicity. Previous biochemical and structural studies have demonstrated that BLM can mediate ss cleavage through multiple binding modes. However, our studies have suggested that ds cleavage occurs by partial intercalation of BLM's bithiazole tail 3′ to the first cleavage site that facilitates its re-activation and re-organization to the second strand without dissociation from the DNA where the second cleavage event occurs. To test this model, a BLM A5 analog (CD-BLM) with β-cyclodextrin attached to its terminal amine was synthesized. This attachment presumably precludes binding via intercalation. Cleavage studies measuring ss:ds ratios by two independent methods were carried out. Studies using [³²P]-hairpin technology harboring a single ds cleavage site reveal a ss:ds ratio of 6.7 ± 1.2:1 for CD-BLM and 3.4:1 and 3.1 ± 0.3:1 for BLM A2 and A5, respectively. In contrast with BLM A5 and A2, however, CD-BLM mediates ds-DNA cleavage through cooperative binding of a second CD-BLM molecule to effect cleavage on the second strand. Studies using the supercoiled plasmid relaxation assay revealed a ss:ds ratio of 2.8:1 for CD-BLM in comparison with 7.3:1 and 5.8:1, for BLM A2 and A5, respectively. This result in conjunction with the hairpin results suggest that multiple binding modes of a single BLM can lead to ds-DNA cleavage and that ds cleavage can occur using one or two BLM molecules. The significance of the current study to understanding BLM's action in vivo is discussed.     

Mg2+-induced triplex formation of an equimolar mixture of poly(rA) and poly(rU)

Magnesium ions strongly influence the structure and biochemical activity of RNA. The interaction of Mg²⁺ with an equimolar mixture of poly(rA) and poly(rU) has been investigated by UV spectroscopy, isothermal titration calorimetry, ultrasound velocimetry and densimetry. Measurements in dilute aqueous solutions at 20°C revealed two differ ent processes: (i) Mg²⁺ binding to unfolded poly(rA)·poly(rU) up to [Mg²⁺]/[phosphate] = 0.25; and (ii) poly(rA)·2poly(rU) triplex formation at [Mg²⁺]/[phosphate] between 0.25 and 0.5. The enthalpies of these two different processes are favorable and similar to each other, ~–1.6 kcal mol^–1 of base pairs. Volume and compressibility effects of the first process are positive, 8 cm³ mol^–1 and 24 × 10^–4 cm³ mol^–1 bar^–1, respectively, and correspond to the release of water molecules from the hydration shells of Mg²⁺ and the polynucleotides. The triplex formation is also accompanied by a positive change in compressibility, 14 × 10^–4 cm³ mol^–1 bar^–1, but only a small change in volume, 1 cm³ mol^–1. A phase diagram has been constructed from the melting experiments of poly(rA)·poly(rU) at a constant K⁺ concentration, 140 mM, and various amounts of Mg²⁺. Three discrete regions were observed, corresponding to single-, double- and triple-stranded complexes. The phase boundary corresponding to the transition between double and triple helical conformations lies near physiological salt concentrations and temperature.     

Mechanistic aspects of CoII(HAPP)(TFA)2 in DNA bulge-specific recognition

A novel octahedral complex Co^II(HAPP)(TFA)₂ [hexaazaphenantholine-cyclophane (HAPP), trifluoroacetate (TFA)] is a DNA bulge-specific probe with single-strand DNA cleavage activity in the presence of H₂O₂. This complex exhibits low affinity towards double-stranded DNA and low reactivity toward single-stranded DNA. Metal–HAPP complexes with different coordination number and ring size were synthesized and their selectivity and reactivity for DNA bulges were compared. The DNA sequence at the bulge site influences the intensity of cleavage at the bulge and the flanking sites after piperidine treatment. Cleavage specificity of Co^II(HAPP)(TFA)₂ was characterized extensively using scavenger reagents to quench the cleavage reaction and high-resolution polyacrylamide gel electrophoresis. In addition, 3′-phosphoglycolate cleavage products were trapped and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. These data were used to deduce that the DNA cleavage pathway for Co^IIHAPP²⁺ in the presence of H₂O₂ involves 4′-H abstraction of the deoxyribose moiety.     

