Reversible detection of proteases and their inhibitors by a pulsed chronopotentiometric polyion-sensitive electrode

Polymer membrane electrodes operated by pulsed chronopotentiometry have recently been introduced to replace traditional ion-selective electrodes for a number of applications. While ion-selective electrodes for the polycation protamine have been reported, for instance, a pulsed chronopotentiometric readout mode (called here pulstrode) provides improved stability and reproducibility while exhibiting sufficient selectivity for the direct detection of protamine in undiluted whole blood samples. Here, such protamine-sensitive pulstrodes are applied for the real-time detection of the activity of the protease trypsin and its soybean inhibitor. This is possible because small fragments produced by the trypsin digestion are not detectable by the protamine-sensing membrane. The real-time response to the proteolytic reaction is shown to exhibit good reproducibility and reversibility, and the initial reaction rate is dependent on the concentration of the protease and its inhibitor.     

Determination of [Mg2+]i – an update on the use of Mg2+-selective electrodes

Since their invention, ion-selective microelectrodes have become an indispensable tool for investigations of intracellular ion regulation and transport. While highly selective sensors for all major intracellular monovalent ions have been available for decades, the development of sensors for divalent cations seems to have presented more difficulties. As ion-selective microelectrodes typically have time-constants in the range of 0.5 to several seconds they turned out to be inapt for the investigation of intracellular Ca²⁺. The development of sensors for Mg²⁺-selective electrodes has made its most striking progress only over the past few years. While the first Mg²⁺ sensor, ETH 1117, was barely able to detect physiological Mg²⁺ concentrations in the presence of other mono- and divalent cations, the newest sensors allow measurements in the micromolar range. When used in macroelectrodes, the most recent developments, ETH 5506 and ETH 5504, have even been reported to do so in the presence of millimolar Ca²⁺ concentrations. Although there is still room for improvement to make these sensors applicable in microelectrodes, some preliminary data look extremely promising and indicate that a new era for Mg²⁺-selective microelectrodes is about to start.     

Ion-selective Microelectrodes: Their Use and Importance in Modern Plant Cell Biology

The exact ion gradients across cellular membranes and their changes due to metabolic or transport processes can be best studied with the use of ion-selective microelectrodes. The last decade of research using ion-selective microelectrodes in intact cells has proven this technique to be indispensable for the investigation of a variety of physiological questions of regulatory processes, membrane transport, cellular signalling, developmental biology and plant nutrition. Their application to selected problems has led to numerous exciting observations, many of which have changed our view concerning cellular responses to environmental stimuli and in many instances have led to a new understanding of plant cell physiology. Since, with these electrodes, intracellular as well as extracellular free ion concentrations can be simultaneously detected with electrical transport parameters such as membrane potential and membrane conductance, they can be powerful tools in the hands of many plant cell biologists.     

Fabrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes.

A simple method of enzyme immobilization was investigated which is useful for fabrication of enzyme sensors based on polymeric ion-selective membranes. The enzyme membrane was built by coating a thin hydrophilic polyurethane (HPU) film directly mixed with an enzyme over an underlying polyurethane (PU)-based ion-selective membrane. This highly simple method of enzyme immobilization was applied to the fabrication of a potentiometric butyrylcholinesterase-based biosensor for the determination of organophosphorus pesticides. The enzyme was well entrapped within the HPU film and the intrinsic potentiometric response of the underlying ion-selective PU membrane was not influenced significantly by the outer HPU/enzyme membrane. The enzyme electrode was optimized by changing systematically the composition of the enzyme membrane to evaluate the effect of the changes on sensor response. The sensor was successfully applied to the analysis of paraoxon, an organophosphorus pesticide.     

The evolution of pheromone diversity

Pheromones are chemical signals whose composition varies enormously between species. Despite pheromones being a nearly ubiquitous form of communication, particularly among insects, our understanding of how this diversity has arisen, and the processes driving the evolution of pheromones, is less developed than that for visual and auditory signals. Studies of phylogeny, genetics and ecological processes are providing new insights into the patterns, mechanisms and drivers of pheromone evolution, and there is a wealth of information now available for analysis. Future research could profitably use these data by employing phylogenetic comparative techniques to identify ecological correlates of pheromone composition. Genetic analyses are also needed to gain a clearer picture of how changes in receivers are associated with changes in the signal.     

