Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 2. Fiber-optic microbial biosensor

A fiber-optic microbial biosensor suitable for direct measurement of organophosphate nerve agents was developed. The unique features of this novel microbial biosensor were the recombinant Escherichia coli cells expressing the enzyme organophosphorus hydrolase on the cell surface and the optical detection of the products of enzyme-catalyzed organophosphate hydrolysis. The use of cells with the metabolic enzyme expressed on the cell surface as a biological sensing element provides advantages of no resistance to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively. The use of an optical transducer allows the detection of different organophosphates in a mixture, presently not feasible with acetylcholinesterase-based biosensors. E. coli cells expressing organophosphorus hydrolase (OPH) on the cell surface were immobilized in low melting temperature agarose on a nylon membrane and attached to the common end of a bifurcated fiber-optic bundle. OPH-expressing E. coli cells catalyzed the hydrolysis of organophosphorus pesticides to form stoichiometric amounts of chromophoric products that absorb light at specific wavelengths. The backscattered radiation of the specific wavelength incident light was measured using a photomultiplier detector and correlated to the organophosphate concentration. The best sensitivity and response time were obtained using a sensor constructed with 1.5 mg of cells operating in pH 9, 50 mM HEPES buffer with 100 mM NaCl and 0.05 mM CoCl2 at 30 degrees C. At optimized conditions, the biosensor measured paraoxon, parathion, and coumaphos pesticides with high selectivity against triazine and carbamate pesticides in approximately 10 min. The lower detection limits were 3 microM for paraoxon and parathion and 5 microM for coumaphos. When stored in the buffer at 22 degrees C, the biosensor was stable for over a 1-month period and showed no decline in the response for over 75 repeated usages. The new fiber-optic microbial biosensor is an ideal tool for on-line monitoring of the detoxification process for organophosphate pesticides-contaminated wastewaters but may not be suitable for environmental monitoring.     

Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 1. Potentiometric microbial electrode

A potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents was developed by modifying a pH electrode with an immobilized layer of Escherichia coli cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosporus pesticides to release protons, the concentration of which is proportional to the amount of hydrolyzed substrate. The sensor signal and response time were optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and weight of cells immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 2.5 mg of cells and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, methyl parathion, and diazinon. The biosensor had very good storage and multiple use stability. The use of cells with the metabolic enzyme expressed on cell surface as a biological transducer provides advantages of no resistances to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively.     

Effect of isradipine on in-vivo platelet function

A new flow-injection amperometric biosensor based on immobilized tryptophan-2-monooxygenase (TMO) has been developed for reagentless L-tryptophan determination. Concentrations of L-tryptophan between 0.1 and 50 mM could be measured with the linear part of the calibration curve between 0.1 and 2 mM. The response time was 30 s and the total analysis time was less than 3 min. The biosensor retained activity for greater than 4 months, when operated daily at 25 degrees C and stored at 8 degrees C. The biosensor was characterized by a relatively high sensitivity to phenylalanine (54% that of L-tryptophan), a modest response to L-methionine (less than 6%) and virtually no response to other amino acids. However, the biosensor selectivity to L-tryptophan could be dramatically increased when indoleacetamide (IA), a competitive inhibitor of TMO, was introduced. In the presence of 10 microM IA, the biosensor response to L-phenylalanine decreased to 7-4% of the unaffected rate for L-tryptophan. In the absence of L-tryptophan and IA the biosensor could be used for L-phenylalanine determination in the concentration range from 1 to 50 mM. The biosensor was successfully used for L-tryptophan determination in nutritional broth.     

Platinum-catalyzed enzyme electrodes immobilized on gold using self-assembled layers

The bonding of enzymes to self-assembled monolayers (SAMs) of alkanethiols onto gold electrode surfaces is exploited to produce an enzyme biosensor. The attachment of glucose oxidase to a SAM of 3-mercaptopropionic acid was achieved using carbodiimide coupling. The resultant biosensor showed good sensitivity to glucose and a large dynamic range when measured amperometrically via the p-benzoquinone mediator. On the other hand, subsequent platinization of the enzyme-SAM electrode allowed hydrogen peroxide produced in the enzyme reaction to be detected directly, thus obviating the need for an artificial redox mediator. The performance of such sensors constructed on bulk gold electrodes was evaluated and finally compared to that of some preliminary thin-film gold electrodes. Biosensors constructed using the two alternative electrode surfaces have quite different sensitivities, thus reflecting the influence of the anchoring surface on the performance of the biosensor.     

