RNA cleavage and inhibition of protein synthesis by bleomycin

Bleomycin is a clinically used antitumor antibiotic long thought to function therapeutically at the level of DNA cleavage. Recently, it has become clear that bleomycin can also cleave selected members of all major classes of RNA. Using the computer program COMPARE to search the database established by the Anticancer Drug Screening Program of the National Cancer Institute, a possible mechanism-based correlation was found between onconase, an antitumor ribonuclease currently being evaluated in phase III clinical trials, and the chemotherapeutic agent bleomycin. Following these observations, experimentation revealed that bleomycin caused tRNA cleavage and DNA-independent protein synthesis inhibition in rabbit reticulocyte lysate and when microinjected into Xenopus oocytes. The correlation of protein synthesis inhibition to the previously reported site-specific RNA cleavage caused by bleomycin supports the thesis that RNA cleavage may constitute an important element of the mechanism of action of bleomycin.     

Weakly and strongly associated nonfreezable water bound in bones

Water bound in bone of rat tail vertebrae was investigated by ¹H NMR spectroscopy at 210–300 K and by the thermally stimulated depolarization current (TSDC) method at 190–265 K. The ¹H NMR spectra of water clusters were calculated by the GIAO method with the B3LYP/6-31G(d,p) basis set, and the solvent effects were analyzed by the HF/SM5.45/6-31G(d) method. The ¹H NMR spectra of water in bone tissue include two signals that can be assigned to typical water (chemical shift of proton resonance δ_H = 4–5 ppm) and unusual water (δ_H = 1.2–1.7 ppm). According to the quantum chemical calculations, the latter can be attributed to water molecules without the hydrogen bonds through the hydrogen atoms, e.g., interacting with hydrophobic environment. An increase in the amount of water in bone leads to an increase in the amount of typical water, which is characterized by higher associativity (i.e., a larger average number of hydrogen bonds per molecule) and fills larger pores, cavities and pockets in bone tissue.     

Analysis of hairpin polyamide complexes having DNA binding sites in close proximity

The binding of two hairpin polyamide ligands at adjacent sites on DNA has been studied using NMR spectroscopy. The ligands ImPyPy-gamma-PyPyPy-Gly-Dp and Ac-ImPyPy-gamma-PyPyPy-Gly-Dp were studied binding to oligomers containing one or two matched binding sites: 5'-XGTTA-3' and 5'-TAACXNGTTA-3', where X is G, C, or A and N = 0, 1 or 2. At these sites the C-terminal ring shows an equilibrium between normal and inverted conformations. Better binding was observed with the ligand running 5' to 3' along the contacted strand than in the opposite direction. Complexes of DNAs with two binding sites indicated that at least one spacing base pair was required, and that the identity of this base pair was not critical. Binding with 5' to 3' contact is again preferred. Demonstrated binding at adjacent sites indicates that it may be possible to engineer cooperative binding for enhanced specificity or affinity.     

DNA-bleomycin interaction: Nucleotide sequence-specific binding and cleavage of DNA by bleomycin

Biosynthetic intermediates and synthetic analogues of bleomycin (BLM) have been investigated for their metal binding, dioxygen activation, and DNA cleavage. Molecular O₂ was activated by the Fe(II) complex of a synthetic model ligand. Nucleotide sequence specificities in DNA cleavage by the BLM-Fe(II) and deglyco-BLM-Fe(II) complexes were almost identical. It has been shown that (1) the β-aminoalanine-pyrimidine-β-hydroxyhistidine portion of BLM is essential for the metal binding and dioxygen activation and (2) the bithiazole moiety contributes to the specific binding to guanine base of DNA.     

