Structure and interactions of aspirin-like drugs

A pre-requisite for the elucidation of the mechanism of action of aspirin-like drugs, which are believed to exert their pharmacological effects through the inhibition of prostaglandin biosynthesis, is an understanding of their molecular geometry, the non-covalent interactions they are likely to be involved in, and the geometrical and the electronic consequences of such interactions. This has been sought to be achieved through the x-ray analysis of these drug molecules and their crystalline complexes with other suitable molecules. The results obtained from such studies have been discussed in terms of specific typical examples. For instance, antipyrine can form metal and hydrogen-bonded complexes; phenylbutazone can form ionic complexes with basic molecules. Complex formation is accompanied by characteristic changes in the molecular geometry and the electronic structure in both the cases. The results obtained so far appear to indicate that the important common invariant structural features of the fenamates, deduced from crystal structures, are retained even when complexation takes place.

Expression Profiling of Nucleotide Metabolism-Related Genes in Human Breast Cancer Cells After Treatment with 5-Fluorouracil

5-Fluorouracil (5-FU) is one of the most widely used drugs for treatment of cancers, including breast cancer that exhibits its anticancer activity by inhibiting DNA synthesis and also incorporated into DNA and RNA. The objective of this investigation was to find out the total nucleotide metabolism genes regulated by 5-FU in breast cancer cell line. The breast cancer cell line MCF-7 was treated with the drug 5-FU. To analyze the expression of genes, we have conducted the experiment using 1.7k and 19k human microarray slide and confirmed the expression of genes by semiquantitative reverse transcription-polymerase chain reaction. The expression of 44 genes involved in the nucleotide metabolism pathway was quantified. Of these 44 genes analyzed, transcription of 6 genes were upregulated and 9 genes were downregulated. Earlier studies revealed that the transcription of genes for key enzymes like thymidylate synthase, thymidinekinase, and dihydropyrimidine dehydrogenase are regulated by 5-FU. This study identified some novel genes like thioredoxin reductase, ectonucleotide triphosphate dephosphorylase, and CTP synthase are regulated by 5-FU. The data also reveal large-scale perturbation in transcription of genes not involved directly in the known mechanism of action of 5-FU.

The Evolution of Guanylyl Cyclases as Multidomain Proteins: Conserved Features of Kinase-Cyclase Domain Fusions

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signa transduction.

Self-Assembly of Bile Steroid Analogues: Molecules, Fibers, and Networks

Structural and rheological features of a series of molecular hydrogels formed by synthetic bile salt analogues have been scrutinized. Among seven gelators, two are neutral compounds, while the others are cationic systems among which one is a tripodal steroid derivative. Despite the fact that the chemical structures are closely related, the variety of physical characteristics is extremely large in the structures of the connected fibers (either plain cylinders or ribbons), in the dynamical modes for stress relaxation of the associated SAFINs, in the scaling laws of the shear elasticity (typical of either cellular solids or fractal floc-like assemblies), in the micron-scale texture and the distribution of ordered domains (spherulites, crystallites) embedded in a random mesh, in the type of nodal zones (either crystalline-like, fiber entanglements, or bundles), in the evolution of the distribution and morphology of fibers and nodes, and in the sensitivity to added salt. SANS appears to be a suitable technique to infer all geometrical parameters defining the fibers, their interaction modes, and the volume fraction of nodes in a SAFIN. The tripodal system is particularly singular in the series and exhibits viscosity overshoots at the startup of shear flows, an “umbrella-like” molecular packing mode involving three molecules per cross section of fiber, and scattering correlation peaks revealing the ordering and overlap of 1d self-assembled polyelectrolyte species.

A hybrid clustering procedure for concentric and chain-like clusters

K-means algorithm is a well known nonhierarchical method for clustering data. The most important limitations of this algorithm are that: (1) it gives final clusters on the basis of the cluster centroids or the seed points chosen initially, and (2) it is appropriate for data sets having fairly isotropic clusters. But this algorithm has the advantage of low computation and storage requirements. On the other hand, hierarchical agglomerative clustering algorithm, which can cluster nonisotropic (chain-like and concentric) clusters, requires high storage and computation requirements. This paper suggests a new method for selecting the initial seed points, so that theK-means algorithm gives the same results for any input data order. This paper also describes a hybrid clustering algorithm, based on the concepts of multilevel theory, which is nonhierarchical at the first level and hierarchical from second level onwards, to cluster data sets having (i) chain-like clusters and (ii) concentric clusters. It is observed that this hybrid clustering algorithm gives the same results as the hierarchical clustering algorithm, with less computation and storage requirements.

Studies on micro explosive driven blast wave propagation in confined domains using NONEL tubes

Explosive driven micro blast waves are generated in the laboratory using NONEL tubes. The explosive mixture coated to the inner walls of the plastic Nonel tube comprises of HMX and Aluminum ( 18mg/m). The detonation is triggered electrically to generate micro blast waves from the open end of the tube. Flow visualization and over pressure measurements have been carried out to understand the propagation dynamics of these micro-blast waves in both confined and unconfined domains. The classical cubic root law used for large scale blast correlation appears to hold good even for these micro-blasts generated in the laboratory.

