Characterization of the sequential non-covalent and covalent interactions of the antitumour antibiotic hedamycin with double stranded DNA by NMR spectroscopy

Hedamycin, a member of the pluramycin class of antitumour antibiotics, consists of a planar anthrapyrantrione chromophore to which is attached two aminosugar rings at one end and a bisepoxide-containing sidechain at the other end, Binding to double-stranded DNA is known to involve both reversible and non-reversible modes of interaction. As a part of studies directed towards elucidating the structural basis for the observed 5'-pyGT-3' sequence selectivity of hedamycin, we conducted one-dimensional NMR titration experiments at low temperature using the hexadeoxyribonucleotide duplexes d(CACGTG)(2) and d(CGTACG)(2). Spectral changes which occurred during these titrations are consistent with hedamycin initially forming a reversible complex in slow exchange on the NMR timescale and binding through intercalation of the chromophore. Monitoring of this reversible complex over a period of hours revealed a second type of spectral change which corresponds with formation of a non-reversible complex. Copyright (C) 1999 John Wiley & Sons, Ltd.

Biophysical characterization of interactions involving importin-alpha during nuclear import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer. Importin-alpha contains the NLS binding site, whereas importin-beta mediates the translocation through the nuclear pore. We characterized the interactions involving importin-alpha during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-beta (stoichiometry, 1:1; K-D = 1.1 x 10(-8) m) increases the affinity for NLSs; the importin-alpha/beta complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K-D = 3.5 x 10(-8) m and 4.8 x 10(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K-D = 1.7 x 10(-8) m; nucleoplasmin NLS, K-D = 1.4 x 10(-8) m). The autoinhibitory domain (as a separate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-alpha and provide a quantitative description of the binding and regulatory steps during nuclear import.

Studies of solute self-association by sedimentation equilibrium: allowance for effects of thermodynamic non-ideality beyond the consequences of nearest-neighbor interactions

A sedimentation equilibrium study of a-chymotrypsin self-association in acetate-chloride buffer, pH 4.1 I 0.05, has been used to illustrate determination of a dimerization constant under conditions where thermodynamic non-ideality is manifested beyond the consequences of nearest-neighbor interactions. Because the expressions for the experimentally determinable interaction parameters comprise a mixture of equilibrium constant and excluded volume terms, the assignment of reasonable magnitudes to the relevant virial coefficients describing non-associative cluster formation is essential for the evaluation of a reliable estimate of the dimerization constant. Determination of these excluded volume parameters by numerical integration over the potential-of-mean-force is shown to be preferable to their calculation by approximate analytical solutions of the integral for this relatively small enzyme monomer with high net charge (+ 10) under conditions of low ionic strength (0.05 M). (C) 2001 Elsevier Science B.V. All rights reserved.

Regulatory interactions in Arabidopsis thaliana acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) contains catalytic and regulatory subunits, the latter being required for sensitivity to feedback regulation by leucine, valine and isoleucine. The regulatory subunit of Arabidopsis thaliana AHAS possesses a sequence repeat and we have suggested preciously that one repeat binds leucine while the second binds valine or isoleucine, with synergy between the two sites. We have mutated four residues in each repeat, based on a model of the regulatory subunit. The data confirm that there are separate leucine and valine/isoleucine sites, and suggest a complex pathway for regulatory signal transmission to the catalytic subunit. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Behaviour of the black flying fox Pteropus alecto: 1. An ethogram of behaviour, and preliminary characterisation of mother-infant interactions

The black flying fox Pteropus alecto is one of four species of flying fox found on the Australian mainland. Little information exists about the specific behaviour of this species, and no framework for the study of its behaviour has yet been constructed. In the study reported here, two P alecto colonies were observed at two day roosts in South East Queensland, Australia, between 1998-2000. Observations focused on solitary and social actions in general and on mother-infant interactions in some detail and led to the construction of an ethogram that defines each action structurally and functionally, describing accompanying vocalisations where appropriate. Diurnal activity patterns of P. alecto throughout the year consisted predominantly of roosting, grooming and sleeping, and involved little social activity. Social interactions were largely restricted to the seasonal contexts of the birthing/rearing period of October to March and the subsequent courtship/mating season of February to April. In all, 74 behavioural units were defined with the aim of facilitating further research and the implementation of effective conservation strategies for the species.

