Measurement of intracellular and extracellular free calcium in apple fruit cells using calcium-selective microelectrodes

Abstract. Intracellular and extracellular free calcium was measured in cortical cells from apple fruit ( Malus domestica Borkh cv. Cox's Orange Pippin) using calcium-selective microelectrodes. It was difficult to position microelectrodes in the cytoplasm, and thus most intracellular measurements reflected vacuolar concentrations of about 0.6mol m⁻³ free calcium. Extracellular free calcium was measured in wells (0.2mm³) that had been cut through the epidermis of the fruit, then filled with osmoticum. Concentrations of free calcium in the sampling well varied between 0.02 and 1.3 mol m⁻³, and were related to the calcium content of the tissue. Functioning of the plasma membrane, and perhaps the supply of calcium for intracellular needs during fruit ripening and storage, may require mol m⁻³ concentrations of extracellular free calcium. Thus, it is suggested that low levels of extracellular free calcium in calcium-deficient fruit may be partly responsible for development of deficiency disorders.     

Oxygen and pH microprofiles above corroding mild steel covered with a biofilm

O₂ and pH microprofiles were measured above corroding mild steel covered with a biofilm. The pH in the anodic areas (tubercles) ranged from 5 to 7 and was always 9.45 at the surface of the cathodic areas. After 1 month of biofilm development, O₂ was depleted at the anodic area but could reach the cathodic surface where it was reduced. Consequently, differential O₂ concentration cells were the driving force for corrosion. The O₂ microprofiles indicated that O₂ was consumed in the tubercles, probably by microbial activity, while O₂ was reduced electrochemically in the cathodic areas. It was concluded that O₂ transfer to the cathodic surface was the rate limiting step for the corrosion process.     

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications^1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings^5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain^7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties^10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo¹⁵, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control^13,16,17.To this end, a custom microtensile tester was designed to accommodate microscale samples^13,17 with widely-varying Young''s moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling¹⁷. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young''s modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.     

Steady-state gradient in calcium ion activity across the intercellular bridges connecting oocytes and nurse cells in Hyalophora cecropia

Intracellular activities of K⁺, H⁺, Mg²⁺, Ca²⁺, and Cl^?, measured with ion selective microelectrodes in the oocyte and the nurse cells in ovarian follicles of Hyalophora cecropia, indicated that a Ca²⁺ current is a key component of the electrical potential that is maintained across the intercellular bridges connecting these two cells. In vitellogenic follicles, Ca²⁺ activity averaged 650 nM in the oocyte and 190 nM in the nurse cells, whereas activities of the other ions studied differed between these cells by no more than 6%. Incubation in 200 μM ammonium vanadate caused a reversal of electrical potential from 8.3 mV, nurse cell negative, to 3.0 mV, oocyte negative, and at the same time the Ca²⁺ gradient was reversed: activities rose to an average 3.0 μM in the nurse cells and 1.6 μM in the oocyte, whereas transbridge ratios of the other cations remained at 0–3%. In immature follicles that had not yet initiated their transbridge potentials, Ca²⁺ activities averaged ～? 2 μM in both oocyte and nurse cells. The results suggest that vitellogenic follicles possess a vanadatesensitive Ca²⁺ extrusion mechanism that is more powerful in the nurse cells than in the oocyte. © 1994 Wiley-Liss, Inc.     

Background-subtraction of fast-scan cyclic staircase voltammetry at protein-modified carbon-fiber electrodes

Background-subtraction techniques were applied to the voltammetry of nicotinamide adenine dinucleotide (NADH) at protein-modified carbon-fiber microelectrodes. The background currents at carbon-fiber electrodes were stable and voltammetric scans immediately before or after the analyte were effectively used for background subtraction. Digital step-potential waveforms were used to excite these carbon-fiber electrodes, where the resulting voltammetric analysis assessed the optimal switching and initial potentials and the electrochemical response time was determined. The initial potential was 0.0 V and the switching potential 1.1 V (versus Ag/AgCl) and the response time was approximately 300 ms. Some sensitivity to NADH was lost and voltammetric prescans were required at protein-modified electrodes to obtain a stable baseline. Current versus time was assessed by the average current of the faradaic region from each voltammogram and by differential current; the average current minus the current from a non-faradaic potential range. Differential current assessments discriminated against artifacts caused by pH (as high as 1.0 pH unit) and ionic strength flux (100 mM). These background-subtraction techniques allowed the faradaic information to be obtained quickly and conveniently while maximizing sensitivity and maintaining selectivity.     

Smart hydrogels for bioseparation

Smart hydrogels are hydrogels which alter their dimension (i.e., either swell or shrink) dramatically upon a small change in an environmental condition, such as temperature, pH, ionic strength, salt type, solvent, etc. Due to large changes in the swelling ratio, the smart hydrogels have been used widely in the separation of various molecules including proteins. Bioseparation using smart hydrogels is convenient, cost effective, and operable in mild conditions. The use of mild conditions during separation is critical for proteins which can be easily denatured or degraded. Smart hydrogels currently used in bioseparation and their limitations as well as improvements to be made are described here. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a pH gradient within a biofilm is dependent upon the limiting nutrient

Monospecies Citrobacter sp. biofilms were grown in a laminar flow cell using a carbon-limiting medium. Microelectrode measurements showed no change in pH between the bulk fluid and biofilm when the flow cell was supplied with the carbon-limiting medium under static or flowing conditions. When the biofilm was supplied with a phosphate-limiting medium the biofilm became more acidic than the bulk fluid and developed a gradient within. The implications for metals-bioremediation processes are discussed.     

Structures of LRP2 reveal a molecular machine for endocytosis

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