Optical trap as a tool for studying motor proteins

The review briefs the optical trap method, a modern experimental tool based on the recently discovered ability of light to trap and hold micron and submicron particles in a focused beam. The physical principle underlying the optical trap and the opportunities that it provides for studying the molecular nature of biological motility are considered. Several studies into the physical characteristics and functions of single motor protein molecules performed using the optical trap and recording nanometer displacements and piconewton forces are analyzed.     

The Y chromosome as a tool for studying human evolution.

The use of the Y chromosome in human evolutionary research has only recently begun to gain momentum, partly because of a paucity of polymorphism. Differences in male/female behaviour patterns and the unique mode of inheritance of the Y chromosome also complicate interpretation of the data on Y chromosome variation.     

Tetanus toxin as a tool for studying epilepsy

The use of tetanus toxin, injected into the hippocampus of the rat, to produce an "animal model" of chronic limbic epilepsy is described. This model has yielded information complementary to that derived from other animal models and has several important advantages: while it involves spontaneous seizures, it occurs without gross damage to the brain ; it is eventually reversible in terms of fits and the overall reappearance of the EEG. It can therefore be used to look both at the effects of ongoing epilepsy and also at the long-term changes in brain function induced by previous epilepsy. Evidence is presented that the toxin probably remains localised at the site of injection. The information which has so far been obtained with this model on the relation between epilepsy and abnormal behaviour is summarised. In particular, it appears that the epilepsy produces long-term deficits in the animals' ability to learn and remember of a sort which suggest that an enduring malfunction has been induced in the hippocampus. The significance of the findings for clinical research and for future investigation of the nature of epilepsy are described. It is emphasised that the neurotoxins may be usefully exploited not only for investigating the molecular basis of neuronal mechanisms but also for inducing long-lasting plastic changes in integrated brain function.     

Non-electrolyte permeability as a tool for studying membrane fluidity

1. The reflection coefficient for the permeation of thiourea through bilayers of phosphatidylcholine is a function of the fatty-acid composition of the lipid molecules. By means of these reflection coefficients an index for membrane fluidity has been given to each of those lipids, relative to that of egg phosphatidylcholine. 2. The maximum number of water molecules that can copermeate with each molecule of solute by means of solute-solvent interaction is a function of the packing of the lipid molecules in the bilayer. This parameter has been used in this paper for characterizing the fluidity of cholesterol-containing membranes and for membranes with their lipids in the gel state.     

Analytical ultracentrifugation as a tool for studying protein interactions

The last two decades have led to significant progress in the field of analytical ultracentrifugation driven by instrumental, theoretical, and computational methods. This review will highlight key developments in sedimentation equilibrium (SE) and sedimentation velocity (SV) analysis. For SE, this includes the analysis of tracer sedimentation equilibrium at high concentrations with strong thermodynamic non-ideality, and for ideally interacting systems, the development of strategies for the analysis of heterogeneous interactions towards global multi-signal and multi-speed SE analysis with implicit mass conservation. For SV, this includes the development and applications of numerical solutions of the Lamm equation, noise decomposition techniques enabling direct boundary fitting, diffusion deconvoluted sedimentation coefficient distributions, and multi-signal sedimentation coefficient distributions. Recently, effective particle theory has uncovered simple physical rules for the co-migration of rapidly exchanging systems of interacting components in SV. This has opened new possibilities for the robust interpretation of the boundary patterns of heterogeneous interacting systems. Together, these SE and SV techniques have led to new approaches to study macromolecular interactions across the entire spectrum of affinities, including both attractive and repulsive interactions, in both dilute and highly concentrated solutions, which can be applied to single-component solutions of self-associating proteins as well as the study of multi-protein complex formation in multi-component solutions.     

Comparative sequence analysis as a tool for studying the secondary structure of mRNAs.