Perylene-naphthalene: restricted IVR in a strongly bound van der waals molecule

The study of the spectroscopic properties of molecular complexes of perylene has been extended to naphthalene. Calculations extrapolated from earlier work predict a deviation from center-of-mass, parallel-plane geometry, and this prediction is supported by the experimental data. The complexes show a very large red-shift, of 748 cm⁻¹, which is almost twice that of benzene. A 2 : 1 complex was also observed, exhibiting a nearly harmonic shift, to 1443 cm⁻¹. The spectra show little perturbation of the in-plane modes from the free perylene case, but strong new low-frequency bands are seen, which may be attributed to complexation-induced deformation of the perylene ground state. The most important result is that strong resonance fluorescence, which was suppressed in small molecule complexes of perylene, is observed for the perylene-naphthalene 353 cm⁻¹in-plane mode. By contrast, excitation into combinations of out-of-plane deformation modes in the same energy region effectively eliminated the resonance fluorescence. This is, therefore, an important case where a mode of a big molecule is decoupled from the quasi-continuum, and develops small-molecule character.     

Chemical synthesis of an artificially branched hairpin ribozyme variant with RNA cleavage activity

Due to the development in the field of RNA synthesis over the past decade of years, preparation of RNA oligonucleotides longer than 50 nucleotides is possible today. In this report, we describe the chemical preparation of a branched RNA molecule with RNA cleavage activity consisting of 81 nucleotides. It is derived from the hairpin ribozyme, a small catalytic RNA occurring in nature. The hairpin ribozyme consists of two separately folded domains (loop A and loop B domain), which can be joined in a number of different ways without loss of activity. In the construct presented here, 2′-deoxy-N4-(6-hydroxyhexyl)-5-methylcytidine was introduced to connect the loop B domain with the loop A domain via an artificial branch. The synthesized branched RNA is able to catalyze the cleavage of a number of suitable substrates. Compared with the corresponding non-branched reverse-joined ribozyme it cleaves its substrates only 5-fold slower. Surprisingly, no ligation activity could be detected.     

Structure of mesh phases in a cationic surfactant system with strongly bound counterions

We report the observation of an intermediate mesh phase with rhombohedral symmetry, corresponding to the space group Rm, in a mixed surfactant system formed by the cationic surfactant cetyltrimethylammonium bromide (CTAB) and the organic salt 3-sodium-2-hydroxy naphthoate (SHN). It occurs between a random mesh phase (L(alpha)(D)) and a lamellar phase (L(alpha)) at low temperatures; at higher temperatures, the (L(alpha)(D)) phase transforms continuously into the (L(alpha)) phase with an increasing surfactant concentration (phi(s)). To separate the effects of salt and phi(s) on the phase behavior, the ternary system consisting of cetyltrimethylammonium 3-hydroxy-naphthalene-2-carboxylate (CTAHN), sodium bromide (NaBr), and water was studied. The intermediate mesh phase is found in this system at high NaBr concentrations. The micellar aggregates, both in the intermediate and random mesh phases, are found to be made up of a two-dimensional network of rod-like segments, with three rods meeting at each node. The average mesh size increases with phi(s), and the transition from the random mesh phase to the intermediate phase is found to occur when it is approximately 1.5 times the lamellar periodicity. The intermediate mesh phase is absent in the equimolar dodecyltrimethylammonium bromide (DTAB)-SHN system, indicating the role of the surfactant chain length in the formation of this phase. This system exhibits a random mesh phase over a very wide range of water content, with the average mesh size decreasing upon an increasing phi(s), contrary to the trend seen in the CTAB-SHN system.     

Syntheses of anthraquinone capped hairpin DNAs and electrochemical redox responses from their self-assembled monolayers on gold electrode

An anthraquinone (AQ) based DNA linker and hairpin-forming DNAs linked by the AQ linker with variable A-T base pairs were synthesized for the investigation of electron transfer through double helical DNA (DNA-ET) in self-assembled monolayers (SAMs). The spectroscopic analysis of absorption spectra indicated that the AQ of the hairpin DNA stacked with adjacent A-T base pair. Electrochemical redox response due to the AQ was observed from the hairpin DNA immobilized on gold electrode, thus the hairpin DNA is suitable for the investigation of DNA-ET in SAMs.     

