NAD-dependent Recovery of Cell Division in UV-irradiated Escherichia coli B and Lon- Mutant of Escherichia coli K-12

We found that both benzyl isothiocyanate (ITC) and phenyl ITC inhibited respiration in the mitochondria in an electrophilic reaction-dependent manner. ITC-induced mitochondrial swelling and cytochrome c release were prevented by cyclosporin A, indicating that they are mediated through the ITC moiety-dependent reaction to critical thiol groups for the opening of membrane permeability transition-dependent pores.     

Reactive oxygen species modulate itraconazole-induced apoptosis via mitochondrial disruption in Candida albicans

Itraconazole (ITC), a well-known fungistatic agent, has potent fungicidal activity against Candida albicans. However, its mechanism of fungicidal activity has not been elucidated yet, and we aimed to identify the mechanism of ITC against C. albicans. ITC caused cell shrinkage via potassium leakage through the ion channel. Since shrunken cells could indicate apoptosis, we investigated apoptotic features. Annexin V-FITC and TUNEL assays indicated that fungicidal activity of ITC was involved in apoptosis. Subsequently, we confirmed an intracellular factor that could cause apoptosis. ITC treatment caused reactive oxygen species (ROS) accumulation. To confirm whether ROS is related with ITC-triggered cell death, cell viability was examined using the ROS scavenger N-acetylcysteine (NAC). NAC pretreatment recovered ITC-induced cell death, indicating that antifungal activity of ITC is associated with ROS, which is also confirmed by impaired glutathione-related antioxidant system and oxidized intracellular lipids. Moreover, ITC-induced mitochondrial dysfunction, in turn, triggered cytochrome c release and metacaspase activation, leading to apoptosis. Unlike the only ITC-treatment group, cells with NAC pretreatment did not show significant damage to mitochondria, and attenuated apoptotic features. Therefore, our results suggest that ITC induces apoptosis as fungicidal mechanism, and intracellular ROS is major factor to trigger the apoptosis by ITC in C. albicans.     

Development and utility of an internal threshold control (ITC) real-time PCR assay for exogenous DNA detection

Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct) for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct) or negative (i.e. Ct greater than the ITC Ct). The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.     

Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies

Isothermal titration calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equilibrium constants and thermodynamics of molecular binding events. Thus, important information about the interaction of metal ions with biological macromolecules can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodynamics of metal-protein interactions.     

Does molecular docking reveal alternative chemopreventive mechanism of activation of oxidoreductase by sulforaphane isothiocyanates?

Isothiocyanates (ITC) are well-known chemopreventive agents extracted from vegetables. This activity results from the activation of human oxidoreductase. In this letter, the uncompetitive activatory mechanism of ITC was investigated using docking and molecular dynamics simulations. This indicates that NAD(P)H:quinone oxidoreductase can efficiently improve enzyme-substrate recognition within the catalytic site if the ITC activator supports the interaction in the uncompetitive binding site.     

Applications of isothermal titration calorimetry in protein science

During the past decade, isothermal titration calorimetry (ITC) has developed from a specialist method for understanding molecular interactions and other biological processes within cells to a more robust, widely used method. Nowadays, ITC is used to investigate all types of protein interactions, including protein-protein interactions, protein-DNA/RNA interactions, protein-small molecule interactions and enzyme kinetics; it provides a direct route to the complete thermodynamic characterization of protein interactions. This review concentrates on the new applications of ITC in protein folding and misfolding, its traditional application in protein interactions, and an overview of what can be achieved in the field of protein science using this method and what developments are likely to occur in the near future. Also, this review discusses some new developments of ITC method in protein science, such as the reverse titration of ITC and the displacement method of ITC.     

Isothermal titration calorimetry for chiral chemistry

One of the most powerful techniques that are currently available to measure thermodynamic parameters such as enthalpy (ΔH), Gibbs free energy (ΔG), entropy changes (ΔS), and binding affinity in chemical reactions is isothermal titration calorimetry (ITC). Recent advances in instrumentation have facilitated the development of ITC as a very essential analytical tool in biology and chemistry. In this article, we will focus on a review of the literature on the application of ITC for the study of chiral systems and chiral interactions. We present studies in which the ITC technique is used to study chiral interactions, for instance in chiral solutions, chiral organometallic complexes, guest‐host chiral binding interactions, and biological macromolecules. Finally, we put strong emphasis on the most recent application of ITC for the study of chirality in nanosystems and at the nanoscale.     

