Role of the Central Metal Ion and Ligand Charge in the DNA Binding and Modification by Metallosalen Complexes

Several metal complexes of three different functionalized salen derivatives have been synthesized. The salens differ in terms of the electrostatic character and the location of the charges. The interactions of such complexes with DNA were first investigated in detail by UV−vis absorption titrimetry. It appears that the DNA binding by most of these compounds is primarily due to a combination of electrostatic and other modes of interactions. The melting temperatures of DNA in the presence of various metal complexes were higher than that of the pure DNA. The presence of additional charge on the central metal ion core in the complex, however, alters the nature of binding. Bis-cationic salen complexes containing central Ni(II) or Mn(III) were found to induce DNA strand scission, especially in the presence of co-oxidant as revealed by plasmid DNA cleavage assay and also on the basis of the autoradiogram obtained from their respective high-resolution sequencing gels. Modest base selectivity was observed in the DNA cleavage reactions. Comparisons of the linearized and supercoiled forms of DNA in the metal complex-mediated cleavage reactions reveal that the supercoiled forms are more susceptible to DNA scission. Under suitable conditions, the DNA cleavage reactions can be induced either by preformed metal complexes or by in situ complexation of the ligand in the presence of the appropriate metal ion. Also revealed was the fact that the analogous complexes containing Cu(II) or Cr(III) did not effect any DNA strand scission under comparable conditions. Salens with pendant negative charges on either side of the precursor salicylaldehyde or ethylenediamine fragments did not bind with DNA. Similarly, metallosalen complexes with net anionic character also failed to induce any DNA modification activities.

Mapping of assembled epitopes with microgram quantities of antigen: Identification of an epitope at the receptor binding region of human follicle stimulating hormone

Identification of epitopes by modification studies has been reported by us recently. The method requires milligram quantities of antigen and since several proteins are not available in large quantities they are not amenable for such an investigation. One such protein is human follicle stimulating hormone (hFSH) whose mapping of epitopes is of importance in reproductive biology. Here we report a method that uses microgram quantities of hFSH to map a beta-specific epitope located at the receptor binding region. This identification has also been validated by the chemical modification method using heterologous antigen ovine follicle stimulating hormone (oFSH).

Oestrogen-induced synthesis of thiamin-binding protein in immature chicks. Kinetics of induction, hormonal specificity and modulation

A specific radioimmunoassay procedure was developed to monitor the plasma concentrations of thiamin-binding protein, a minor yolk constituent of the chicken egg. By using this sensitive assay, the kinetics of oestrogen-induced elaboration of this specific protein in immature chicks was investigated. After a single injection of the steroid hormone, with an initial lag period of 4–5h the thiamin-binding protein rapidly accumulated in the plasma, attaining peak concentrations around 75h and declining thereafter. A 4-fold amplification of the response was noticed during the secondary stimulation, and this increased to 9-fold during the tertiary stimulation with the steroid hormone. The magnitude of the response was dependent on the hormone dose, and the initial latent period and the duration of the ascending phase of induction were unchanged for the hormonal doses tested during both the primary and secondary stimulations. The circulatory half-life of the protein was 6h as calculated from the measurement of the rate of disappearance of the exogenously administered 125I-labelled protein. Simultaneous administration of progesterone, dihydrotestosterone or corticosterone did not alter the pattern of induction. On the other hand, hyperthyroidism markedly decreased the oestrogenic response, whereas propylthiouracil-induced hypothyroidism had the opposite effect. The anti-oestrogen E- and Z-clomiphene citrates, administered 30min before oestrogen, effectively blocked the hormonal induction. α-Amanitin and cycloheximide administered along with or shortly after the sex steroid severely curtailed the protein elaboration. A comparison of the kinetics of induction of thiamin- and riboflavin-binding proteins by oestrogen revealed that, beneath an apparent similarity, a clear-cut difference exists between the two vitamin-binding proteins, particularly with regard to hormonal dose-dependent sensitivity of induction and the half-life in circulation. The steroid-mediated elaboration of the two yolk proteins thus appears to be not strictly co-ordinated, despite several common regulatory features underlying their induction.