Cleavage of the glycosidic linkage of pyrimidine ribonucleosides by the bisulfite-oxygen system

When a solution containing 2 mM uridine, 20 mM sodium bisulfite, 0.1 mM MnCl₂, and 100 mM sodium phosphate buffer of pH 7.0 was incubated aerobically at 37° or 0°, partial cleavage of the glycosidic linkage of uridine took place. About 20% of the uridine was converted to uracil by the incubation for 4 hrs. Cytosine was produced from cytidine by similar treatment with bisulfite. These reactions were caused by free radicals generated by Mn²⁺-catalyzed autoxidation of bisulfite. Glycosidic bond cleavage by the bisulfite-oxygen system was not detected for adenosine, AMP, guanosine, GMP, thymidine, TMP, deoxyuridine, dCMP, dAMP, and dGMP. When poly(U) and poly(C) were treated with 20 mM sodium bisulfite in the same manner, chain fission of the polymer occurred as judged by the elution-pattern change in gel filtration through Sephadex columns. No change in the elution pattern was observed for bisulfite-treated poly(A), poly(U)· poly(A) or tRNA.     

Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers

The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) · poly (dT), poly (dA-dT) · poly(dA-dT) and poly (dI-dC) · poly (dI-dC) but poorly, if at all, to poly (dG) · poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K_a=2.9 · 10⁵ M⁻¹); corresponding values for distamycin A are one site per 6.1 nucleotides with K_a= 11.6 · 10⁵ M⁻¹. Binding sites apparently involve predominantly A·T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3° per bound antibiotic molecule.     

Biochemical characterization of Santalum album (Chandan) leaf peroxidase

The Santalum peroxidase was extracted from the leaves and precipitated with double volume of chilled acetone. The optimum percent relative activity for the Santalum peroxidase was observed at pH 5.0 and 50 °C temperature. The Santalum peroxidase per cent relative activity was stimulated in the presence of phenolic compounds like ferrulic acid and caffeic acids; however, indole-3-acetic acid (IAA) and protocatechuic acid act as inhibitors. All divalent cations Fe²⁺, Mn²⁺, Mg²⁺, Cu²⁺ and Zn²⁺ stimulate the relative activity of the Santalum peroxidase at concentration of 2.0 μM. Amino acids like L-alanine and L-valine activate the per cent relative activity, while L-proline and DL-methionine showed moderate inhibition for the Santalum peroxidase. However, a very low a concentration of cysteine acts as a strong inhibitor of Santalum peroxidase at the concentration of 0.4 mM. Native polyacrylamide gel electrophoresis (Native-PAGE) was performed for isoenzyme determination and two bands were observed. K_m and V_max values were calculated from Lineweaver-Burk graph. The apparent V_max/K_m value for O-dianisidine and H₂O₂ were 400 and 5.0 × 105 Units/min/mL respectively.     

Relative Stabilities and Transitions of DNA Conformations in 1:1 Electrolytes: A Theoretical Study

Abstract

We use a recently developed formalism (1) to calculate the salt dependent part of the free energy determining DNA conformational stability in 1:1 electrolytes. The conformations studied are the A,B,C and alternating-B right-handed forms and the Z₁Z_II left-handed forms of DNA. In the case of the B-Z₁ transition of d(G-C) · d(G-C) helices in NaCl solution, the free energy contribution considered suffices to describe the transition in a quantitative manner. The theory also predicts the occurrence of salt-induced B-A transitions which have been recently observed with poly[d(n²A-T)| and poly[d(G-C)|. In other cases, additional terms in the free energy balance, particularly due to hydration effects, must be at least as important as salt effects in determining conformational stability and structural transitions in solution. If diffuse ionic cloud electrostatic effects alone would dominate in all cases, the relative helical stabilities at 0.2 M monovalent salt would decrease in the order C > B > A > Z_II > Z₁ > alternating-B. At high salt concentrations (2.0 M - 5.0 M), the order would be alternating-B > Z, > A > Z_II > B > C.     

Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding

Transfer RNA structure involves complex folding interactions of the TΨC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TΨC domain, rT₅₄, Ψ₅₅ and m⁵C₄₉, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA^Phe T-half molecules (nucleosides 40–72) with the corresponding unmodified D-half molecule (nucleosides 1–30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg²⁺ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K_ds (rT₅₄ = 6 ± 2, m⁵C₄₉ = 11 ± 2, Ψ₅₅ = 14 ± 5, and rT₅₄,Ψ₅₅ = 11 ± 3 µM) significantly lower than that of the unmodified T-half (40 ± 10 µM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA^Phe and native yeast tRNA^Phe, as determined by lead cleavage patterns at U₁₇ and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA’s TΨC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.     

CO2-induced ion and fluid transport in human retinal pigment epithelium

In the intact eye, the transition from light to dark alters pH, [Ca²⁺], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO₂ and H₂O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO₂ from 5 to 13%; this maneuver decreased cell pH from 7.37 ± 0.05 to 7.14 ± 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (≈10-fold; n = 7) to CO₂ than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO₂ diffusion at the basolateral membrane promotes carbonic anhydrase–mediated HCO₃ transport by a basolateral membrane Na/nHCO₃ cotransporter. The activity of this transporter was increased by elevating apical bath CO₂ and was reduced by dorzolamide. Increasing apical bath CO₂ also increased intracellular Na from 15.7 ± 3.3 to 24.0 ± 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO₂-induced acidification also inhibited the basolateral membrane Cl/HCO₃ exchanger and increased net steady-state fluid absorption from 2.8 ± 1.6 to 6.7 ± 2.3 µl × cm⁻² × hr⁻¹ (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO₂ and H₂O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.     

Interactions of metallic ions with DNA. V. DNA renaturation mechanism in the presence of Cu 2+

New experimental data were obtained by means of circular dichroism, melting, renaturation, and kinetic experiments, upon Cu²⁺ binding to DNA, poly dAT, and poly dGdC. They enable us to propose a model of binding giving a satisfactory explanation to all of the data found in the literature. Two types of binding sites are proposed: (a) a “sandwich” of Cu²⁺ between two adjacent G-C pairs giving a charge-transfer complex, and (b) a chelate between a phosphate group and a nitrogen atom of the bases (N₇ of guanine and N₃ of cytosine at room temperature, N₃ of adenine after thermal opening of A-T pair). Type (a) stabilizes the helix and keeps the two strands linked. Type (b) destabilizes the helix and explains why the kinetic rate of renaturation is the same as that of copper release.     

Lactobacillus rhamnosus Strain GG Reduces Aflatoxin B1 Transport, Metabolism, and Toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B₁ (AFB₁) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB₁ availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB₁ levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB₁ transport from the apical to the basolateral chamber was reduced from 11.1% ± 1.9% to 6.4% ± 2.5% (P = 0.019) and to 3.3% ± 1.8% (P = 0.002) within the first hour in monolayers coincubated with GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml, respectively). GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml) bound 40.1% ± 8.3% and 61.0% ± 6.0% of added AFB₁ after 1 h, respectively. AFB₁ caused significant reductions of 30.1% (P = 0.01), 49.4% (P = 0.004), and 64.4% (P < 0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1 × 10¹⁰ CFU/ml GG after 24 h protected against AFB₁-induced reductions in transepithelial resistance at both 24 h (P = 0.002) and 48 h (P = 0.04). DNA fragmentation was apparent in cells treated only with AFB₁ cells but not in cells coincubated with either 1 × 10¹⁰ or 5 × 10¹⁰ CFU/ml GG. GG reduced AFB₁ uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.