Microbial sensor for new-generation cephalosporins based in a protein-engineered beta-lactamase

A protein-engineered beta-lactamase, constructed by site-directed mutagenesis in Escherichia coli (E104M/G238S), and having broadened specificity, was able to degrade cephalosporins of first, second, and third generations. Manipulations of culture conditions allowed an increase in beta-lactamase specific activity by up to twofold. The resultant bacteria were used to construct an immersable whole-cell biosensor for the detection of new-generation cephalosporins. Cells were immobilized on agar membranes, which in turn were attached to the surface of a flat pH electrode, thus constituting a biosensor based on the detection of pH changes. The sensor was able to detect second- and third-generation cephalosporins: cefamandole (0.4-4 mM), cefotaxime (0.4-3.5 mM), and cefoperazone (0.3-1.85 mM). Response times were between 3.5 and 11 min, depending on the kind of cephalosporin tested. The biosensor was stable for at least 7 d, time during which up to 100 tests were performed.     

Utility of wiring nitrate reductase by alkylpyrroleviologen-based redox polymers for electrochemical biosensor and bioreactor applications

The purpose of this work was to see if the alkylpyrroleviologen redox polymer technology previously developed for a reagentless nitrate biosensor based on nitrate reductase (NaR) from Escherichia coli (Cosnier, S.; Innocent, C.; Jouanneau, Y. Anal. Chem. 1994, 66, 3198-3201) could be applied to the isozyme from Aspergillus niger. In particular, the enzyme viability after immobilization was of great interest, as Cosnier et al. reported a residual activity of only 0.33% of the amount initially applied. The present work showed that A. niger NaR lost 99.2% of soluble activity on vacuum-drying in the presence of 2.5 nM N-methyl-N'-(12-[pyrrol-1-yl]dodecyl)-4,4'-bipyridinium ditetrafluoroborate monomer (C12V2+) and that most of this loss was due to monomer inhibition (91%). The loss due to dehydration was only 8%. In the biosensor configuration, the enzyme gave a residual activity of 0.18% of the amount originally applied and a specific response of 1.7 mA M-1 cm-2, but all activity was lost after 4 d storage at 4 degrees C in phosphate buffer. It was concluded that for practical biosensors and bioreactors, modification of the redox polymer format was needed, for example by covalent immobilization, to effect higher loading of viable NaR and improved enzyme stability.     

Analysis of lecithin in pharmaceutical products and diet integrators using a new biosensor operating directly in non aqueous solvent

One of the first examples of a bienzymatic organic phase enzyme electrode (OPEE) is described. It was obtained using two enzymes (phospholipase D and choline oxidase), both immobilised in kappa-Carrageenan gel and, as electrochemical transducer, an amperometric gas diffusion electrode for oxygen. The response of the biosensor was recorded and its sensitivity, linearity range, response time evaluated. Lastly drugs and diet products containing lecithin were analysed using the new biosensor device working in an organic mixture.     

Design of a stable charge transfer complex electrode for a third-generation amperometric glucose sensor

A novel approach to prepare a stable charge transfer complex (CTC) electrode for the direct oxidation of flavoproteins and the fabrication of a third generation amperometric biosensor (Koopal, C.G.J.; Feiters, M.C.; Nolte, R.J.M. Bioelectrochem. Bioenerg. 1992, 29, 159-175) system is described. Tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ), an organic CTC, is grown at the surface of a shapable electroconductive (SEC) film (a polyanion-doped polypyrrole film) in such a way that it makes a tree-shaped crystal structure standing vertically on the surface. Glucose oxidase (GOx) is adsorbed and cross-linked with glutaraldehyde to fix at the surface of the CTC structure. The space between crystals is filled with cross-linked gelatin to ensure the stability of the treelike crystal structure as well as the stability of the enzyme. Because of the close proximity and the favorable orientation of the enzyme at the CTC surface, the enzyme is directly oxidized at the crystal surface, which leads to a glucose sensor with remarkably improved performance. It works at a potential from 0.0 to 0.25 V (vs Ag/AgCl). The maximum current density at 0.25 V reaches 1.8 mA/cm2, with an extended linear range. The oxygen in the normal buffer solution has little effect on the sensor output. The current caused by interference contained in the physiological fluids is negligible. The working life as well as the shelf life of the sensor is substantially prolonged. The sensor was continuously used in a flow injection system with a continuous polarization at 0.1 V, and the samples (usually 10 mM glucose) were injected at 30 min intervals. After 100 days of continuous use, the current output dropped to 40% of the initial level. No change in the output of the sensor was observed over a year when the sensor was stored dry in a freezer. The electrochemical rate constants and the effective Michaelis constant of the system are reported.     

