Molecular surface comparison. 2. Similarity of electrostatic vector fields in drug design

In the first article of this series¹ a real-time graphics method was described for molecular similarity of scalar properties. This has now been extended for the comparison of molecular vector properties, most notably electrostatic field. A comparison of the various techniques of calculating fields is presented that includes a new method based on natural orbital fitted point charges. In the two examples described, namely, a series of benzodiazepine agonists and a set of serotonin 5-HT₃ antagonists, the program has been shown to produce useful pharmacophoric overlaps that can be used in the design of novel therapeutic agents.     

Molecular drive

The review of the concept of molecular drive developed by Dover is presented. The ideas on the possible role of non-coding DNA are described and the duplication of genes as the fundamental factor preceding the appearance of the gene, which possesses a new function are characterized. The non-Mendelian processes--the non-equal crossing-over and the conversion of genes are considered. Molecular drive includes both these phenomena and the transposition of genes. The examples of drive are presented. Drive can be considered as the fundamental molecular mechanism of speciation and macroevolution. Evolution at molecular level is directly connected with the non-constancy of the genome and is non-Mendelian and non-Darwinian. Both the coding and non-coding DNA can serve as material for evolution.     

Simulations of melting of polyatomic solids and nanoparticles

Molecular dynamics (MD) methods for calculating the melting point of complex molecular and ionic solids and nanoparticles are described. Various approaches for simulating melting and computing the thermodynamic melting point are discussed along with some force fields that have been used in simulations of the melting of molecular and ionic solids. The different structural, energetic and dynamical quantities used to characterize the melting transition are described. The article ends with a discussion of selected examples of melting point calculations of bulk solids and nanoparticles. Pointers on how each method can be implemented in DL_POLY are given.     

Quantitative comparative analysis of complex two-dimensional electropherograms

A protocol is described to detect and assess differences between complex electrophoretic patterns. A semiautomated method is used to collect accurate absolute mobility data from many two-dimensional electropherograms and a computer algorithm has been developed which normalizes and averages these data. The program generates refined numerical maps consisting of the mean electrophoretic mobilities and corresponding confidence limits for each component protein represented in the original two-dimensional electrophoretic pattern. Tests of statistical significance of apparent differences between averaged numerical maps are carried out to evaluate electrophoretic polymorphisms between the ribosomal proteins of two different plant species. Furthermore, using a nonlinear function relating log molecular weight to mobility, precise molecular weight estimates are obtained from measurements of electrophoretic mobilities of proteins in the presence of sodium dodecyl sulfate. Several examples are presented which demonstrate application of these semiautomated analyses to quantitative comparison and interpretation of two dimensional gel electropherograms.     

A Cladistic Outline of the Eumycota

Abstract— A cladistic classification of fungi determined by a parsimony method with 21 terminal taxa and 51 characters is presented. Outgroup comparison with Oomycetes determined polarity assessments. The group Eumycota, including the traditional taxa Hyphochytriomycetes, Chytridio-mycetes, Zygomycetes, Ascomycetes and Basidiomycetes, is defined by two synapomorphies, molecular weight of 25S RNA, and chitin cell walls. Some groups are supported as monophyletic; Eumycota, Amastigomycota, Dicaryomycotina, Ascomycetes, Protobasidiomycetes, Basidiomycetes, Euascomy-cetidae, Hymenomycetidae and Homobasidiomycetales. The Hyphochytriomycota is the sister group to remaining groups. The Taphrinaceae and Saccharomycetaceae are more closely related to the Basidiomycetes than to any of the ascomycetous groups. In the absence of unique character sets groups such as the Mastigomycotina, Hemiascomycetes, Ustomycetes, Holobasidiomycetes, Heterobasidio-mycetes, Phragmobasidiomycetes and the Teliomycetes cannot be maintained and are abandoned as paraphyletic. Characters and terminal taxa used for the analysis are defined and discussed.     

Navigation in Marine Protected Areas: National and International Law

Abstract

World fish resources, fishing methods, and processing operations in the seafood industry are described. The fishery situation in developing countries (LDCs) is discussed, with particular reference to artisanal and other local fisheries, and examples are cited to illustrate the structure of the industry. Technology transfer from developed countries to LDCs is discussed and recommendations are presented for future technology transfer programs. It is concluded that an integrated complex of small projects with defined, attainable objectives and immediate impact on income and food supply of the LDC populations is likely to be more successful than large‐scale programs with little immediate payoff. A case study of fisheries in two developing countries, Thailand and Peru, and extensive tabulation of statistical data on catches, value of catch, and unit value of fish species groups for selected countries, with a discussion of the significance of the data, are presented in an appendix.     

