Hierarchical sampling for metastable conformers determines biomolecular recognition: the case of malectin and diglucosylated N-glycan interactions

Structural information over the entire course of binding interactions based on the analyses of energy landscapes is described, which provides a framework to understand the events involved during biomolecular recognition. Conformational dynamics of malectin's exquisite selectivity for diglucosylated N-glycan (Dig-N-glycan), a highly flexible oligosaccharide comprising of numerous dihedral torsion angles, are described as an example. For this purpose, a novel approach based on hierarchical sampling for acquiring metastable molecular conformations constituting low-energy minima for understanding the structural features involved in a biologic recognition is proposed. For this purpose, four variants of principal component analysis were employed recursively in both Cartesian space and dihedral angles space that are characterized by free energy landscapes to select the most stable conformational substates. Subsequently, k-means clustering algorithm was implemented for geometric separation of the major native state to acquire a final ensemble of metastable conformers. A comparison of malectin complexes was then performed to characterize their conformational properties. Analyses of stereochemical metrics and other concerted binding events revealed surface complementarity, cooperative and bidentate hydrogen bonds, water-mediated hydrogen bonds, carbohydrate-aromatic interactions including CH-pi and stacking interactions involved in this recognition. Additionally, a striking structural transition from loop to beta-strands in malectin CRD upon specific binding to Dig-N-glycan is observed. The interplay of the above-mentioned binding events in malectin and Dig-N-glycan supports an extended conformational selection model as the underlying binding mechanism.

Sequence specific recognition and cleavage of DNA by synthetic molecules

DNA recognition is an essential biological process responsible for the regulation of cellular functions including protein synthesis and cell division and is implicated in the mechanism of action of some anticancer drugs. Studies directed towards defining the elements responsible for sequence specific DNA recognition through the study of the interactions of synthetic organic ligands with DNA are described.

DNA recognition by poly-N-methylpyrrolecarboxamides was studied by the synthesis and characterization of a series of molecules where the number of contiguous N-methylpyrrolecarboxamide units was increased from 2 to 9. The effect of this incremental change in structure on DNA recognition has been investigated at base pair resolution using affinity cleaving and MPE•Fe(II) footprinting techniques. These studies led to a quantitative relationship between the number of amides in the molecule and the DNA binding site size. This relationship is called the n + 1 rule and it states that a poly-N methylpyrrolecarboxamide molecule with n amides will bind n + 1 base pairs of DNA. This rule is consistent with a model where the carboxamides of these compounds form three center bridging hydrogen bonds between adjacent base pairs on opposite strands of the helix. The poly-N methylpyrrolecarboxamide recognition element was found to preferentially bind poly dA•poly dT stretches; however, both binding site selection and orientation were found to be affected by flanking sequences. Cleavage of large DNA is also described.

One approach towards the design of molecules that bind large sequences of double helical DNA sequence specifically is to couple DNA binding subunits of similar or diverse base pair specificity. Bis-EDTA-distamycin-fumaramide (BEDF) is an octaamide dimer of two tri-N methylpyrrolecarboxamide subunits linked by fumaramide. DNA recognition by BEDF was compared to P7E, an octaamide molecule containing seven consecutive pyrroles. These two compounds were found to recognize the same sites on pBR322 with approximately the same affinities demonstrating that fumaramide is an effective linking element for Nmethylpyrrolecarboxamide recognition subunits. Further studies involved the synthesis and characterization of a trimer of tetra-N-methylpyrrolecarboxamide subunits linked by β-alanine ((P4)_(3)E). This trimerization produced a molecule which is capable of recognizing 16 base pairs of A•T DNA, more than a turn and a half of the DNA helix.

DNA footprinting is a powerful direct method for determining the binding sites of proteins and small molecules on heterogeneous DNA. It was found that attachment of EDTA•Fe(II) to spermine creates a molecule, SE•Fe(II), which binds and cleaves DNA sequence neutrally. This lack of specificity provides evidence that at the nucleotide level polyamines recognize heterogeneous DNA independent of sequence and allows SE•Fe(II) to be used as a footprinting reagent. SE•Fe(II) was compared with two other small molecule footprinting reagents, EDTA•Fe(II) and MPE•Fe(II).

A Aplicação Errônea do Direito Brasileiro pelo Árbitro: Uma Análise à Luz do Direito Comparado