Fiber-optic enzyme biosensor for direct determination of organophosphate nerve agents

A fiber-optic enzyme biosensor for the direct measurement of organophosphate nerve agents was developed. The basic element of this biosensor is organophosphorus hydrolase immobilized on a nylon membrane and attached to the common end of a bifurcated optical fiber bundle. The enzyme catalyzes the hydrolysis of organophosphate compounds to form stoichiometric amounts of chromophoric products that absorb light at specific wavelengths. The back-scattered radiation of the specific incident radiation was measured using a photomultiplier detector and correlated to the organophosphate concentration. The effects of buffer pH, temperature, and the units of enzyme immobilized on the steady-state and kinetic response of the biosensor were investigated to optimize the operating conditions for the fiber-optic enzyme biosensor. These conditions were then used to measure parathion, paraoxon, and coumaphos selectively without interference from carbamates and triazines. Concentrations as low as 2 microM can be measured in less than 2 min using the kinetic response. When stored in buffer at 4 degreesC the biosensor shows long-term stability.     

A nitrite biosensor based on a maltose binding protein nitrite reductase fusion immobilized on an electropolymerized film of a pyrrole-derived bipyridinium

The preparation and electrochemical characterization of glassy carbon electrodes (GCEs) modified with electropolymerized films of the cation N-(3-pyrrol-1-yl-propyl)-4,4'-bipyridine (PPB) are described. The behavior of a new biosensor, which exhibits a high catalytic activity for nitrite reduction and which consists of a maltose binding protein nitrite reductase fusion (MBP-Nir) immobilized on an electropolymerized film of PPB as an electrocatalyst, is also described. The insoluble perchlorate salt of the poly(benzyl viologen) dication was used to immobilize MBP-Nir onto an electrode previously modified with an electropolymerized film of PPB. The electropolymerized film of PPB on the GCE is redox active and exhibits special electron-transfer properties toward the MBP-Nir layer but not toward Nir (Nir without MBP fusion attached), suggesting an intimate interaction between the PPB film and the MBP-Nir layer. The kinetics of the catalytic reaction between the biosensor and nitrite anion were characterized using cyclic voltammetry and rotated disk electrode techniques, and a value of (4.6 +/- 0.5) x 10(3) M-1 S-1 was obtained for the rate constant.     

A conducting salt-based amperometric biosensor for measurement of extracellular lactate accumulation in ischemic myocardium

In this paper, fabrication, characterization, and physiological application of a miniaturized amperometric lactate biosensor are described. The sensor is based on cross-linked lactate oxidase and tetrathiafulvalene-tetracyano-quinodimethane (TTF-TCNQ) charge transfer complex. The sensor was developed for continuous quantitative measurement of the lactate accumulation in ischemic myocardium under severe depletion of oxygen. The sensor was evaluated in vitro at an applied potential of 0.15 V vs Ag/AgCl; it proved to combine all the performance characteristics desired for the present application, such as proper response in absence of oxygen, good operational stability, good accuracy and precision (103.5 +/- 1.2%), adequate response time (t95% = 80 s), and wide linear dynamic range up to 27 mM (r = 0.9998) in N2-saturated solutions and at 37 degrees C. The prepared sensors (n = 12) showed sensitivity of 380 +/- 90 nA/mM, and a background current of 240 +/- 50 nA. The lower limit of detection is 0.4 +/- 0.15 mM with a S/N ratio equal to 3. Results obtained for direct lactate monitoring in ischemic rabbit papillary muscle under no-flow conditions and PO2 < 6 mm Hg are presented.     

Direct Electrochemistry Transfer and Electrocatalysis of Hemoglobin Immobilized in Porous Mn2O3

Novel porous Mn2O3 with good crystallinity was synthesized via hard-template method. Hb-Mn2O3 na nocomposite was prepared and used for biosensor construction. The Hb-Mn2O3-Nafion modified electrode shows fast direct electron transfer and displays good electrocatalytic response to the reduction of H2O2. The response time is less than 5 s, the sensitivity is as high as 493 μA·L·mmol-1·cm-2 in a linear range of 1-100 μmol/L, and the detection limit is 0.16 μmol/L. This modified electrode also shows good stability and reproducibility. This indicates that the porous Mn2O3 provides a good matrix for enzyme immobilization and biosensor construction.     