A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage

Background: Ribozymes are biological catalysts that promote the hydrolysis and transesterification of phosphate diesters of RNA. They typically require divalent magnesium ions for activation, although it has proven difficult to differentiate structural from catalytic roles for the magnesium ions and to identify the molecular mechanism of catalysis. Direct inner-sphere coordination is usually invoked in the catalytic step, although there is no evidence to support the generality of such a pathway for all ribozymes.Results: We studied the catalytic pathway for the hairpin class of ribozyme. The substitutionally inert transition metal complex cobalt hexaammine [Co(NH₃)₆³⁺) was shown to be as active as Mg²⁺(aq) in promoting hairpin ribozyme activity, demonstrating that inner-sphere pathways are not used by this class of ribozyme. These results were confirmed by studies with R_P- and S_P-phosphorothioate substrate analogs which show a similar reactivity to that of the native substrate towards the magnesium-activated ribozyme. Monovalent cations enhance the activity of Co(NH₃)₆³⁺-promoted reactions, but inhibit Mg²⁺-activated catalysis, demonstrating a requirement for hydrated cations at several key sites in the ribozyme.Conclusions: These results provide clear support for a model of RNA catalysis that does not involve direct coordination of magnesium to the phosphate ester, nor activation of a bound water molecule. A mechanism in which catalysis is carried out by functional groups on the RNA ribozyme itself is possible; such functional groups are likely to have pK_a values that are appropriate for carrying out this catalysis. The metal cofactor would then serve to define the architecture of the catalytic pocket and contribute to the stabilization of transient species, as has been described earlier. Hydrolytic pathways in nucleic acid reactions are apparently more diverse than was previously thought, and the hairpin ribozyme falls into a mechanistically distinct class from the Tetrahymena and the hammerhead ribozymes.     

Synthesis and DNA cleavage activity of a novel bleomycin A(5) glycoconjugate

[structure in text] To explore the possibility of modifying bleomycin in a fashion that could alter its physiological distribution in a therapeutic setting, a new analogue of bleomycin has been prepared. This analogue is intended to target the asialoglycoprotein receptor on liver cells. Critically, despite the large C-substituent, the bleomycin conjugate was found to degrade DNA in the same fashion as bleomycin A(5) itself, and with only modestly decreased efficiency.     

Effect of infection withVerticillium dahliae on the bound and strongly bound lipids of cotton seeds of varieties with contrasting resistance

The lipids of the kernels of ripe cotton seeds of varieties resistant and susceptible to verticillium wilt have been determined. Intra- and intervariety changes in these indices for the infected plants have been revealed. Different directions of the post-infection changes in the lipids according to the degree of resistance of the variety to wilt have been shown. Differences have been observed both in healthy and in infected plants.     

Soluble polymer bound cleavage reagents: a multipolymer strategy for the cleavage of tertiary amines from REM resin

Soluble polymer bound reagent 1 has been prepared to cleave tertiary amines from REM resin. Normally, amines cleaved from REM resin require extraction or chromatography to remove excess cleavage reagent and its byproducts. The solubility profile of non-crosslinked polystyrene (NCPS) based reagent 1 eliminates the need for such purification and allows for the direct isolation of a library of pure tertiary amines through simple filtration and concentration operations.     

Support Vector Machines for predicting HIV protease cleavage sites in protein

Knowledge of the polyprotein cleavage sites by HIV protease will refine our understanding of its specificity, and the information thus acquired is useful for designing specific and efficient HIV protease inhibitors. The pace in searching for the proper inhibitors of HIV protease will be greatly expedited if one can find an accurate, robust, and rapid method for predicting the cleavage sites in proteins by HIV protease. In this article, a Support Vector Machine is applied to predict the cleavability of oligopeptides by proteases with multiple and extended specificity subsites. We selected HIV-1 protease as the subject of the study. Two hundred ninety-nine oligopeptides were chosen for the training set, while the other 63 oligopeptides were taken as a test set. Because of its high rate of self-consistency (299/299 = 100%), a good result in the jackknife test (286/299 = 95%) and correct prediction rate (55/63 = 87%), it is expected that the Support Vector Machine method can be referred to as a useful assistant technique for finding effective inhibitors of HIV protease, which is one of the targets in designing potential drugs against AIDS. The principle of the Support Vector Machine method can also be applied to analyzing the specificity of other multisubsite enzymes.     