A simple system for the measurement of ion activities with solvent polymeric membrane electrodes.

A simple device for the application of ion-selective liquid membrane electrodes to biochemistry is described. K⁺- and Ca²⁺-selective membranes were sealed on polyvinyl chloride (PVC) tubing and inserted into the side wall of a Plexiglas reaction vessel, enabling simultaneous measurements of K⁺ and Ca²⁺. The vessel also permits the simultaneous detection of changes in pH and oxygen and is predisposed to work under a controlled atmosphere. The system is particularly suitable for the measurement of fast changes in ion activities, since the stirring system described has a mixing time below 0.1 s. The 95% response times of the ion-selective electrodes and of the oxygen electrode were below 0.5 s. The volume of the reaction vessel is 0.5–1.0 ml.     

In vivo calibration of the subcutaneous amperometric glucose sensors using a non-enzyme electrode

A new two-point calibration method for the subcutaneous amperometric continuous glucose sensor is reported. The proposed method is based on direct measurement of the background current (I(o)) using a non-enzyme electrode. For in vivo test, three electrodes were implanted in rabbits. Two of the three were identical needle-type enzyme electrodes with perfluorinated polymer outer layers (Pt/enzyme layer/Kel-F/PTFE/Kel-F/Nafion) that were placed in subcutaneous tissue and in a vessel (ear artery), respectively. And one non-enzyme electrode with exactly the same membrane composition as those of other two was in the subcutaneous layer to measure the background current. Implantation in the subcutaneous layer generated many crevices on the protecting layers of the electrodes. The signals from enzyme electrodes were effectively corrected by the measured background current from the non-enzyme electrode. In addition, a telemetric monitoring system was developed and evaluated for in vivo continuous glucose monitoring in order to alleviate the problems of motion artifact.     

Elemental Composition and Water Content of Neuron and Glial Cells in the Central Nervous System of the North American Medicinal Leech (Macrobdella decora)

Elemental (Na, P, S, Cl, K, Ca, Mg) composition and water content of neurons and glial cells of the leech (Macrobdella decora) were determined by x-ray microanalysis of frozen hydrated and dried section techniques. Results are reported as elemental mass fractions (mass/mass) and water content as percent mass. Specific cell compartments and cell types had distinct elemental patterns and water content which suggests that chemical composition of specific cell types is unique and may represent an expression of cell differentiation analogous to morphological specialization. Water content of cells was also cell specific and ranged from 55% (neurons) to 90% (vacuolated zone of glial cells). K and Na were present in concentrations greater than predicted by ion-selective microelectrode measurements, indicating that not all the K and Na were simultaneously accessible to such electrodes.     

Bacterial spore structures and their protective role in biocide resistance

The structure and chemical composition of bacterial spores differ considerably from those of vegetative cells. These differences largely account for the unique resistance properties of the spore to environmental stresses, including disinfectants and sterilants, resulting in the emergence of spore-forming bacteria such as Clostridium difficile as major hospital pathogens. Although there has been considerable work investigating the mechanisms of action of many sporicidal biocides against Bacillus subtilis spores, there is far less information available for other species and particularly for various Clostridia. This paucity of information represents a major gap in our knowledge given the importance of Clostridia as human pathogens. This review considers the main spore structures, highlighting their relevance to spore resistance properties and detailing their chemical composition, with a particular emphasis on the differences between various spore formers. Such information will be vital for the rational design and development of novel sporicidal chemistries with enhanced activity in the future.     