Analysis of phylogenetically conserved secondary structure has been important in the development of models for the secondary structure of structural RNAs. In this paper, we apply this type of analysis to several families of informational RNAs to evaluate its usefulness in developing secondary structure models for mRNAs and mRNA precursors. We observed many conserved helices in all mRNA groups analyzed. Three criteria were used to identify potential helices which were not conserved solely because of coding sequence constraints, and may therefore be important for the structure and function of the RNA. These results suggest that this approach will be useful in deriving secondary structure models for informational RNAs when used in conjunction with other complementary techniques, and in designing experiments to determine the functional significance of conserved base pairing interactions.     

Protein microarrays as a discovery tool for studying protein-protein interactions

Exploring the function of the genome and the encoded proteins has emerged as a new and exciting challenge in the postgenomic era. Novel technologies come into view that promise to be valuable for the investigation not only of single proteins, but of entire protein networks. Protein microarrays are the innovative assay platform for highly parallel in vitro studies of protein-protein interactions. Due to their flexibility and multiplexing capacity, protein microarrays benefit basic research, diagnosis and biomedicine. This review provides an overview on the basic principles of protein microarrays and their potential to multiplex protein-protein interaction studies.     

Peroxidase activity as a tool for studying the folding of c-type cytochromes

The peroxidase activity of c-type cytochromes increases substantially by unfolding. This phenomenon was used to study the equilibrium unfolding of ferricytochrome c. The peroxidase activity is already enhanced at low denaturant concentrations. The lowest free energy folding intermediate is easily detected by this method, while it is invisible using fluorescence or optical spectroscopy. The free energy difference between this folding intermediate and the native state depends on the strength of the sixth ligand of the heme-iron and the increase in peroxidase activity upon unfolding is shown to be a sensitive indicator of the strength of this ligand. Under fully denaturing conditions, the peroxidase activity is inhibited by protein-based ligands. It is shown that at least three different ligand groups can be responsible for this inhibition, and that at neutral or alkaline pH, the predominant ligand is not histidine. The use of peroxidase activity assays as a method to study the unfolding of cytochrome c is evaluated.     

Cluster conservation as a novel tool for studying protein-protein interactions evolution

Protein-protein interactions networks has come to be a buzzword associated with nets containing edges that represent a pair of interacting proteins (e.g. hormone-receptor, enzyme-inhibitor, antigen-antibody, and a subset of multichain biological machines). Yet, each such interaction composes its own unique network, in which vertices represent amino acid residues, and edges represent atomic contacts. Recent studies have shown that analyses of the data encapsulated in these detailed networks may impact predictions of structure-function correlation. Here, we study homologous families of protein-protein interfaces, which share the same fold but vary in sequence. In this context, we address what properties of the network are shared among relatives with different sequences (and hence different atomic interactions) and which are not. Herein, we develop the general mathematical framework needed to compare the modularity of homologous networks. We then apply this analysis to the structural data of a few interface families, including hemoglobin alpha-beta, growth hormone-receptor, and Serine protease-inhibitor. Our results suggest that interface modularity is an evolutionarily conserved property. Hence, protein-protein interfaces can be clustered down to a few modules, with the boundaries being evolutionarily conserved along homologous complexes. This suggests that protein engineering of protein-protein binding sites may be simplified by varying each module, but retaining the overall modularity of the interface.     

Hydrogel microchip as a tool for studying exosomes in human serum

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.     

Metaproteomics as a tool for studying the protein landscape of human-gut bacterial species

Host-microbiome interactions and the microbial community have broad impact in human health and diseases. Most microbiome based studies are performed at the genome level based on next-generation sequencing techniques, but metaproteomics is emerging as a powerful technique to study microbiome functional activity by characterizing the complex and dynamic composition of microbial proteins. We conducted a large-scale survey of human gut microbiome metaproteomic data to identify generalist species that are ubiquitously expressed across all samples and specialists that are highly expressed in a small subset of samples associated with a certain phenotype. We were able to utilize the metaproteomic mass spectrometry data to reveal the protein landscapes of these species, which enables the characterization of the expression levels of proteins of different functions and underlying regulatory mechanisms, such as operons. Finally, we were able to recover a large number of open reading frames (ORFs) with spectral support, which were missed by de novo protein-coding gene predictors. We showed that a majority of the rescued ORFs overlapped with de novo predicted protein-coding genes, but on opposite strands or in different frames. Together, these demonstrate applications of metaproteomics for the characterization of important gut bacterial species.     