Biomolecule–nanoparticle interactions: Elucidation of the thermodynamics by isothermal titration calorimetry

BackgroundNanomaterials (NMs) are often exposed to a broad range of biomolecules of different abundances. Biomolecule sorption driven by various interfacial forces determines the surface structure and composition of NMs, subsequently governs their functionality and the reactivity of the adsorbed biomolecules. Isothermal titration calorimetry (ITC) is a nondestructive technique that quantifies thermodynamic parameters through in-situ measurement of the heat absorption or release associated with an interaction.Scope of reviewThis review highlights the recent applications of ITC in understanding the thermodynamics of interactions between various nanoparticles (NPs) and biomolecules. Different aspects of a typical ITC experiment that are crucial for obtaining accurate and meaningful data, as well as the strengths, weaknesses, and challenges of ITC applications to NP research were discussed.Major conclusionsITC reveals the driving forces behind biomolecule–NP interactions and the effects of the physicochemical properties of both NPs and biomolecules by quantifying the crucial thermodynamics parameters (e.g., binding stoichiometry, ΔH, ΔS, and ΔG). Complimentary techniques would strengthen the interpretation of ITC results for a more holistic understanding of biomolecule–NP interactions.General significanceThe thermodynamic information revealed by ITC and its complimentary characterizations is important for understanding biomolecule–NP interactions that are fundamental to the biomedical and environmental applications of NMs and their toxicological effects. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Administration of isothiocyanates enhances heat tolerance in Arabidopsis thaliana

Although it has been documented that plants generate isothiocyanates (ITCs) through the glucosinolate-myrosinase system to defend against biotic stresses, the roles of ITCs in defending against abiotic stresses have scarcely been studied. Here, we report that exogenously applied ITCs enhance the heat tolerance of Arabidopsis thaliana. Pre-administration of phenethyl ITC to Arabidopsis plants mitigated growth inhibition after heat stress at 55?°C for 1?h. Although methyl ITC and allyl ITC also tended to reduce the growth inhibition that the same heat treatment caused, the reduction effects were weaker. The expression levels of heat shock protein 70 genes in Arabidopsis were elevated after phenethyl ITC treatment. These results suggest that ITCs may act as heat-tolerance enhancers in plants.     

Applications of isothermal titration calorimetry in pure and applied research--survey of the literature from 2010

Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for macromolecular assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein α-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method.     

Enzymatic activity of immobilized enzyme determined by isothermal titration calorimetry

The activity of adsorbed β-glucosidase onto spherical polyelectrolyte brushes (SPBs) is investigated by UV-Vis spectroscopy and isothermal titration calorimetry (ITC). By comparing the results of these two methods, we demonstrate that ITC is a precise method for the study of the activity of immobilized enzymes. The carrier particles used for immobilization here consist of a polystyrene core onto which poly(acrylic acid) chains are grafted. High amounts of enzyme can be immobilized in the brush layer at low ionic strength by the polyelectrolyte-mediated protein adsorption (PMPA). Analysis of the activity of β-glucosidase was done in terms of Michaelis-Menten kinetics. Moreover, the enzymatic activity of immobilized enzyme is studied by ITC using cellobiose as substrate. All data show that ITC is a general method for the study of the activity of immobilized enzymes.     

Making cool drugs hot: isothermal titration calorimetry as a tool to study binding energetics

Characterization of the thermodynamics of binding interactions is important in improving our understanding of bimolecular recognition and forms an essential part of the rational drug design process. Isothermal titration calorimetry (ITC) is rapidly becoming established as the method of choice for undertaking such studies. The power of ITC lies in its unique ability to measure binding reactions by the detection of the heat change during the binding interaction. Since heat changes occur during many physicochemical processes, ITC has a broad application, ranging from chemical and biochemical binding studies to more complex processes involving enthalpy changes, such as enzyme kinetics. Several features of ITC have facilitated its preferential use compared to other techniques that estimate affinity. It is a sensitive, rapid, and direct method with no requirement for chemical modification or immobilization. It is the only technique that directly measures enthalpy of binding and so eliminates the need for van't Hoff analysis, which can be time consuming and prone to uncertainty in parameter values. Although ITC has facilitated the measurement of the thermodynamics governing binding reactions, interpretation of these parameters in structural terms is still a major challenge.     