Review of continuum, finite element and hybrid techniques in the analysis of stress concentrations in structures

When a uniform flow of any nature is interrupted, the readjustment of the flow results in concentrations and rare-factions, so that the peak value of the flow parameter will be higher than that which an elementary computation would suggest. When stress flow in a structure is interrupted, there are stress concentrations. These are generally localized and often large, in relation to the values indicated by simple equilibrium calculations. With the advent of the industrial revolution, dynamic and repeated loading of materials had become commonplace in engine parts and fast moving vehicles of locomotion. This led to serious fatigue failures arising from stress concentrations. Also, many metal forming processes, fabrication techniques and weak-link type safety systems benefit substantially from the intelligent use or avoidance, as appropriate, of stress concentrations. As a result, in the last 80 years, the study and and evaluation of stress concentrations has been a primary objective in the study of solid mechanics. Exact mathematical analysis of stress concentrations in finite bodies presents considerable difficulty for all but a few problems of infinite fields, concentric annuli and the like, treated under the presumption of small deformation, linear elasticity. A whole series of techniques have been developed to deal with different classes of shapes and domains, causes and sources of concentration, material behaviour, phenomenological formulation, etc. These include real and complex functions, conformal mapping, transform techniques, integral equations, finite differences and relaxation, and, more recently, the finite element methods. With the advent of large high speed computers, development of finite element concepts and a good understanding of functional analysis, it is now, in principle, possible to obtain with economy satisfactory solutions to a whole range of concentration problems by intelligently combining theory and computer application. An example is the hybridization of continuum concepts with computer based finite element formulations. This new situation also makes possible a more direct approach to the problem of design which is the primary purpose of most engineering analyses. The trend would appear to be clear: the computer will shape the theory, analysis and design.

Numerical integration over arbitrary polygonal domains based on Schwarz-Christoffel conformal mapping

This paper presents a new numerical integration technique oil arbitrary polygonal domains. The polygonal domain is mapped conformally to the unit disk using Schwarz-Christoffel mapping and a midpoint quadrature rule defined oil this unit disk is used. This method eliminates the need for a two-level isoparametric mapping Usually required. Moreover, the positivity of the Jacobian is guaranteed. Numerical results presented for a few benchmark problems in the context of polygonal finite elements show that the proposed method yields accurate results.

Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1

The genome of the human pathogen Entamoeba histolytica, a primitive protist, contains non-long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1-encoded endonuclease catalyzed reaction, determined under steady-state and single-turnover conditions, revealed a significant burst phase followed by a slower steady-state phase, indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the K-m was 2.6 x 10-8 m and the k(cat) was 1.6 x 10-2 sec-1. For linear E. histolytica DNA substrate the K-m and k(cat) values were 1.3 x 10-8 m and 2.2 x 10-4 sec-1 respectively. Single-turnover reaction kinetics suggested a noncooperative mode of hydrolysis. The enzyme behaved as a monomer. While Mg2+ was required for activity, 60% activity was seen with Mn2+ and none with other divalent metal ions. Substitution of PDX12-14D (a metal-binding motif) with PAX(12-14)D caused local conformational change in the protein tertiary structure, which could contribute to reduced enzyme activity in the mutated protein. The protein underwent conformational change upon the addition of DNA, which is consistent with the known behavior of restriction endonucleases. The similarities with bacterial restriction endonucleases suggest that the EhLINE1-encoded endonuclease was possibly acquired from bacteria through horizontal gene transfer. The loss of strict sequence specificity for nicking may have been subsequently selected to facilitate spread of the retrotransposon to intergenic regions of the E. histolytica genome.

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.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

A characterization of domains in C 2 with noncompact automorphism group

Let D be a bounded domain in C 2 with a non-compact group of holomorphic automorphisms. Model domains for D are obtained under the hypotheses that at least one orbit accumulates at a boundary point near which the boundary is smooth, real analytic and of finite type.

Characterization of the Entamoeba histolytica Ornithine Decarboxylase-Like Enzyme

Background: The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme. Methodology and Results: To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC)-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF) of similar to 1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa) from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size similar to 46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids-aspartate-332, aspartate-361, and tyrosine-323-by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity. Conclusion: To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from E. histolytica. Computer modeling revealed that three of the critical residues required for binding of DFMO to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO.

Delamination of Surfactant-Intercalated Brucite-Like Hydroxy Salts of Cobalt and Copper and Solvothermal Decomposition of the Resultant Colloidal Dispersions

Surfactant anion intercalated hydroxy salts of copper and cobalt of the formula M(OH)(2-x)(surf)(x)center dot mH(2)O [M = Cu, Co; surf = dodecyl sulfate. dodecyl benzene sulfonate. and x = 0.5 for Cu and 0.67 for Co] delaminate readily in 1-butanol to give translucent colloidal dispersions that are stable for months. The extent of delamination and the colloidal dispersion observed in these solids is higher than what had been observed for layered double hydroxides. The dispersions yield the corresponding nanoparticulate oxides on solvothermal decomposition. While the copper hydroxy salt forms similar to 300 nm dendrimer-like CuO nanostructures comprising nanorods of similar to 10 nm diameter, the cobalt analogue forms similar to 20 nm superparamagnetic particles of Co3O4.