Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (Mstopol) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage m the absence of Mg2+ but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim. (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+, in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike Mstopol and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.

Novel 3-dimensional sixfold interpenetrating diamondoid networks of copper(I) coordination polymers of polypyridyl ligands - Syntheses,characterization and crystal structures

Two new coordination polymers [Cu(L-1)(2)](n)(ClO4)(n)center dot 2nH(2)O (1), [Cu(L-2)(2)](n)(ClO4)(n)center dot 2nH(2)O (2) of polydentate imine/pyridyl ligands, L-1 and L-2 with Cu(I) ion have been synthesized and characterized by single crystal X-ray diffraction studies, elemental analyses, IR' UV-vis and NMR spectroscopy. They represent 3-dimensional, sixfold interpenetrating diamondoid network structures having large pores of dimension, 35 x 21 angstrom(2) in 1 and 38 x 19 angstrom(2) in 2, respectively.

Catalytic cyclopropanation of olefins using copper(I) diphosphinoamines

Catalytic cyclopropanation reactions of olefins with ethyl diazoacetate were carried out using copper(I) diphosphinoamine (PPh2)(2)N(R) (R = Pr-i, H, Ph and -CH2-C6H4-CH=CH2) complexes at 40 degrees C in chloroform. High yields of the cyclopropanes were obtained in all cases. The rate of the reaction was influenced by the nuclearity of the complex and the binding mode of the ligand which was either bridging or chelating. Comparison of isostructural complexes shows that the rate follows the order R = Pr-i > H > Ph, where R is the substituent on the N. However, cyclopropane formation versus dimerization of the carbene, and trans to cis ratios of cyclopropane was similar in all cases. The nearly identical selectivity for different products formed was indicative of a common catalytic intermediate. A labile "copper-olefin" complex which does not involve the phosphine or the counterion is the most likely candidate. The differences in the reaction rates for different complexes are attributed to differences in the concentration of the catalytically active species which are in equilibrium with the catalytically inactive copper-phosphinoamine complex. To test the hypothesis a diphosphinoamine polymer complexed to copper(I) was used as a heterogeneous catalyst. Leaching of copper(I) and deactivation of the catalyst confirmed the proposed mechanism. (C) 2008 Elsevier B. V. All rights reserved.

Chelating and bridging diphosphinoamine (PPh2)(2)N(Pr-i) complexes of copper(I)

The ligand bis(diphenylphosphino) isopropylamine (dppipa) has been shown to be a versatile ligand sporting different coordination modes and geometries dictated by copper(I). Most of the molecular structures were confirmed by X-ray crystallography. It is found in a chelating mode, in a monomeric complex when the ligand to copper ratio is 2:1. A tetrameric complex is formed when low ratios of ligand to metal (1: 2) were used. But with increasing ratios of ligand to metal (1: 1 and 2: 1), a trimer or a dimer was obtained depending on the crystallization conditions. Variable temperature P-31{H-1} NMR spectra of these complexes in solution showed that the Cu-P bond was labile and the highly strained 4-membered structure chelate found in the solid state readily converted to a bridged structures. On the other hand, complexes with the ligand in a bridging mode in the solid state did not form chelated structures in solution. The effect of adding tetra-alkylammonium salts to solutions of various complexes of dppipa were probed by P-31{H-1} NMR and revealed the effect of counter ions on the stability of complexes in solution. (C) 2008 Elsevier B.V. All rights reserved.

Perpetuation of immunological memory through common MHC-I binding modes of peptidomimic and antigenic peptides

Understanding the molecular mechanisms of immunological memory assumes importance in vaccine design. We had earlier hypothesized a mechanism for the maintenance of immunological memory through the operation of a network of idiotypic and anti-idiotypic antibodies (Ab2). Peptides derived from an internal image carrying anti-idiotypic antibody are hypothesized to facilitate the perpetuation of antigen specific T cell memory through similarity in peptide-MHC binding as that of the antigenic peptide. In the present work, the existence of such peptidomimics of the antigen in the Ab2 variable region and their similarity of MHC-I binding was examined by bioinformatics approaches. The analysis employing three known viral antigens and one tumor-associated antigen shows that peptidomimics from Ab2 variable regions have structurally similar MHC-I binding patterns as compared to antigenic peptides, indicating a structural basis for memory perpetuation. (C)) 2007 Elsevier Inc. All rights reserved.