Learning to Attend: Modeling the Shaping of Selectivity in Infero-temporal Cortex in a Categorization Task

Recent experiments on behaving monkeys have shown that learning a visual categorization task makes the neurons in infero-temporal cortex (ITC) more selective to the task-relevant features of the stimuli (Sigala and Logothetis in Nature 415 318–320, 2002). We hypothesize that such a selectivity modulation emerges from the interaction between ITC and other cortical area, presumably the prefrontal cortex (PFC), where the previously learned stimulus categories are encoded. We propose a biologically inspired model of excitatory and inhibitory spiking neurons with plastic synapses, modified according to a reward based Hebbian learning rule, to explain the experimental results and test the validity of our hypothesis. We assume that the ITC neurons, receiving feature selective inputs, form stronger connections with the category specific neurons to which they are consistently associated in rewarded trials. After learning, the top-down influence of PFC neurons enhances the selectivity of the ITC neurons encoding the behaviorally relevant features of the stimuli, as observed in the experiments. We conclude that the perceptual representation in visual areas like ITC can be strongly affected by the interaction with other areas which are devoted to higher cognitive functions. Electronic Supplementary Material: Supplementary material is available in the online: version of this article at http://dx.doi.org/10.007/s00422-006-0054-z     

Advances in the analysis of isothermal titration calorimetry data for ligand-DNA interactions

Isothermal titration calorimetry (ITC) is a well established technique for the study of biological interactions. The strength of ITC is that it directly measures enthalpy changes associated with interactions. Experiments can also yield binding isotherms allowing quantification of equilibrium binding constants, hence an almost complete thermodynamic profile can be established. Principles and application of ITC have been well documented over recent years, experimentally the technique is simple to use and in ideal scenarios data analysis is trivial. However, ITC experiments can be designed such that previously inaccessible parameters can be evaluated. We outline some of these advances, including (1) exploiting different experimental conditions; (2) low affinity systems; (3) high affinity systems and displacement assays. In addition we ask the question: What if data cannot be fit using the fitting functions incorporated in the data-analysis software that came with your ITC? Examples where such data might be generated include systems following non 1:n binding patterns and systems where binding is coupled to other events such as ligand dissociation. Models dealing with such data are now appearing in literature and we summarise examples relevant for the study of ligand-DNA interactions.     

User-friendly models of the costs and efficacy of tsetse control: application to sterilizing and insecticidal techniques

An interactive programme, incorporating a deterministic model of tsetse (Diptera: Glossinidae) populations, was developed to predict the cost and effect of different control techniques applied singly or together. Its value was exemplified by using it to compare: (i) the sterile insect technique (SIT), involving weekly releases optimized at three sterile males for each wild male, and (ii) insecticide-treated cattle (ITC) at 3.5/km(2). The isolated pre-treatment population of adults was 2500 males and 5000 females/km(2); if the population was reduced by 90%, its growth potential was 8.4 times per year. However, the population expired naturally when it was reduced to 0.1 wild males/km(2), due to difficulties in finding mates, so that control measures then stopped. This took 187 days with ITC and 609 days with SIT. If ITC was used for 87 days to suppress the population by 99%, subsequent control by SIT alone took 406 days; the female population increased by 48% following the withdrawal of ITC and remained above the immediate post-suppression level for 155 days; the vectorial capacity initially increased seven times and remained above the immediate post-suppression level for 300 days. Combining SIT and ITC after suppression was a little faster than ITC alone, provided the population had not been suppressed by more than 99.7%. Even when SIT was applied under favourable conditions, the most optimistic cost estimate was 20-40 times greater than for ITC. Modelling non-isolated unsuppressed populations showed that tsetse invaded approximately 8 km into the ITC area compared to approximately 18 km for SIT. There was no material improvement by using a 3-km barrier of ITC to protect the SIT area. In general, tsetse control by increasing deaths is more appropriate than reducing births, and SIT is particularly inappropriate. User-friendly models can assist the understanding and planning of tsetse control. The model, freely available via http://www.tsetse.org, allows further exploration of control strategies with user-specified assumptions.     

Intertubular cells and their relation to the renal cambium]

The intertubular cells (ITC) of the cortex of the human kidney (from the fetus, newborns, children aged from 1 to 5 years, adults aged from 30 to 70) were studied. The ITC in the kidneys with an undeveloped structure (fetus, newborns) amounted 21.4--12.6% of the epithelial cells of the convoluted tubules and for children over one year and adults--7.6%. When the kidney was affected by nephrotoxic poison (ethylene-glycol) and deep hypoxia and ITC were activated, began to multiply and made up 14.5% of the epithelium of the convoluted tubules. It is suggested that the ITC represented a peculiar cambium of the renal tubules which formed due to the transferring of a part of the cells of the metaneophrogenic tissue into position of rest (R-period).     