Glycodelin A triggers T cell apoptosis through a novel calcium-independent galactose-binding lectin activity

Glycodelin A (GdA) is one of the progesterone inducible endometrial factors that protect the fetal semiallograft from maternal immune rejection. The immumoregulatory effects of GdA are varied, with diverse effects on the fate and function of most immune cell types. Its effects on T cells are particularly relevant as it is capable of regulating T cell activation, differentiation, as well as apoptosis. We have previously reported that GdA triggers mitochondrial stress and apoptosis in activated T cells by a mechanism that is distinct and independent of its effects on T cell activation. In this study we describe the characterization of a cell surface receptor for GdA on T cells. Our results reveal a novel calcium-independent galactose-binding lectin activity of GdA, which is responsible for its apoptogenic function. This discovery adds GdA to a select group of soluble immunoregulatory lectins that operate within the feto-placental compartment, the only other members being the galectin family proteins. We also report for the first time that both CD4(+) and CD8(+) T cell subsets are equally susceptible to inhibition with GdA, mediated by its novel lectin activity. We demonstrate that GdA selectively recognizes complex-type N-linked glycans on T cell surface glycoproteins. and propose that the galectin-1 glycoprotein receptor CD7 maybe a novel target for GdA on T cells. This study, for the first time, links the lectin activity of GdA to its biological function.

Computing magnetic anisotropy constants of single molecule magnets

We present here a theoretical approach to compute the molecular magnetic anisotropy parameters, D (M) and E (M) for single molecule magnets in any given spin eigenstate of exchange spin Hamiltonian. We first describe a hybrid constant M (S) valence bond (VB) technique of solving spin Hamiltonians employing full spatial and spin symmetry adaptation and we illustrate this technique by solving the exchange Hamiltonian of the Cu6Fe8 system. Treating the anisotropy Hamiltonian as perturbation, we compute the D (M)and E(M) values for various eigenstates of the exchange Hamiltonian. Since, the dipolar contribution to the magnetic anisotropy is negligibly small, we calculate the molecular anisotropy from the single-ion anisotropies of the metal centers. We have studied the variation of D (M) and E(M) by rotating the single-ion anisotropies in the case of Mn12Ac and Fe-8 SMMs in ground and few low-lying excited states of the exchange Hamiltonian. In both the systems, we find that the molecular anisotropy changes drastically when the single-ion anisotropies are rotated. While in Mn12Ac SMM D (M) values depend strongly on the spin of the eigenstate, it is almost independent of the spin of the eigenstate in Fe-8 SMM. We also find that the D (M)value is almost insensitive to the orientation of the anisotropy of the core Mn(IV) ions. The dependence of D (M) on the energy gap between the ground and the excited states in both the systems has also been studied by using different sets of exchange constants.

Cu-II-Azide Polymers of Cu-3 and Cu-6 Building Units: Synthesis,Structures, and Magnetic Exchange Mechanism

Two new neutral copper-azido polymers [Cu-3(N-3)(6)(tmen)(2)](n)(1)and [Cu-6(N-3)(12)(deen)(2)](n) (2) [tmen = N,N,N, N-tetramethylethylenediamine and deen = N,N-diethylethylenediamine] have been synthesized by using lower molar equivalents of the chelating diamine ligands with Cu(NO3)(2)center dot 3H(2)O and an excess of NaN3. The single crystal X-ray structure shows that in the basic unit of the 1D complex 1, the three Cu-II ions are linked by double end-on azido bridges with Cu-N-EO-Cu angles on both sides of the magnetic exchange critical angle of 108 degrees. Complex 2 is a 3D framework of a basic u-6 cluster. Cryomagnetic susceptibility measurements over a wide range of temperature exhibit dominant ferromagnetic behavior in both the complexes. Density functional theory calculations (B3LYP functional) have been performed on the trinuclear unit to provide a qualitative theoretical interpretation of the overall ferromagnetic behavior shown by the complex 1.

Nonspecifically bound proteins spin while diffusing along DNA

It is known that DNA-binding proteins can slide along the DNA helix while searching for specific binding sites, but their path of motion remains obscure. Do these proteins undergo simple one-dimensional (1D) translational diffusion, or do they rotate to maintain a specific orientation with respect to the DNA helix? We measured 1D diffusion constants as a function of protein size while maintaining the DNA-protein interface. Using bootstrap analysis of single-molecule diffusion data, we compared the results to theoretical predictions for pure translational motion and rotation-coupled sliding along the DNA. The data indicate that DNA-binding proteins undergo rotation-coupled sliding along the DNA helix and can be described by a model of diffusion along the DNA helix on a rugged free-energy landscape. A similar analysis including the 1D diffusion constants of eight proteins of varying size shows that rotation-coupled sliding is a general phenomenon. The average free-energy barrier for sliding along the DNA was 1.1 +/- 0.2 k(B)T. Such small barriers facilitate rapid search for binding sites.