Microbial O2- and H2O2-electrode sensors for alcohol assays based on the use of permeabilized mutant yeast cells as the sensitive bioelements

Two types of alcohol-specific microbial/electrochemical biosensors have been developed using specially constructed mutant cells of the methylotrophic yeast Hansenula polymorpha. The cells were immobilized in a calcium alginate gel, and placed between two membranes on the surface of oxygen or hydrogen peroxide-electrodes. The O2 electrode based biosensor contained mutant cells with strongly elevated alcohol oxidase activity. The peroxide electrode based biosensor consisted of catalase-defective mutant cells which produce hydrogen peroxide in the presence of alcohol. Both types of mutant cells were used in permeabilized form in order to release some components of the cellular respiration system, thus increasing the selectivity of the cellular respiration response to alcohol (cell/O2-biosensor) Permeabilization also increased sensitivity of the signal and shortened the response time (cell/H2O2-biosensor). Cell/O2 biosensors were linear up to 1.2 mM for ethanol and 0.35 mM for methanol, cell/H2O2 biosensors were linear up to 4.0 mM for ethanol, and 1.2 mM for methanol. Results were reproducible, sample pretreatment was not required, and the sensors exhibited good operational and storage stability. The use of sucrose, dulcitol or inositol during the preparation of the sensors resulted in increased stability of cells during their liophilization and storage in the dried state. Both biosensors had similar selectivity towards alcohols in the order of methanol (100%), ethanol (21%), and formaldehyde (12%). No signal was observed with glucose or glycerol as substrates.     

Tiagabine: a novel antiepileptic drug

Reproductive management is a major financial concern of the dairy industry, with missed estrus detection a main cause of lost income. A biosensor was developed for on-line measurement of progesterone in bovine milk and detection of estrus. The biosensor used an enzyme immunoassay format for molecular recognition, which was developed to run in approximately eight minutes. The sensor was designed to operate on-line in a dairy parlor using microinjection pumps and valves for fluid transport, fiber optics and photodiodes for light measurement, and a control computer for sequencing. Calibration showed a dynamic response between 0.1 and 5 ng/ml progesterone in milk. The reusability of the test well was evaluated. Thiocyanate (0.5 M, pH 5.1) quickly regenerated the antibody surface while maintaining antibody activity for 15-20 cycles, but noise from the residual enzyme limited reusability.     

Development of a Sulfamic Acid-Based Chemical Formulation for Effective Cleaning of Modified 9Cr–1Mo Steel Steam Generator Tubes

A new sulfamic acid-based formulation is developed for the effective chemical cleaning of modified 9Cr–1Mo steel as a replacement for the conventional corrosive nitric acid (HNO3) and hydrofluoric acid (HF) mixture. The effect of sulfamic acid concentrations and inhibitor (2-mercaptobenzimidazole, MBI) on the metal loss during cleaning of modified 9Cr–1Mo steel was studied using weight loss and electrochemical impedance spectroscopic methods. The metal loss was found to increase with increase in acid concentration, but it was significantly lower with increasing inhibitor concentration, with an efficiency of > 90%. The corrosion rates of modified 9Cr–1Mo steel with 10% sulfamic acid + 2 mM MBI, 10% sulfamic acid without inhibitor and HNO3 + HF mixture were 1290, 6426, and 303,515 μm year−1 respectively. The optimal composition for efficient cleaning, with least base metal loss, was found to be 10% sulfamic acid + 2 mM MBI. Laser Raman spectroscopic (LRS) analysis of the corrosion products obtained during chemical cleaning process revealed that a protective chromium oxide film was formed during the cleaning with sulfamic acid + inhibitor as compared to iron oxide-based films with HNO3 + HF mixture and 10% sulfamic acid solutions.     

Dichloroacetic acid induction of peroxisome proliferation in cultured hepatocytes