Conformational analysis of polypropylene chains bound to model catalytic sites

The models of catalytic sites for isospecific Ziegler-Natta polymerization proposed previously (edges and reliefs on lateral surfaces of violet TiCl₃) are compared in order to verify their validity in the presence of long alkyl groups and of analyzing the conformational constraints imposed by the TiCl₃ surface on the growing chain. Our results indicate that sites on edges of lateral surfaces parallel or nearly parallel to the c axis may be not suitable for the coordination of long alkyl chains; the coordination of long alkyl chains at less hindered sites, such as edges of inclined lateral surfaces or reliefs, do not involve any increase of repulsive interactions for an isobutyl group. Further, these latter sites allow the optimum spiralization of the chain at a very early stage of growth, and their isospecific behaviour is not affected by the presence of configuration defects in the growing chain.     

Effect of infection withVerticillium dahliae on the bound and strongly bound lipids of cotton seeds of varieties with contrasting resistance

The lipids of the kernels of ripe cotton seeds of varieties resistant and susceptible to verticillium wilt have been determined. Intra- and intervariety changes in these indices for the infected plants have been revealed. Different directions of the post-infection changes in the lipids according to the degree of resistance of the variety to wilt have been shown. Differences have been observed both in healthy and in infected plants.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 89 14 75. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 609–612, September–October, 1994.     

Classical and quantum dynamical regimes in the bound space projected dynamics of strongly driven H Rydberg atoms

The bound space projected dynamics of the one dimensional model of H Rydberg atoms subjected to strong microwave radiation exhibits three dynamical regimes:(i) perturbatively localized,(ii) chaotic,(iii) external field dominated. This classification holds classically as well as quantum mechanically. The spectral properties of the bound space projected dipole operator dominating regime(iii) are studied analytically. A semiclassical analysis shows that its eigenfunctions, projected on the unperturbed basis states |n〉 of the one dimensional model, decay liken ^?7/3.     

The influence of the nuclear shape and of the muonic vacuum polarization on strongly bound electrons

We calculate the energy levels ofK- andL-shell electrons in lead, uranium, fermium and in the super-heavy systemZ=169 taking into account various nuclear charge distributions and the muonic vacuum polarization.     

On the nature of factors determining reactivity of strongly bound surface species as intermediates in heterogeneous catalytic redox reactions

The rate-limiting stage of heterogeneous catalytic redox reactions with participation of strongly bound surface species, such as nitrates, carbonates and sulfites, is the interaction of these heteroatomic oxidants with the reducing agents. It appears to be mainly determined by their bonding strength with the surface, being independent of the nature of the central atom (carbon, sulfur, nitrogen) and the energy of its bond with oxygen.     

Characterization of ligand sites on natural sediment particles

Organic and inorganic ligand sites on sediment particles were alkalimetrically titrated using a glass electrode as indicating device. Data obtained for suspensions containing known masses of sediment were used to calculate the concentration of surface ligand sites and their stability constants for complex formation with proton and copper(II) ion. The relationship between the concentration of ligand sites and the concentrations of metals (Cd, Cr, Cu, Fe, Mn, Pb, and Zn) and of C, N, and S was used to try to discriminate between the contributions of organic and inorganic components to the total ligand capacity of the sediment. The reliability of the chemical model deduced from potentiometric data was checked by comparing calculated values for aqueous copper(II) as a function of pH with values experimentally determined via atomic absorption spectrometry. The procedure proposed might contribute to the modeling of sediment-contaminant interaction, providing information on the nature of the ligands involved.     

A strategy for the development of small-molecule-based sensors that strongly fluoresce when bound to a specific RNA

We report a broadly applicable approach for the development of small-molecule-based RNA sensors. Our photoinduced electron transfer (PET) sensor consists of a fluorescein derivative as the fluorophore and two aniline derivatives as electron donors (quenchers). The isolation of electron-donor-binding RNA by in vitro selection (also known as SELEX) yielded an RNA aptamer that could increase the fluorescence intensity of the sensor by 13-fold. This result shows that RNA-electron-donor interactions can be used to develop modular RNA chemosensors.