The Mycobacterial MsDps2 Protein Is a Nucleoid-Forming DNA Binding Protein Regulated by Sigma Factors sigma(A) and sigma(B)

The Dps (DNA-binding protein from starved cells) proteins from Mycobacterium smegmatis MsDps1 and MsDps2 are both DNA-binding proteins with some differences. While MsDps1 has two oligomeric states, with one of them responsible for DNA binding, MsDps2 has only one DNA-binding oligomeric state. Both the proteins however, show iron-binding activity. The MsDps1 protein has been shown previously to be induced under conditions of starvation and osmotic stress and is regulated by the extra cellular sigma factors sigma(H) and sigma(F). We show here, that the second Dps homologue in M. smegmatis, namely MsDps2, is purified in a DNA-bound form and exhibits nucleoid-like structures under the atomic force microscope. It appears that the N-terminal sequence of Dps2 plays a role in nucleoid formation. MsDps2, unlike MsDps1, does not show elevated expression in nutritionally starved or stationary phase conditions; rather its promoter is recognized by RNA polymerase containing sigma(A) or sigma(B), under in vitro conditions. We propose that due to the nucleoid-condensing ability, the expression of MsDps2 is tightly regulated inside the cells.

Toward the Discovery of Vaccine Adjuvants: Coupling In Silico Screening and In Vitro Analysis of Antagonist Binding to Human and Mouse CCR4 Receptors

Background: Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology: Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cellmigration, including that of CCR4(+) Tregs. Significance: Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Spontaneous and reversible self-assembly of a polypeptide fragment of insulin-like growth factor binding protein-2 into fluorescent nanotubular structures

In this communication, we report the spontaneous and reversible in vitro self-assembly of a polypeptide fragment derived from the C-terminal domain of Insulin-like Growth Factor Binding Protein (IGFBP-2) into soluble nanotubular structures several micrometres long via a mechanism involving inter-molecular disulfide bonds and exhibiting enhanced fluorescence.

Natively unfolded nucleic acid binding P8 domain of SeMV polyprotein 2a affects the novel ATPase activity of the preceding P10 domain

Open reading frame (ORF) 2a of Sesbania mosaic virus (SeMV) codes for polyprotein 2a (Membrane anchor-protease-VPg-P10-P8). The C-terminal domain of SeMV polyprotein 2a was cloned, expressed and purified in order to functionally characterize it. The protein of size 8 kDa (P8) domain, like viral protein genome linked (VPg), was found to be natively unfolded and could bind to nucleic acids.Interestingly, P10-P8 but not P8 showed a novel Mg2+ dependent ATPase activity that was inhibited in the presence of poly A. In the absence of P8, the ATPase activity of the protein of size 10 kDa (P10) domain was reduced suggesting that the natively unfolded P8 domain influenced the P10 ATPase.

Immunospecific binding of tRNA precursors containing N6-(delta 2-isopentenyl) adenosine

Antibodies specific for N6-(delta 2-isopentenyl) adenosine (i6A) were immobilized on Sepharose and this adsorbent (Sepharose-anti-i6A) was used to selectively isolate bacteriophage T4 tRNA precursors containing i6A/ms2i6A from an unfractionated population of 32P-labeled T4 RNAs. The results showed that antibodies to i6A selectively bound only those tRNA precursors containing i6A/ms2i6A. Binding of tRNA precursors by antibody and specificity of the binding was assessed by membrane binding using 32P-labeled tRNA precursor. Binding was highly specific for i6A/ms2i6A residues in the tRNA precursors. This binding can be used to separate modified from unmodified precursor RNAs and to study the biosynthetic pathways of tRNA precursors.

13C nuclear magnetic resonance studies of the binding of alkali and alkaline earth metal salts to amides

3C resonances of carbonyl and methyl groups in amides are shifted down-field on interaction with alkali and alkaline earth metal salts. The magnitude of the shift depends on the ionic potential of the cation. Ions like Li+ bind to the amide carbonyl group both in neat amide solutions as well as in concentrated salt solutions in water.

Fluorescence polarization as a tool to study lectin-sugar interaction. An investigation of the binding of 4-methylumbelliferyl beta-D-galactopyranoside to Abrus precatorious agglutinin

Polarization of ligand fluorescence was used to study the binding of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmb-Galp) to Abrus precatorious agglutinin. The binding of the fluorescent sugar to the lectin led to considerable polarization of the MeUmb-Galp fluorescence, which was also quenched by about 30% on binding to the lectin. The binding of the fluorescent sugar was carbohydrate-specific, as evidenced by inhibition of both fluorescence polarization and quenching when lectin was preincubated with lactose. The association constant as determined by fluorescence polarization is 1.42 x 10(4) M-1 at 25 degrees C and is in excellent agreement with those determined by fluorescence quenching (Ka = 1.51 x 10(4) M-1) and equilibrium dialysis (Ka = 1.62 x 10(4) M-1) at 25 degrees C. The numbers of binding sites as determined by fluorescence polarization, quenching and equilibrium dialysis agree very well with one another, n being equal to 2.0 +/- 0.05. The consistency between the association constant value determined by fluorescence polarization, quenching and equilibrium dialysis shows the validity of this approach to study lectin-sugar interaction.