Survey of the year 2008: applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is a fast, accurate and label‐free method for measuring the thermodynamics and binding affinities of molecular associations in solution. Because the method will measure any reaction that results in a heat change, it is applicable to many different fields of research from biomolecular science, to drug design and materials engineering, and can be used to measure binding events between essentially any type of biological or chemical ligand. ITC is the only method that can directly measure binding energetics including Gibbs free energy, enthalpy, entropy and heat capacity changes. Not only binding thermodynamics but also catalytic reactions, conformational rearrangements, changes in protonation and molecular dissociations can be readily quantified by performing only a small number of ITC experiments. In this review, we highlight some of the particularly interesting reports from 2008 employing ITC, with a particular focus on protein interactions with other proteins, nucleic acids, lipids and drugs. As is tradition in these reviews we have not attempted a comprehensive analysis of all 500 papers using ITC, but emphasize those reports that particularly captured our interest and that included more thorough discussions we consider exemplify the power of the technique and might serve to inspire other users. Copyright © 2010 John Wiley & Sons, Ltd.     

An easy-to-use tool for planning and modeling a calorimetric titration

Isothermal titration calorimetry (ITC) is widely employed to measure thermodynamic properties of binding interactions between two macromolecules or a macromolecule and a small ligand. No labeling of interacting species is required for ITC, but this advantage is offset by potentially material-consuming experimental optimization complicated by an indirect readout of an ITC titration. Here we present a simple, practical, and portable spreadsheet-based tool for planning and modeling an ITC titration experiment accompanied by basic guidelines.     

Intermolecular chiral assemblies in R(-) and S(+) 2-butanol detected by microcalorimetry measurements

Supramolecular chiral assemblies of R(-) and S(+) 2-butanol, in their neat form or when dissolved in their nonchiral isomer isobutanol, were evaluated by isothermal titration calorimetry (ITC) ensuing mixing. Dilution of 0.5 M solution of R(-) 2-butanol in isobutanol into the latter liberated heat of several calories per mole, which was approximately double than that obtained in parallel dilutions of S(+) 2-butanol in isobutanol. The ITC dilution profiles indicated an estimate of about 100 isobutanol solvent molecules surrounding each of the 2-butanol enantiomers, presumably arranged in chiral configurations, with different adopted order between the isomers. Mixings of neat R and S 2-butanol were followed by endothermic ITC profiles, indicating that, in racemic 2-butanol, both the supramolecular order and the intermolecular binding energies are lower than in each of the neat chiral isomers. The diversion from symmetrical ITC patterns in these mixings indicated again a subtle difference in molecular organization between the neat enantiomers. It should be noted that the presence of impurities, α-pinene and teterhydrofuran, at a level totaling 0.5%, did not influence the ITC heat flow profiles. The findings of this study demonstrate for the first time that chiral solutes in organic solvents are expected to acquire asymmetric solvent envelopes that may be different between the enantiomers, thus broadening this phenomenon beyond the previously demonstrated cases in aqueous solutions.     

Application of ITC in foods: A powerful tool for understanding the gastrointestinal fate of lipophilic compounds

BackgroundIsothermal titration calorimetry (ITC) is a biophysical technique widely used to study molecular interactions in biological and non-biological systems. It can provide important information about molecular interactions (such as binding constant, number of binding sites, free energy, enthalpy, and entropy) simply by measuring the heat absorbed or released during an interaction between two liquid solutions.Scope of the reviewIn this review, we present an overview of ITC applications in food science, with particular focus on understanding the fate of lipids within the human gastrointestinal tract. In this area, ITC can be used to study micellization of bile salts, inclusion complex formation, the interaction of surface-active molecules with proteins, carbohydrates and lipids, and the interactions of lipid droplets.Major conclusionsITC is an extremely powerful tool for measuring molecular interactions in food systems, and can provide valuable information about many types of interactions involving food components such as proteins, carbohydrates, lipids, surfactants, and minerals. For systems at equilibrium, ITC can provide fundamental thermodynamic parameters that can be used to establish the physiochemical origin of molecular interactions.General significanceIt is expected that ITC will continue to be utilized as a means of providing fundamental information about complex materials such as those found in foods. This knowledge may be used to create functional foods designed to behave in the gastrointestinal tract in a manner that will improve human health and well-being. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.