The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations

High-performance electrophoresis chromatography (HPEC) is a recent development that features continuous-elution gel electrophoresis for isolating proteins or peptides in range of 1 to 300 microgram quantities. Column gel electrophoresis is conducted under thermostated conditions, and the field voltage can be varied within a run with a programmable power supply. Applications of this apparatus in protein purification are presented to demonstrate the utility of the (Model 230A) HPEC. These examples include on-line detection with direct analyte recovery of highly purified sample, which mimics high-performance liquid chromatography, for subsequent structure-function characterization. A method to remove salts from sodium dodecyl sulfate electrophoresed samples for subsequent sequencing or amino acid analysis is described. This desalting procedure recovers from 90%-95% of the sample and employs a low molecular weight cut-off membrane during sample centrifugation onto a polyvinylidene difluoride membrane. Subsequent washings are performed to efficiently remove salts, free amino acids and detergents that are known to interfere with sequence analysis. Sequence information such as initial recovery, repetitive yields and chromatogram comparisons are presented to demonstrate the utility of this procedure when used following isolation of sample with HPEC.     

A micromethod for estimation of adenosine deaminase and adenosine nucleosidase with modified cellulose nitrate membranes

Modified cellulose nitrate membrane strips were applied in a new chromatographic procedure for rapid and sensitive estimation of adenosine deaminase (EC 3.5.4.4) and adenosine nucleosidase (EC 3.2.2.7). In this method the enzymes serve each other as reagents. The products of their subsequent action are adenine and inosine, well separable on membrane strips, thanks to the different adsorptive affinities of these two compounds to the cellulose nitrate membranes. Employing adenine-labeled adenosine, microgram amounts of wet biological material may be used for estimation of the enzymes. The method has been applied to routine estimations of these two enzymes in various biological materials and examples are presented. A simple method is described for preparative purification and stabilization of adenosine nucleosidase of barley leaves used as reagent for adenosine deaminase assay.     

phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta

The combination of algorithms from the structure-modeling field with those of crystallographic structure determination can broaden the range of templates that are useful for structure determination by the method of molecular replacement. Automated tools in phenix.mr_rosetta simplify the application of these combined approaches by integrating Phenix crystallographic algorithms and Rosetta structure-modeling algorithms and by systematically generating and evaluating models with a combination of these methods. The phenix.mr_rosetta algorithms can be used to automatically determine challenging structures. The approaches used in phenix.mr_rosetta are described along with examples that show roles that structure-modeling can play in molecular replacement.     

THE DISTRIBUTION OF SELECTED DIAGNOSTIC CHARACTERS IN THE LECANORALES

Traditional classification concepts in lichenology are often, but not always, supported by molecular results. Molecular data should be compared and correlated with micro-morphological and ultrastructural information before systematic rearrangements are undertaken. Visualization of the distribution of morphological and other characters in specified groups is considered as a desirable resultper se, but it is also important to discover whether correlating characters are dependent on each other or not; and if not, whether their distribution in a group might support existing classification concepts. A data set for lecanoralean and other lichenized and lichenicolous genera, comprising 90—mostly multi-state—characters was used to store morphological, chemical and ecological data, and to test character correlations. Several examples of such analyses are presented. The following pairs of characters show some degree of dependence: ascospore septation and number per ascus, ascospore wall type and pigmentation, ascospore and epihymenium pigmentation. Several authors postulated that ascus types are good phylogenetic markers. Ascus types have been widely used for classification concepts of the Lecanorales. Two-dimensional correlation queries of ascus types with the following morphologcal characters were made: substratum preference, thallus growth form and ascospore septation. These correlations supply characteristic profiles for the various ascus types, which have to be compared with forthcoming phylogenetic hypotheses based on molecular data.     

Development of a technical arsenal of the method of fluorescent probes

A brief history of the development of the method of fluorescent probes and examples of its application are presented. Works done at the 2nd Moscow medical institute and institute of physical chemical medicine in collaboration with other institutes on: (1) detection of T- and B-lymphocytes in immune pathology; (2) investigation of the structure and clinical estimation of lipoproteins from blood plasma or serum in relation to the assessment of risk factors for the development of cardiovascular diseases; (3) detection of changes in album molecule in a series of pathological processes improving the prognosis of the development of such diseases as peritonitis, pancreatitis, poisoning with psychotropic preparations etc.; (4) intravital measurement of the potentials in electric fields in leukocytes and changes of these fields in the course of immunological diseases are described. With these approaches it is possible to study molecular events in the course of pathogenesis and also obtain diagnostically significant information on physical chemical aspect of these events. This information is not a conventional method used in the clinical laboratory.     

Method for electroporation for the early chick embryo

In vitro whole-embryo culture of chick embryos, originally invented by New, has been widely used for studies of early embryogenesis. Here, a method for electroporation using the New culture and its derivatives is described, to achieve misexpression of exogenous gene in a temporally and spatially controlled manner in gastrulating chick embryos. Detailed information for the devices and procedures, and some experimental examples are presented.     

