Fluorescent probe studies of biomembranes and model systems. The design and use of anionic site reporters

The fluorescence properties of a homologous series of fluorescent alkylamines are described. The binding of the probes to crythrocyte membranes increases with the length of the alkyl chain. The probes are shown to interact more strongly with membranes than with protein and lipid model systems. The binding of the probes to the membrane is sensitive to the cation concentration of the medium.

Use of structure-directed DNA ligands to probe the binding of recA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing

We have used circular dichroism and structure-directed drugs to identify the role of structural features, wide and narrow grooves in particular, required for the cooperative polymerization, recognition of homologous sequences, and the formation of joint molecules promoted by recA protein. The path of cooperative polymerization of recA protein was deduced by its ability to cause quantitative displacement of distamycin from the narrow groove of duplex DNA. By contrast, methyl green bound to the wide groove was retained by the nucleoprotein filaments comprised of recA protein-DNA. Further, the mode of binding of these ligands and recA protein to DNA was confirmed by DNaseI digestion. More importantly, the formation of joint molecules was prevented by distamycin in the narrow groove while methyl green in the wide groove had no adverse effect. Intriguingly, distamycin interfered with the production of coaggregates between nucleoprotein filaments of recA protein-M13 ssDNA and naked linear M13 duplex DNA, but not with linear phi X174 duplex DNA. Thus, these data, in conjunction with molecular modeling, suggest that the narrow grooves of duplex DNA provide the fundamental framework required for the cooperative polymerization of recA protein and alignment of homologous sequences. These findings and their significance are discussed in relation to models of homologous pairing between two intertwined DNA molecules.

Anion directed template synthesis of Cu(II) complexes of a N,N,O donor mono-condensed Schiff base ligand: A molecular scaffold forming highly ordered H-bonded rectangular grids

Anion directed, template syntheses of two dinuclear copper(II) complexes of mono-condensed Schiff base ligand Hdipn (4-[(3-aminopentylimino)-methyl]-benzene-1,3-diol) involving 2,4- dihydroxybenzaldehyde and 1,3-diaminopentane were realized in the presence of bridging azide and acetate anions. Both complexes, [Cu-2(dipn)(2)(N-3)(2)] (1) and [Cu-2(dip(n))(2)(OAc)(2)] (2) have been characterized by X-ray crystallography. The two mononuclear units are joined together by basal-apical, double end-on azido bridges in complex 1 and by basal-apical, double mono-atomic acetate oxygen-bridges in 2. Both complexes form rectangular grid-like supramolecular structures via H-bonds connecting the azide or acetate anion and the p-hydroxy group of 2,4- dihydroxybenzaldehyde. Variable-temperature (300-2 K) magnetic susceptibility measurements reveal that complex 1 has antiferromagnetic coupling (J = -2.10 cm (1)) through the azide bridge while 2 has intra-dimer ferromagnetic coupling through the acetate bridge and inter-dimer antiferromagnetic coupling through H-bonds (J = 2.85 cm (1), J' = -1.08 cm (1)). (C) 2009 Elsevier B. V. All rights reserved.

Expression profiling of lymph nodes in tuberculosis patients reveal inflammatory milieu at site of infection

Extrapulmonary manifestations constitute 15 to 20% of tuberculosis cases, with lymph node tuberculosis (LNTB) as the most common form of infection. However, diagnosis and treatment advances are hindered by lack of understanding of LNTB biology. To identify host response, Mycobacterium tuberculosis infected lymph nodes from LNTB patients were studied by means of transcriptomics and quantitative proteomics analyses. The selected targets obtained by comparative analyses were validated by quantitative PCR and immunohistochemistry. This approach provided expression data for 8,728 transcripts and 102 proteins, differentially regulated in the infected human lymph node. Enhanced inflammation with upregulation of T-helper1-related genes, combined with marked dysregulation of matrix metalloproteinases, indicates tissue damage due to high immunoactivity at infected niche. This expression signature was accompanied by significant upregulation of an immunoregulatory gene, leukotriene A4 hydrolase, at both transcript and protein levels. Comparative transcriptional analyses revealed LNTB-specific perturbations. In contrast to pulmonary TB-associated increase in lipid metabolism, genes involved in fatty-acid metabolism were found to be downregulated in LNTB suggesting differential lipid metabolic signature. This study investigates the tissue molecular signature of LNTB patients for the first time and presents findings that indicate the possible mechanism of disease pathology through dysregulation of inflammatory and tissue-repair processes.