Tuning the beta-turn segment in designed peptide beta-hairpins: Construction of a stable type I ' beta-turn nucleus and hairpin-helix transition promoting segments

Designed octapeptides Boc-Leu-Val-Val-Aib-(D)Xxx-Leu- Val-Val-OMe ((D)Xxx = (D)Ala, 3a; (D)Val, 3c and (D)Pro, 5a) and Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (3b) have been investigated to construct models of a stable type I' beta-turn nucleated hairpin and to generate systems for investigating helix-hairpin conformational transitions. Peptide 5a, which contains a central Aib-(D)Pro segment, is shown to adopt a stable type I' beta-turn nucleated hairpin structure, stabilized by four cross-strand hydrogen bonds. The stability of the structure in diverse solvents is established by the observation of all diagnostic NOEs expected in a beta-hairpin conformation. Replacement of (D)Pro5 by (D)Ala/(D)Val (3a-c) results in sequences that form beta-hairpins in hydrogen bonding solvents like CD3OH and DMSO-d(6). However, in CDCl3 evidence for population of helical conformations is obtained. Peptide 6b (Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe), which contains a centrally positioned Aib-Aib segment, provides a clear example of a system, which exhibits a helical conformation in CDCl3 and a significant population of both helices and hairpins in CD3OH and DMSO-d(6). The coexistence of multiple conformations is established by the simultaneous observation of diagnostic NOEs. Control over stereochemistry of the central beta-turn permits generation of models for robust beta-hairpins and also for the construction of systems that may be used to probe helix-hairpin conformational transitions. (c) 2006 Wiley Periodicals, Inc.

Conformation of di-n-propylglycine residues (Dpg) in peptides: crystal structures of a type I 'beta-turn forming tetrapeptide and an alpha-helical tetradecapeptide

The crystal structures of two oligopeptides containing di-n-propylglycine (Dpg) residues, Boc-Gly-Dpg-Gly-Leu-OMe (1) and Boc-Val-Ala-Leu-Dpg-Val-Ala-Leu-Val-Ala-Leu-Dpg-Val-Ala-Leu-OMe (2) are presented. Peptide 1 adopts a type I' beta-turn conformation with Dpg(2)-Gly(3) at the corner positions. The 14-residue peptide 2 crystallizes with two molecules in the asymmetric unit, both of which adopt alpha-helical conformations stabilized by 11 successive 5 -> 1 hydrogen bonds. In addition, a single 4 -> 1 hydrogen bond is also observed at the N-terminus. All live Dpg residues adopt backbone torsion angles (phi, psi) in the helical region of conformational space. Evaluation of the available structural data on Dpg peptides confirm the correlation between backbone bond angle N-C-alpha-C' (tau) and the observed backbone phi,psi values. For tau > 106 degrees, helices are observed, while fully extended structures are characterized by tau < 106 degrees. The mean r values for extended and folded conformations for the Dpg residue are 103.6 degrees +/- 1.7 degrees and 109.9 degrees +/- 2.6 degrees, respectively. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

The syntheses, characterizations, X-ray crystal structures and properties of Cu(I) complexes of a bis-bidentate schiff base ligand

Bis-bidentate Schiff base ligand L and its two mononuclear complexes [CuL(CH3CN)(2)]ClO4 (1)and [CuL(PPh3)(2)]ClO4 (2)have been prepared and thoroughly characterized by elemental analyses, IR, UV-Vis, NMR spectroscopy and X-ray diffraction analysis. In both the complexes the metal ion auxiliaries adopt tetrahedral coordination environment. Their reactivity, electrochemical and photophysical behavior have been studied. Complex 1 shows reversible Cu-II/I couple with potential 0.74 V versus Ag/AgCl in CH2Cl2. At room temperature L is weakly fluorescent in CH2Cl2, however in Cu(I)complexes 1 and 2 the emission in quenched. (C) 2009 Elsevier B. V. All rights reserved.

Studies of the polarographic and coulometric behaviour of aromatic nitro-compounds: I. Nitrobenzene in ethanol

A detailed polarographic (a.c. and d.c.) and coulometric investigation of nitrobenzene has been made at various pH values in the presence of different concentrations of ethanol. Below pH 4.7, two waves are apparent but above this pH, the second wave does not appear. Coulometric evidence indicates that the first and second waves correspond to the four-and two-electron processes, respectively. The coulometric method was not applicable in sodium hydroxide and sodium acetate solutions. When the diffusion coefficients (from the diaphragm cell) are used in the Ilkovic equation, no reliable conclusions can be reached for the number of electrons involved in the reduction process in alkaline solutions. The a.c. polarographic method gives evidence for the formation of species such as: C6H5NO2H22+, C6H5NO2− and C6H5NO22−. Analysis of d.c. polarographic data by Delahay's treatment of irreversible waves, indicates that the number of electrons involved in the rate-determining step is 2. In sodium hydroxide solutions, however, the first main wave is split indicating more than one rate-determining step. The results presented in this paper indicate that the first wave in the reduction of nitrobenzene is a four-electron process at all pH values. The second wave, which appears below pH 4.7, corresponds to a two-electron process irrespective of wave heights. The difference in the a.c. polarographic behaviour in acid and alkaline solutions has given evidence for the formation of species like C6H5NO2H2, C6H5NO2−, and C6H5NO22.