Inferring biogeographic history from molecular phylogenies

The present study illustrates a method for analysing the biogeography of a group that is based on the group's phylogeny but does not invoke founder dispersal or centre of origin. The case studies presented include groups from many different parts of the world, but most are from the south‐west Pacific. The idea that basal groups are ancestral is not valid as a generalization. Neither the basal group, nor the oldest fossil represents the centre of origin, the time of origin or the ancestral ecology. Basal groups comprise less diverse sister groups and their distributions occur around centres of differentiation in already widespread ancestors, and not centres of origin for the whole group. Thus, the sequence of nodes in a phylogeny may indicate the spatial sequence of differentiation in a widespread ancestor rather than a series of founder dispersal events. Allocation of clades to a priori geographic areas, such as the continents, in the initial stages of biogeographic analysis has often involved incorrect assumptions of sympatry. This has led to the idea that the ‘areas of sympatry’ were centres of origin. Areas other than those defined by the taxa themselves need not be used in analysis. The fossil‐calibrated molecular clock, with dates transmogrified from minimum to maximum dates, has been used to test for vicariance. Recent work in population genetics, however, indicates that allopatry is caused by vicariance rather than founder dispersal, and so vicariance can instead be used to test the clock. Deriving evolutionary chronology by calibrating spatial vicariance in molecular clades with associated tectonic events is more reasonable than relying on the fossil record to give maximum (absolute) dates. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 757–774.     

Direct molecular haplotyping by melting curve analysis of hybridization probes: beta 2-adrenergic receptor haplotypes as an example

Direct determination of the association of multiple genetic polymorphisms, or haplotyping, in individual samples is challenging because of chromosome diploidy. Here, we describe the ability of hybridization probes, commonly used as genotyping tools, to establish single nucleotide polymorphism (SNP) haplotypes in a single step. Three haplotypes found in the beta 2-adrenergic receptor (β2AR) gene and characterized by three different SNPs combinations are presented as examples. Each combination of SNPs has a unique stability, recorded by its melting temperature, even when intervening sequences from the template must loop out during probe hybridization. In the course of this study, two haplotypes in β2AR not described previously were discovered. This approach provides a tool for molecular haplotyping that should prove useful in clinical molecular genetics diagnostics and pharmacogenetic research where methods for direct haplotyping are needed.     

Enzyme-assisted semisynthesis of polypeptide active esters and their use.

A method is described for the preparation of polypeptides activated uniquely at the C-terminus. The polypeptide is incubated in a concentrated solution of an amino acid active ester, the latter having its amino group free but adequately protected by protonation. The amino acid ester is coupled via its amino group to the C-terminus of the polypeptide by enzymic catalysis (reverse proteolysis). The resulting polypeptide C-terminal active ester is then isolated and coupled to a suitable amino component (generally a polypeptide) in a subsequent chemical coupling. The method appears to be generally applicable; fragments of horse heart cytochrome c, and porcine insulin, are used as examples. Two new analogues of cytochrome c have been prepared by using this method, with yields of up to 60% in the final coupling. Scope and limitations of the method are discussed.     

Mechanography: a non-invasive technique for the evaluation of cardiac function in children

Experience in the pediatric age group with mechanography, an indirect method of cardiovascular investigation, is described with emphasis on the recording technique and on the analysis of the tracings. A few examples are presented with comments on the morphological aspects and the time characteristics of the pulse curves, showing how much information about cardiac disease and especially myocardial function in children may be obtained.     

Bayes's Theorem and the Analysis of Binomial Random Variables

A very practical application of Bayes's theorem, for the analysis of binomial random variables, is presented. Previous papers (Walters, 1985; Walters, 1986a) have already demonstrated the reliability of the technique for one, or two random variables, and the extension of the approach to several random variables is described. Two biometrical examples are used to illustrate the method.     

Identification and characterization of proteins binding A + U-rich elements

A + U-Rich elements (AREs) have been extensively investigated as cis-acting determinants of rapid mRNA turnover. Recently, a number of RNA-binding proteins interacting with AREs have been described. This article presents strategies and techniques used by our laboratory to identify and characterize a family of ARE-binding proteins collectively termed AUF1. However, these techniques may be applied to the study of any protein displaying sequence-specific RNA binding activity. The techniques described here include the purification of native AUF1 from cultured cells as well as the preparation of recombinant AUF1 proteins using a bacterial expression system. Analyses of RNA-protein interactions are also described, including the use of gel mobility shift assays with synthetic RNA probes to monitor specific RNA binding activity in cell extracts or with recombinant proteins. Variations of this technique are also described to evaluate the RNA binding affinity of recombinant proteins and the use of specific RNA competitors to assess RNA determinants of protein binding specificity. Other techniques presented include the identification of specific proteins in RNA:protein complexes using antibody supershifts and the estimation of molecular weights of RNA-binding proteins by UV crosslinking. Results of individual experiments are presented as examples of some techniques. Throughout the article, suggestions are included to avoid commonly encountered problems and to assist in the optimization of these techniques for the study of other RNA-binding proteins.