Molecular modelling as a tool for studying the disassembly of potentially leishmanicide-targeted dendrimer

Molecular modelling studies were carried out to analyse which carbonyl group is the most vulnerable to the disassembly of potentially leishmanicide-targeted dendrimer. The dendrimers were designed using myo-inositol (core and directing group), d-mannose (directing group), l-malic acid (spacer and dendron) and hydroxymethylnitrofurazone (NFOH) as a bioactive agent. The molecular models were built containing one, two and three branches. For this preliminary analysis, physicochemical properties, such as electronic density distribution and steric hindrance regarding the carbonyl groups were evaluated, and the carbon atoms in the following carbonyl groups were considered: near the core (C₁), close to the directing group (C₂), in l-malic acid (C₃) and near the bioactive agent (C₄). The most probable targeted dendrimers showed the carbonyl close to the core as the most susceptible to a nucleophilic attack, except those molecular systems containing two branches with d-mannose as the directing group, which displayed the carbonyl group near NFOH as the most likely ester breaking point.     

PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp.

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp. Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L. sativa and 13 accessions of the wild species L. serriola, L. saligna, L. virosa, L. perennis, and L. indica. The same genotypes were analyzed as in a previous study that had utilized RFLP markers. The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data. The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis. There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data. AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

A bioinspired autonomous swimming robot as a tool for studying goal-directed locomotion

The bioinspired approach has been key in combining the disciplines of robotics with neuroscience in an effective and promising fashion. Indeed, certain aspects in the field of neuroscience, such as goal-directed locomotion and behaviour selection, can be validated through robotic artefacts. In particular, swimming is a functionally important behaviour where neuromuscular structures, neural control architecture and operation can be replicated artificially following models from biology and neuroscience. In this article, we present a biomimetic system inspired by the lamprey, an early vertebrate that locomotes using anguilliform swimming. The artefact possesses extra- and proprioceptive sensory receptors, muscle-like actuation, distributed embedded control and a vision system. Experiments on optimised swimming and on goal-directed locomotion are reported, as well as the assessment of the performance of the system, which shows high energy efficiency and adaptive behaviour. While the focus is on providing a robotic platform for testing biological models, the reported system can also be of major relevance for the development of engineering system applications.     

Somatic mutations as a useful tool for studying clonal dynamics in trees

The seemingly eternal cycles of clonal growth in many tree species, with members of Populus (aspen, poplars, cottonwoods and the like) featuring most prominently, provoke a number of questions on the interface between ecology, genetics and forestry. In this issue, two groups present their approaches to clonal dynamics ( Ally et al. 2008 and Mock et al. 2008 ), using microsatellite (or simple sequence repeat, SSR) variation in P. tremuloides. Ally et al. developed and applied a model for using microsatellites to estimate clone age and infer other community characteristics. Mock et al. used fewer microsatellites but in more individuals, to examine clone size and distribution across the landscape.     

Receptor-Galpha fusion proteins as a tool for ligand screening

We have prepared fusion proteins of muscarinic M1-M5 receptors with alpha subunits of G proteins Gi1, Gi2, Gs, G11, G16 and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M2-Gi1alpha and M4-Gi1alpha, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [35S]GTPgammaS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of Gq/G11-coupled receptors such as M1-G11alpha, M3-G11alpha, and M5-G11alpha. The effect of agonists could be observed for fusion proteins with G16alpha of muscarinic M1, M2 and adrenergic beta2 receptors, but the extent of the effect was much less than that for fusion proteins with Gi1alpha of Gi/Go-coupled receptors. Fusion proteins of M1 receptors with Gi1alpha or chimera of G16alpha and Gi2alpha were also not effective in detecting the action of agonists.