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.     

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.     

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.     

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.     

A laboratory method for studying zooplankton swimming behaviors

A laboratory method is presented for studying zooplankton swimming behaviors such as phototaxis and photokinesis. The method attempts to standardize laboratory conditions and to minimize the effects of several phenomena which modify zooplankton behavior. The role of angular light distribution in zooplankton behavior is discussed, and an apparatus which simulates a natural underwater light environment is described. The procedure minimizes the fluctuations in zooplankton swimming speed and vertical distribution that are caused by large light stimuli, noise, food deprivation, endogenous rhythms, and other factors. The experimental animals were viewed remotely with the aid of a light amplifier and video camera. A mathematical equation and computer program for calculating three-dimensional swimming speeds of zooplankton from video recordings are described in detail.     

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.     

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.     

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.     

Lipid-impregnated filters as a tool for studying the electric current-generating proteins.

Porous filters and collodion film impregnated with decane solution of phospholipids, were used for measurements of electric potential differences generated by bacteriorhodopsin, chromatophore redox chain, H⁺-ATPase, pyrophosphatase, and mitochondrial respiratory chain. It was shown that reconstituted proteoliposomes, containing e.g., bacteriorhodopsin or natural coupling membrane vesicles, such as Rhodospirillum rubrum chromatophores, can be attached to a filter surface by means of Ca²⁺ or Mg²⁺ ions. Addition of the respective energy source was found to result in electric potential difference being generated across the filter. This effect was measured directly by $\text{Ag}\text{AgCl}$ electrodes immersed into electrolyte solutions on both sides of the filter. Using a phospholipid-impregnated collodion film one can measure electric responses as fast as 300 nsec. The phospholipid-impregnated filters turned out to be sensitive and reliable electrodes for measuring the concentration of synthetic penetrating ions, such as phenyldicarbaundecaborane, tetraphenylborate, tetrapentylammonium, and tetraphenylphosphonium. By measuring changes in the concentration of these ions in the suspension of proteoliposomes, chromatophores, mitochondria, or bacterial cells, one can follow the formation and dissipation of transmembrane potential differences in these systems. It is shown that the phospholipid-impregnated filters are much more reliable and handy than planar phospholipid membranes previously used for studying electrogenic activity of electric current-producing membrane proteins.     

A new paramagnetic analogue of cholesterol as a tool for studying molecular interactions of genuine cholesterol

The synthesis of a new paramagnetic (nitroxide) analogue of cholesterol is described. This compound (called CNO) contains a doxyl group in the lateral chain at position 25. Our results show that CNO retains three molecular interactions which characterize authentic cholesterol: It assumes an orientation perpendicular to the phospholipid bilayer with the doxyl group buried in the membrane core, as seen by ESR spectroscopy. It widens the transition temperature of dimyristoylphosphatidylcholine, to the same extent as cholesterol, as measured by Raman and ESR spectroscopies. It interacts with polyene antibiotics, such as amphotericin B and filipin, in the same manner as its model. This was proved on the one hand by the change in fluorescence of self quenched vesicle-entrapped calcein, after dilution in the external medium, provoked by filipin, and on the other hand by fluorescence quenching provoked by cobalt ions entering the vesicles under the influence of amphotericin B. We concluded that CNO, although it has a side chain different from genuine cholesterol, can help to solve many physiologically meaningful questions related to the distribution and movement rate of cholesterol itself.     

Dynamic fluorescence of extrinsic fluorophores as a tool for studying protein conformational substates

Summary The fluorescence lifetime distribution of 2-p-toluidinyl-6-naphthalene sulfonic acids (TNS) bound to the heme site of apomyoglobin has been examined. The results were compared to those observed for the free fluorophore in isotropic nonviscous solvent. Two different excitation wavelengths were used, i.e. 290 and 350 nm. The results showed that the distribution of TNS bound to apomyoglobin is wider than that of the free fluorophore, thus indicating the existence of a large number of conformational substates originating from the interaction between TNS and the protein matrix. The comparison of the distribution obtained at two different excitation wavelengths allowed the emission arising from conformational substates, in which the excited state of fluorophore moiety has a higher probability to be populated by Forster energy transfer mechanism, to be distinguished.     

Evolving locomotion for a 12-DOF quadruped robot in simulated environments

We demonstrate the power of evolutionary robotics (ER) by comparing to a more traditional approach its performance and cost on the task of simulated robot locomotion. A novel quadruped robot is introduced, the legs of which – each having three non-coplanar degrees of freedom – are very maneuverable. Using a simplistic control architecture and a physics simulation of the robot, gaits are designed both by hand and using a highly parallel evolutionary algorithm (EA). It is found that the EA produces, in a small fraction of the time that takes to design by hand, gaits that travel at two to four times the speed of the hand-designed one. The flexibility of this approach is demonstrated by applying it across a range of differently configured simulators.     

A reevaluation of veratridine as a tool for studying the depolarization-induced release of neurotransmitters from nerve endings

The effect of veratridine on neurotransmitter release was studied using rat brain synaptosomes superfused at 37°C. Veratridine (5–75 M) caused a concentration-dependent release of [³H]GABA from prelabeled synaptosomes in the presence of 2.7 mM Ca²⁺. In the whole range of veratridine concentrations, the release of [³H]GABA elicited by the drug was substantially increased rather than decreased in the absence of Ca²⁺ or with Ca²⁺ concentrations of 0.45 and 0.9 mM. The release of the amino acid was inhibited more by 5.4 mM than by 2.7 mM Ca²⁺. The effect on endogenous (chemically measured) GABA was similar to that on [³H]GABA. The inhibitory effect of Ca²⁺ on the veratridine-induced release of [³H]GABA was consistently seen in a variety of experimental conditions except one, namely when the experiment was run at room temperature (22–23°C) rather than at physiological temperature (37°C). In fact, at 22–23°C the release of GABA evoked by the alkaloid was somewhat potentiated by Ca²⁺. At 37°C, glutamate appeared to behave similarly to GABA, whereas the veratridine-induced release of [³H]noradrenaline and [³H]dopamaine was largely Ca²⁺-dependent. The mechanism of the release of transmitters elicited by veratridine is discussed. It is concluded that the evoked release of GABA and glutamate is due more to the veratridine-induced depolarization (Na⁺ influx) than to the accompanying influx of Ca²⁺, and it is suggested that the inhibitory effect of Ca²⁺ on the overall release of amino acids is due to the antagonism exerted by the divalent cation on the veratridine action at the Na⁺ channel. In contrast, in the case of catecholamines, the influx of Ca²⁺ would have a prominent role in triggering exocytotic release, whereas the depolarization itself would have slight or no importance.     

Isolated neurosecretory nerve endings as a tool for studying the mechanism of stimulus-secretion coupling

In the present paper we discuss the properties of a recently developed preparation of isolated neurosecretory nerve endings obtained from the rate neurohypophysis. These nerve terminals release two neurohormones, oxytocin and vasopressin, which are easily assayed by radioimmunoassay. Depolarization-induced secretion is dependent on the same parameters as those regulating release from the whole neural lobe. The isolated nerve endings can be permeabilized by means of digitonin; a treatment which gives direct access to the cytoplasm allowing the study of the minimal requirements for inducing neuropeptide release. Furthermore, some nerve endings are large enough to allow the use of the patch-clamp technique. In the present paper we present evidences which show that the isolated neurohypophysial nerve terminals represent a protent tool for studying the mechanism of stimulus-secretion.     

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.