Structures and molecular-dynamics studies of three active-site mutants of bovine pancreatic phospholipase A(2)

Phospholipase A(2) hydrolyzes phospholipids at the sn-2 position to cleave the fatty-acid ester bond of L-glycerophospholipids. The catalytic dyad (Asp99 and His48) along with a nucleophilic water molecule is responsible for enzyme hydrolysis. Furthermore, the residue Asp49 in the calcium-binding loop is essential for controlling the binding of the calcium ion and the catalytic action of phospholipase A2. To elucidate the structural role of His48 and Asp49, the crystal structures of three active-site single mutants H48N, D49N and D49K have been determined at 1.9 angstrom resolution. Although the catalytically important calcium ion is present in the H48N mutant, the crystal structure shows that proton transfer is not possible from the catalytic water to the mutated residue. In the case of the Asp49 mutants, no calcium ion was found in the active site. However, the tertiary structures of the three active-site mutants are similar to that of the trigonal recombinant enzyme. Molecular-dynamics simulation studies provide a good explanation for the crystallographic results.

Studies on the ionic transport and structural investigations of La0.5Li0.5TiO3 perovskite synthesized by wet chemical methods and the effect of Ce, Zr substitution at Ti site

La0.5Li0.5TiO3 perovskite was synthesized by various wet chemical methods. By adopting low temperature methods of preparation lithium loss from the material is prevented. La0.5Li0.5TiO3 (LLTO) was formed with cubic symmetry at 1473 K. LLTO was formed at relatively lower temperature by using hydrothermal preparation method. PVA gel-decomposition route yield tetragonal LLTO on annealing the dried gel at 1473 K. By using gel-carbonate route LiTi2O4 minor phase was found to remain even after heat-treatment at 1473 K. The hydroxylation of LLTO was done in deionized water as well as in dilute acetic acid medium. By hydroxylation process incorporation of hydroxyls and leaching out of Li+ was observed from the material. The Li+ concentration of these compositions was examined by AAS. The electrical conductivities of these compositions were measured by dc and ac impedance techniques at elevated temperatures. The activation energies of electrical conduction for these compositions were estimated from the experimental results. The measured activation energy of Li+ conduction is 0.34 eV. Unhydroxylated samples exhibit only Li+ conduction, whereas, the hydroxylated LLTO show proton conductivity at 298-550 K in addition to Li+ conductivity. The effect of Zr or Ce substitution in place of Ti were attempted. La0.5Li0.5ZrO3 Perovskite was not formed; instead pyrochlore phase (La2Zr2O7) along with monoclinic ZrO2 phases was observed above 1173 K; below 1173 K cubic ZrO2 is stable. (La0.5Li0.5)(2)CeO4 solid solution was formed in the case of Ce substitution at Ti sublattice on heat-treatment up to 1673 K. (c) 2005 Springer Science + Business Media, Inc.

Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Cloning by genomic PCR and production of peanut agglutinin in Escherichia coli

Using the polymerase chain reaction, the coding sequence for peanut agglutinin (PNA) was cloned and expressed in Escherichia coli. Amplified PNA is identical to previously reported cDNA, suggesting the absence of any introns in PNA gene. Recombinant (re-) PNA forms inclusion bodies in E. coli. Production of PNA was confirmed by probing Western blots with polyclonal anti-PNA immunoglobulin G. Inclusion bodies were solubilized with 6 M guanidine-HCl and renatured by rapid dilution in the presence of metal ions. The renatured lectin was then purified by affinity chromatography. The re-lectin shows carbohydrate-binding properties similar to the natural PNA. This expression system provides a model for future mutagenesis studies of the carbohydrate-binding site and thus facilitates ongoing efforts to explore the molecular basis for the specificity of lectin-carbohydrate interaction.