Application of a taste evaluation system to the monitoring of Kimchi fermentation

As an objective method, taste evaluation with an instrument is able to supplement the subjective sensory evaluation and to be applied to the optimization of food processing. Kimchi, a Korean traditional pickle fermented with lactic acid bacteria, is expanding its consumption worldwide. The fermentation control of it has been routinely done by measuring titratable acidity and pH. In this study, an eight-channel taste evaluation system was prepared, followed by an application to the monitoring of Kimchi fermentation. Eight polymer membranes which individually responded to cationic or anionic substances were prepared by mixing electroactive materials such as tri-n-octylmethylammonium chloride, bis(2-ethylhexyl)sebacate as the plasticizer and polyvinyl chloride in the ratio of 1:66:33. Each membrane prepared was separately installed onto the sensitive area of an ion-selective electrode to produce the respective taste sensor. The eight-channel sensor array and a double junction reference electrode were connected to a 16-channel high input impedance amplifier. The amplified sensor signals were stored to a personal computer via a multi-channel A/D converter. Two sensor groups composed of the cation-selective and anion-selective polymer membrane electrodes showed characteristic concentration-dependency to various artificial taste substances. As a whole, the response potentials of the sensor array increased during the fermentation period at 4, 10 and 25 degrees C. Even the response potentials of the anion-selective taste sensors slightly increased possibly due to the protonation of anions by liberated H+ ions, thereby leading to a decrease in the anion concentration. When the signal data were interpreted by principal component analysis (PCA), the first PC at 4 degrees C explained most of the total data variance. A close correlation was found between the values of titratable acidity and the first PC, which indicated a possible applicability of the multi-channel taste sensor of this study to the process monitoring of various pickle.     

Potassium selective chemically modified field effect transistors based on AlGaN/GaN two-dimensional electron gas heterostructures

We investigate the use of the AlGaN/GaN high electron mobility transistor (HEMT) as a novel transducer for the development of ion-selective chemically modified HEMT sensors (ChemHEMTs). For this, polyvinyl chloride (PVC) membrane doped with ion-selective ionophores is deposited onto the area of the gate for the chemical recognition step, while the AlGaN/GaN HEMT is used as the transducer. In particular, the use of a valinocycin doped membrane with thickness of 50 μm generates a sensor with excellent analytical characteristics for the monitoring of K⁺. The K⁺-ChemHEMT has sensitivity of 52.4 mV/pK⁺in the linear range of 10⁻⁵ to 10⁻² M, while the detection limit is in the order of 3.1 × 10⁻⁶ M. Also, the sensor shows selectivity similar to valinomycin-based ISEs, while the signal stability over time and the measurement to measurement reproducibility are very good.     

The talin wags the dog: new insights into integrin activation

Integrin transmembrane receptors have a unique property that distinguishes them from other signaling receptors. Their affinity for ligands can be modulated from the inside out in response to intracellular signals generated by non-integrin receptors. Recent findings provide novel mechanistic insights into this process by demonstrating that talin, a protein that links integrins to actin, is necessary for the inside-out activation of integrins.     

An intracellular conformational sensor assay for Abl T315I

Conformational change is a common molecular mechanism for the regulation of kinase activities. Small molecule modulators of protein conformations, including allosteric kinase inhibitors, are highly wanted as tools for the interrogation of kinase biology and as selective therapeutic agents. However, straightforward cellular assays monitoring kinase conformations in a manner conducive to high-throughput screening (HTS) are not readily available. Here we describe such an HTS-compatible conformational sensor assay for Abl based on a split luciferase construct. The Abl sensor responds to intramolecular structural rearrangements associated with intracellular Abl deactivation and small molecule inhibition. The intact regulatory CAP-SH3-SH2 domain is required for the full functionality of the sensor. Moreover, a T334I Abl mutant (T315I in Abl1a) was found to be particularly well suited for HTS purposes and mechanistic intracellular studies of T334I mutant inhibitors. We expect that the split luciferase-based conformational sensor approach might be more broadly useful to probe the intracellular activation of other kinases and enzymes in general.     

Mechanisms and inhibition of uracil methylating enzymes

Uracil methylation is essential for survival of organisms and passage of information from generation to generation with high fidelity. Two alternative uridyl methylation enzymes, flavin-dependent thymidylate synthase and folate/FAD-dependent RNA methyltransferase, have joined the long-known classical enzymes, thymidylate synthase and SAM-dependent RNA methyltransferase. These alternative enzymes differ significantly from their classical counterparts in structure, cofactor requirements and chemical mechanism. This review covers the available structural and mechanistic knowledge of the classical and alternative enzymes in biological uracil methylation, and offers a possibility of using inhibitors specifically aiming at microbial thymidylate production as antimicrobial drugs.     