A presente tese versa sobre a aplicação errônea, pelo árbitro, do Direito Brasileiro ao mérito do litígio. Tendo em vista o ineditismo, em nosso País, do estudo das consequências advindas à sentença arbitral prolatada mediante uma aplicação equivocada do Direito que rege o mérito da controvérsia, revelou-se fundamental uma análise de Direito Comparado. Assim, consultou-se o Direito dos Estados Unidos da América, do Reino Unido, da Alemanha, da Itália, da Áustria, da Suíça e da França, a fim de se constatar se é admissível, nos referidos Países, alguma medida judicial contra tal situação. Primeiramente, empreendeu-se um exame das consequências que podem advir, nos 7 (sete) Países citados, a uma sentença arbitral doméstica, assim definida conforme a legislação de cada País. Como a medida habitual para se afastar uma sentença arbitral doméstica é a ação de anulação, buscou-se examinar as hipóteses que ensejam seu ajuizamento em cada País estudado, a fim de se constatar se dentre elas insere-se a aplicação errônea do Direito que rege o fundo da disputa. Ou, em caso negativo, se a violação à ordem pública inclui-se nas hipóteses de anulação. E, por último, se a ofensa à ordem pública compreende a aplicação errônea, pelo árbitro, do Direito de cada País sub examine. Em segundo lugar, examinaram-se as consequências que advêm para uma sentença arbitral estrangeira em que se aplicou erroneamente o Direito que rege o mérito da controvérsia. Como todos os sete Países examinados são Estados-membros da Convenção de Nova Iorque de 10 de junho de 1958 sobre o Reconhecimento e a Execução de Sentenças Arbitrais Estrangeiras, que prevê em seu artigo V, n 2, alínea b, a violação à ordem pública como óbice à homologação da sentença arbitra alienígena, a aplicação do referido dispositivo em cada Estado foi analisado. Tentou-se averiguar se a ofensa à ordem pública consubstanciada na referida Convenção poderia abranger a aplicação errônea do Direito material pelo árbitro. Em seguida, examinou-se a legislação e a doutrina brasileiras tanto no que tange à ação anulatória de laudos arbitrais brasileiros, quanto à homologação dos laudos estrangeiros , a fim de se proporem soluções para esta questão em nosso ordenamento jurídico. Por fim, analisou-se se é possível, no Brasil, homologar uma sentença arbitral estrangeira que tenha sido anulada no País em que foi prolatada com base no argumento da aplicação errônea do Direito do referido Estado ao mérito da arbitragem.

Proton magnetic resonance studies of ribonucleic acid complexes. I. Complexes of biological bases and oligonucleotides with RNA. II. Template recognition and the degeneracy of the genetic code

Part I. Complexes of Biological Bases and Oligonucleotides with RNA

The physical nature of complexes of several biological bases and oligonucleotides with single-stranded ribonucleic acids have been studied by high resolution proton magnetic resonance spectroscopy. The importance of various forces in the stabilization of these complexes is also discussed.

Previous work has shown that purine forms an intercalated complex with single-stranded nucleic acids. This complex formation led to severe and stereospecific broadening of the purine resonances. From the field dependence of the linewidths, T₁ measurements of the purine protons and nuclear Overhauser enhancement experiments, the mechanism for the line broadening was ascertained to be dipole-dipole interactions between the purine protons and the ribose protons of the nucleic acid.

The interactions of ethidium bromide (EB) with several RNA residues have been studied. EB forms vertically stacked aggregates with itself as well as with uridine, 3'-uridine monophosphate and 5'-uridine monophosphate and forms an intercalated complex with uridylyl (3' → 5') uridine and polyuridylic acid (poly U). The geometry of EB in the intercalated complex has also been determined.

The effect of chain length of oligo-A-nucleotides on their mode of interaction with poly U in D₂0 at neutral pD have also been studied. Below room temperatures, ApA and ApApA form a rigid triple-stranded complex involving a stoichiometry of one adenine to two uracil bases, presumably via specific adenine-uracil base pairing and cooperative base stacking of the adenine bases. While no evidence was obtained for the interaction of ApA with poly U above room temperature, ApApA exhibited complex formation of a 1:1 nature with poly U by forming Watson-Crick base pairs. The thermodynamics of these systems are discussed.

Part II. Template Recognition and the Degeneracy of the Genetic Code

The interaction of ApApG and poly U was studied as a model system for the codon-anticodon interaction of tRNA and mRNA in vivo. ApApG was shown to interact with poly U below ~20°C. The interaction was of a 1:1 nature which exhibited the Hoogsteen bonding scheme. The three bases of ApApG are in an anti conformation and the guanosine base appears to be in the lactim tautomeric form in the complex.

Due to the inadequacies of previous models for the degeneracy of the genetic code in explaining the observed interactions of ApApG with poly U, the "tautomeric doublet" model is proposed as a possible explanation of the degenerate interactions of tRNA with mRNA during protein synthesis in vivo.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic leach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

Computer-assisted automatic peak recognition and result evaluation for analysis of chlorinated hydrocarbons in environmental samples

A data manipulation method has been developed for automatic peak recognition and result evaluation in the analysis of organic chlorinated hydrocarbons with dual-column gas chromatography. Based on the retention times of two internal standards, pentachlorotoluene and decachlorobiphenyl, the retention times of chlorinated hydrocarbons can be calibrated automatically and accurately. It is very convenient to identify the peaks by comparing the retention times of samples with the calibrated retention times calculated from the relative retention indices of standards. Meanwhile, with a suggested two-step evaluation method the evaluation coefficients and the suitable quantitative results of each component can be automatically achieved for practical samples in an analytical system using two columns with different polarities and two internal standards. (C) 2002 Elsevier Science B.V. All rights reserved.

View-Based Models of 3D Object Recognition and Class-Specific Invariances

This paper describes the main features of a view-based model of object recognition. The model tries to capture general properties to be expected in a biological architecture for object recognition. The basic module is a regularization network in which each of the hidden units is broadly tuned to a specific view of the object to be recognized.

Fault identification through the combination of symbolic conflict recognition and Markov Chain-aided belief revision

F. Smith and Q. Shen. Fault identification through the combination of symbolic conflict recognition and Markov Chain-aided belief revision. IEEE Transactions on Systems, Man and Cybernetics, Part A: Systems and Humans, 34(5):649-663, 2004.

Christian missions and social progress : a sociological study of foreign missions (vol. 1)

http://www.archive.org/details/socialprogress00dennuoft/