N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yI)colcemid, a probe for different classes of colchincine-binding site on tubulin

The nature of binding of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-colcemid (NBD-colcemid), an environment-sensitive fluorescent analogue of colchicine, to tubulin was tested. This article reports the first fluorometric study where two types of binding site of colchincine analogue on tubulin were detected. Binding of NBD-colcemid to one of these sites equilibrates slsowly. NBD-colcemid competes with colchicine for this site. Binding of NBD-colcemid to this site also causes inhibition of tubulin self-assembly. In contrast, NBD-colcemid binding to the other site is characterised by rapid equilibration and lack of competition with colchicine. Nevertheless, binding to this site is highly specific for the cholchicine nucleus, as alkyl-NBD analogues have no significant binding activity. Fast-reaction-kinetic studies gave 1.76 × 105 M–1 s–1 for the association and 0.79 s–1 for the dissociation rate constants for the binding of NBD-colcemid to the fast site of tubulin. The association rate constants for the two phases of the slow site are 0.016 × 10–4 M–1 s–1 and 3.5 × 10–4 M–1 respectively. These two sites may be related to the two sites of colchicine reported earlier, with binding characteristics altered by the increased hydrophobic nature of NBD-colcemid.

Hybrid moving block bootstrap for stochastic simulation of multi-site multi-season streamflows

The Hybrid approach introduced by the authors for at-site modeling of annual and periodic streamflows in earlier works is extended to simulate multi-site multi-season streamflows. It bears significance in integrated river basin planning studies. This hybrid model involves: (i) partial pre-whitening of standardized multi-season streamflows at each site using a parsimonious linear periodic model; (ii) contemporaneous resampling of the resulting residuals with an appropriate block size, using moving block bootstrap (non-parametric, NP) technique; and (iii) post-blackening the bootstrapped innovation series at each site, by adding the corresponding parametric model component for the site, to obtain generated streamflows at each of the sites. It gains significantly by effectively utilizing the merits of both parametric and NP models. It is able to reproduce various statistics, including the dependence relationships at both spatial and temporal levels without using any normalizing transformations and/or adjustment procedures. The potential of the hybrid model in reproducing a wide variety of statistics including the run characteristics, is demonstrated through an application for multi-site streamflow generation in the Upper Cauvery river basin, Southern India. (C) 2004 Elsevier B.V. All rights reserved.

Unusual dielectric response in B-site size-disordered hexagonal transition metal oxides

We discover that hexagonal holmium copper titanate (Ho2CuTiO6), has a unique and highly desirable combination of high dielectric constant, low losses, very small temperature coefficient, and low frequency dependence. Our first-principles calculations indicate that these exceptional properties result from a size-difference at the Cu/Ti B-site that suppresses the expected ferroelectric transition, combined with the dominance of intermediate-frequency polar vibrational modes in the dielectric response. Our results suggest that the use of such B-site disorder in alloys of hexagonal transition-metal oxides should generally result in similar robust dielectrics.

Site characterization model using least-square support vector machine and relevance vector machine based on corrected SPT data (N-c)

Statistical learning algorithms provide a viable framework for geotechnical engineering modeling. This paper describes two statistical learning algorithms applied for site characterization modeling based on standard penetration test (SPT) data. More than 2700 field SPT values (N) have been collected from 766 boreholes spread over an area of 220 sqkm area in Bangalore. To get N corrected value (N,), N values have been corrected (Ne) for different parameters such as overburden stress, size of borehole, type of sampler, length of connecting rod, etc. In three-dimensional site characterization model, the function N-c=N-c (X, Y, Z), where X, Y and Z are the coordinates of a point corresponding to N, value, is to be approximated in which N, value at any half-space point in Bangalore can be determined. The first algorithm uses least-square support vector machine (LSSVM), which is related to aridge regression type of support vector machine. The second algorithm uses relevance vector machine (RVM), which combines the strengths of kernel-based methods and Bayesian theory to establish the relationships between a set of input vectors and a desired output. The paper also presents the comparative study between the developed LSSVM and RVM model for site characterization. Copyright (C) 2009 John Wiley & Sons,Ltd.

Structural aspects of semantic-directed clusters

Knowledge-based clusters are studied from the structural point of view. Generalized descriptions for such clusters are stated and illustrated. Peculiarities of certain knowledge-based cluster configurations are highlighted. The adequacy of the connectives logical and (“and”) logical or (“exclusive-or”) in describing such clusters is justified. The definition of “concept” is elaborated from the clustering point of view and used to establish the equivalence between, descriptions of clusters and concepts. The order-independence of semantic-directed clustering approach is established formally based on axiomatic considerations.