Interactions among endocrinology, seasonal reproductive cycles and the nesting biology of the female green sea turtle

We collected data on plasma levels of testosterone+5a-dihydrotestosterone (T+DHT) and corticosterone (CORT) from adult female green sea turtles (Chelonia mydas) from southern Queensland during distinct stages of their reproductive cycle. Those females capable of breeding in a given year had elevated plasma steroid levels (T+DHT 0.91 +/- 0.08; CORT 1.05 +/- 0.29 ng/ml), associated with follicular development, until courtship began in October. At the beginning of the nesting season in November plasma levels of 2 CORT were related to when the female first nested (r(2) = 0.06; F = 10.45; P = 0.01). However, they were not correlated with the number of clutches a female laid in that season (F = 3.65; P = 0.08). We repeatedly sampled 23 turtles over the nesting season and profiled changes in steroids immediately following oviposition of each clutch. Levels of T+DHT (range 0.41-0.58 ng/ml) and CORT (range 2.13-2.81 ng/ml) were similar through the early stages of the nesting season and inter-nesting period, and declined to near basal levels (T+DHT 0.37 +/- 0.03 and CORT 1.85 +/- ng/ml) following the last clutch for the season. Steroid hormone levels were also low (T+DHT 0.38 +/- 0.16; CORT 0.46 +/- 0.21 ng/ml) in four independent post-breeding (atretic) females; samples for these females were taken at a time when body condition was presumably at the lowest for the season. Subtle changes in the nesting environment, such as variation in nesting habitat or the time of night that nesting occurred, were associated with a small and slow CORT increase. We suggest CORT is increased in nesting females to assist in lipid transfer to prepare the ovarian follicles and/or the reproductive organs for ovulation.

The E(NK) model: extending the NK model to incorporate gene-by-environment interactions and epistasis for diploid genomes

The haploid NK model developed by Kauffman can be extended to diploid genomes and to incorporate gene-by-environment interaction effects in combination with epistasis. To provide the flexibility to include a wide range of forms of gene-by-environment interactions, a target population of environment types (TPE) is defined. The TPE consists of a set of E different environment types, each with their own frequency of occurrence. Each environment type conditions a different NK gene network structure or series of gene effects for a given network structure, providing the framework for defining gene-by-environment interactions. Thus, different NK models can be partially or completely nested within the E environment types of a TPE, giving rise to the E(NK) model for a biological system. With this model it is possible to examine how populations of genotypes evolve in context with properties of the environment that influence the contributions of genes to the fitness values of genotypes. We are using the E(NK) model to investigate how both epistasis and gene-by-environment interactions influence the genetic improvement of quantitative traits by plant breeding strategies applied to agricultural systems. © 2002 Wiley Periodicals, Inc.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Biological determinants of extinction risk: why are smaller species less vulnerable?

It is becoming increasingly clear that species of smaller body size tend to be less vulnerable to contemporary extinction threats than larger species, but few studies have examined the mechanisms underlying this pattern. In this paper, data for the Australian terrestrial mammal fauna are used to ask whether higher reproductive output or smaller home ranges can explain the reduced extinction risk of smaller species. Extinct and endangered species do indeed have smaller litters and larger home ranges for their body size than expected under a null model. In multiple regressions, however, only litter size is a significant predictor of extinction risk once body size and phylogeny are controlled for. Larger litters contribute to fast population growth, and are probably part of the reason that smaller species are less extinction-prone. The effect of litter size varies between the mesic coastal regions and the and interior of Australia, indicating that the environment a species inhabits mediates the effect of biology on extinction risk. These results suggest that predicting extinction risk from biological traits is likely to be a complex task which must consider explicitly interactions between biology and environment.

The development of chromatography for the characterization of protein interactions: a personal perspective

This article reviews the progress of a personal endeavour to develop chromatography as a quantitative procedure for the determination of reaction stoichiometries and equilibrium constants governing protein interactions. As well as affording insight into an aspect of chromatography with which many protein chemists are unfamiliar, it shows the way in which minor adaptations of conventional chromatographic practices have rendered the technique one of the most powerful methods available for the characterization of interactions. That pathway towards quantification is followed from the introduction of frontal gel filtration for the study of protein self-association to the characterization of ligand binding by the biosensor variant of quantitative affinity chromatography.

Analysis of RNA-protein interactions by flow cytometry

Flow cytometry, in combination with advances in bead coding technologies, is maturing as a powerful high-throughput approach for analyzing molecular interactions. Applications of this technology include antibody assays and single nucleotide polymorphism mapping. This review describes the recent development of a microbead flow cytometric approach to analyze RNA-protein interactions and discusses emerging bead coding strategies that together will allow genome-wide identification of RNA-protein complexes. The microbead flow cytometric approach is flexible and provides new opportunities for functional genomic studies and small-molecule screening.