Nucleotidases in plants : I. Partial purification and properties of the enzyme hydrolyzing flavine adenine dinucleotide from mung bean seedlings (Phaseolus radiatus)

The occurrence of an enzyme hydrolyzing flavine adenine dinucleotide (FAD) was demonstrated in a number of seed extracts. The enzyme from Phaseolus radiatus was purified 104-fold by fractionation with ammonium sulfate and ethanol and by negative adsorption on alumina Cγ gel. The enzyme cleaves the POP bond of FAD to yield flavine mononucleotide and adenosine monophosphate. When reduced glutathione is added to the enzyme, it cleaves FAD at the COP bond to yield riboflavine, adenosine, and pyrophosphate, Both the activities are optimal at a pH of 7.2 and at a temperature of 37 . The Km for both the activities is 1.65 × 10−5 M. The stoichiometry and the identity of the products of both the treated and untreated enzyme were established. The untreated enzyme was not inhibited by pCMB or arsenite, but the treated enzyme was sensitive to both these inhibitors. The inhibition by pCMB could be reversed by monothiols and the inhibition by arsenite by dithiols.

Studies on the structure of the chromosomes - I. Allium cepa-Stain fixatives and their utility

The structure of the mitotic chromosomes of Allium cepa has been elucidated by controlling the temperature and time of exposure of fresh roots to stain fixatives. The details seen in material stained in N HCl-orcein for 8 min. at 60° C. and squashed after varying intervals of storage at room temperature were essentially similar to pictures obtained with 1% aceto-orcein and 1% aceto-orcein-N HCl (10:1) under identical conditions of handling. The chromosomes appear quadri-partite at metaphase and bi-partite at anaphase. A rare instance of the precocious assumption of a quadri-partite condition by two anaphase chromosomes is illustrated. Caduceus coiling of chromonemata was seen in chromosome bridges also. Chromosomes have material easily dissociable from the chromonemata and their removal does not affect the structural integrity of the chromosome.

Structural stability of slender aerospace vehicles: Part I Mathematical modeling

A detailed mechanics based model is developed to analyze the problem of structural instability in slender aerospace vehicles. Coupling among the rigid-body modes, the longitudinal vibrational modes and the transverse vibrational modes due to asymmetric lifting-body cross-section are considered. The model also incorporates the effects of aerodynamic pressure and the propulsive thrust of the vehicle. The model is one-dimensional, and it can be employed to idealized slender vehicles with complex shapes. Condition under which a flexible body with internal stress waves behaves like a perfect rigid body is derived. Two methods are developed for finite element discretization of the system: (1) A time-frequency Fourier spectral finite element method and (2) h-p finite element method. Numerical results using the above methods are presented in Part II of this paper. (C) 2010 Elsevier Ltd. All rights reserved.

Type I seesaw mechanism for quasi-degenerate neutrinos

We discuss symmetries and scenarios leading to quasi-degenerate neutrinos in type I seesaw models. The existence of degeneracy in the present approach is not linked to any specific structure for the Dirac neutrino Yukawa coupling matrix y(D) and holds in general. Basic input is the application of the minimal flavour violation principle to the leptonic sector. Generalizing this principle, we assume that the structure of the right-handed neutrino mass matrix is determined by y(D) and the charged lepton Yukawa coupling matrix y(l) in an effective theory invariant under specific groups G(F) contained in the full symmetry group of the kinetic energy terms. G(F) invariance also leads to specific structure for the departure from degeneracy. The neutrino mass matrix (with degenerate mass m(0)) resulting after seesaw mechanism has a simple form Mv approximate to m(0)(I - py(l)y(l)(T)) in one particular scenario based on supersymmetry. This form is shown tolead to correct description of neutrino masses and mixing angles. The thermal leptogenesis after inclusion of flavour effects can account for the observed baryon asymmetry of the universe within the present scenario. Rates for lepton flavour violating processes can occur at observable levels in the supersymmetric version of the scenario. (c) 2010 Elsevier B.V. All rights reserved.

A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants

Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity. (C) 2010 Elsevier B.V. All rights reserved.