The impact of thiol peroxidases on redox regulation

The biology of glutathione peroxidases and peroxiredoxins is reviewed with emphasis on their role in metabolic regulation. Apart from their obvious function in balancing oxidative challenge, these thiol peroxidases are not only implicated in orchestrating the adaptive response to oxidative stress, but also in regulating signaling triggered by hormones, growth factors and cytokines. The mechanisms presently discussed comprise dampening of redox-sensitive regulatory processes by elimination of hydroperoxides, suppression of lipoxygenase activity, committing suicide to save H₂O₂ for signaling, direct binding to receptors or regulatory proteins in a peroxidase activity-independent manner, or acting as sensors for hydroperoxides and as transducers of oxidant signals. The various mechanistic proposals are discussed in the light of kinetic data, which unfortunately are scarce. Taking into account pivotal criteria of a meaningful regulatory circuit, kinetic plausibility and specificity, the mechanistic concepts implying a direct sensor/transducer function of the thiol peroxidases appear most appealing. With rate constants for the reaction with hydroperoxide of 10⁵–10⁸ M^{? 1} s^{? 1}, thiol peroxidases are qualified as kinetically preferred hydroperoxide sensors, and the ability of the oxidized enzymes to react with defined protein thiols lends specificity to the transduction process. The versatility of thiol peroxidases, however, allows multiple ways of interaction with regulatory pathways.     

Toward a molecular understanding of nanoparticle–protein interactions

Wherever nanoparticles (NPs) come in contact with a living organism, physical and chemical interactions take place between the surfaces of the NPs and biomatter, in particular proteins. When NP are exposed to biological fluids, an adsorption layer of proteins, a “protein corona” forms around the NPs. Consequently, living systems interact with the protein-coated NP rather than with a bare NP. To anticipate biological responses to NPs, we thus require comprehensive knowledge of the interactions at the bio–nano interface. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP–protein interactions. In this brief review, we present the latest findings regarding the composition of the protein corona as it forms on NPs in the blood stream. We also discuss molecular aspects of this adsorption layer and its time evolution. The current state of knowledge is summarized, and issues that still need to be addressed to further advance our understanding of NP–protein interactions are identified.     

Building of an immunosensor: how can the composition and structure of the thiol attachment layer affect the immunosensor efficiency?

Immunosensors, based on the immobilization of a model rabbit antibody on mixed self-assembled monolayers and Protein A as a linking agent on gold transducers, were elaborated and characterized at each step by modulated polarization-infrared spectroscopy (PM-IRRAS) and occasionally by atomic force microscopy (AFM) and quartz crystal microbalance (QCM). By testing two different mixed SAMs comprising 11-mercaptoundecanoic acid (MUA), together with either decanethiol (C9CH3) or mercaptohexanol (C6OH), the role of the chemical composition and structure of the antibody attachment layer upon the sensor performance was demonstrated.     

Detection of myocardial ischemia by epicardial detection of potassium ion activity]

Early detection of myocardial ischaemia is a central problem in cardiological and cardiosurgical intensive care. A new approach is the use of ion-selective electrodes implanted directly on the myocardium, enabling detection of increased potassium activity as an indication of general hypoxia. After a comprehensive study of the electrode parameters, an animal experiment was carried out, in which it was found that respiration-induced hypoxia resulted in an increase in epicardial potassium activity (p < 0.01). Blood gas analysis performed simultaneously revealed reduced arterial pO2, but no acidosis. Haemodynamic data evidenced hypoxic depression of circulatory parameters. Histological examinations of the myocardium beneath the electrodes revealed typical lymphocytic infiltration. Electron microscopy demonstrated crystolysis in the mitochondria as an early sign of hypoxia, thus confirming the sensitivity of these electrodes. This underscores the potential of ion-selective electrodes for the detection of myocardial ischaemia, and they should now be investigated in the clinical setting.