Trichloroethylene is a widespread industrial solvent and one of the most common environmental contaminants. Trichloroethylene causes hepatocarcinoma in the B6C3F1 mouse in a dose-dependent manner. Trichloroethylene's hepatocarcinogenicity is thought to be mediated through its metabolites trichloroacetate and dichloroacetate. Although the mechanism of action is not well understood, hepatic tumors are thought to arise as a result of excessive peroxisome-dependent active oxygen production or secondary to enhanced cell replication. The peroxisome proliferative activity of trichloroacetate has been replicated in cultured rodent hepatocytes, while that of dichloroacetate has not been demonstrated. The present experiments were designed to characterize the peroxisome proliferative response to dichloroacetate in hepatocyte cultures from male B6C3F1 mice and male Long Evans rats. The cultured hepatocytes were treated after attachment with 0.1, 0.5, 1.0, 2.0, or 4.0 mM dichloroacetate for 72 hours. Peroxisome proliferation was assessed by measuring palmitoyl-CoA oxidation and by immunoquantitation of peroxisomal bifunctional enzyme. Palmitoyl CoA oxidation increased in a concentration-dependent manner, with maximal induction of 5.5- and 5-fold in mouse and rat hepatocytes, respectively, after treatment with 2.0 mM dichloroacetate. Peroxisomal bifunctional enzyme protein levels also increased in a concentration-dependent manner in both rat and mouse hepatocytes in response to dichloroacetate exposure. These results indicate that the peroxisomal response observed in vivo in response to dichloroacetate administration can be reproduced in primary cultures of rat and mouse hepatocytes treated with dichloroacetate. Further studies using this model system will help elucidate mechanisms of dichloroacetate-induced hepatocarcinogenesis.     

Drug metabolism biosensors: electrochemical reactivities of cytochrome P450cam immobilised in synthetic vesicular systems

Biosensors containing cytochrome P450cam in a didodecyldimethylammonium bromide vesicular system were prepared by cross-linking onto a glassy carbon electrode (GCE) with glutaraldehyde in the presence of bovine serum albumin. Cyclic voltammetric responses of the sensor in air-free buffer solution showed that the sensor exhibited reversible electrochemistry due to direct electron exchange between the haem Fe(3+/2+) redox system and the GCE surface. In air-saturated solution containing camphor, the biosensor gave an irreversible electrocatalytic current which is compatible with the monooxygenation of the substrate. Steady state amperometric experiments with camphor, adamantanone and fenchone were performed with a biosensor prepared by cross-linking P450cam with glutaraldehyde onto a Pt disc electrode. The sensor was characterised by fast amperometric responses, attaining steady-state in about 20 s in a cobalt sepulchrate mediated electrochemical system. The kinetic parameters of the biosensor were analysed using the electrochemical Michaelis Menten equation. The estimated apparent Michaelis-Menten constant, Km, values for the biosensors were in the range of 1.41-3.9 mM.     

Mechanism of irreversible inactivation of phosphomannose isomerases by silver ions and flamazine

Silver ions and silver-containing compounds have been used as topical antimicrobial agents in a variety of clinical situations. We have previously shown that the enzyme phosphomannose isomerase (PMI) is essential for the biosynthesis of Candida albicans cell walls. In this study, we find that PMI can be inhibited by silver ions. This process is shown to be irreversible, and is a two-step process, involving an intermediate complex with a dissociation constant, Ki, of 59 +/- 8 microM, and a maximum rate of inactivation of 0.25 +/- 0.04 min-1 in 50 mM Hepes buffer, pH 8.0 at 37 degrees C. The enzyme can be protected against this inactivation by the substrate mannose 6-phosphate, with a dissociation constant of 0.31 +/- 0.04 mM, close to its Km value. Flamazine (silver sulfadiazine) is a silver-containing antibiotic which is used clinically as a topical antimicrobial and antifungal agent. We compared the ability of silver sulfadiazine and two other silver-containing compounds to irreversibly inactivate C. albicans PMI. The addition of the organic moiety increased the affinity of the compounds, with silver sulfadiazine showing a Ki of 190 +/- 30 nM. In all cases, the maximum inhibition rate was similar, implying a similar rate-determining step. Silver sulfadiazine does not inhibit Escherichia coli PMI, and this suggests a role of the only free cysteine, Cys-150, in the inactivation process. To confirm this, we mutated this residue to alanine in C. albicans PMI. The resultant Cys150 --> Ala mutant protein showed similar Vm and Km values to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

An immunosensor based on disposable electrodes for rapid estimation of fatty acid-binding protein, an early marker of myocardial infarction

An immunosensor was developed that allows the rapid estimation of fatty acid-binding protein (FABP) in neat plasma samples. FABP is released into the blood following myocardial infarction and elevated levels are found already 3 h after onset of symptoms. The sensor is based on screen-printed graphite working and Ag/AgCl reference electrodes and an immunosandwich procedure for the quantification of FABP. The capture antibodies are bound to the electrode surface by adsorption and will trap FABP from the plasma sample. The sandwich is then completed by a second monoclonal antibody conjugated with alkaline phosphatase. The enzyme converts p-aminophenylphosphate to p-aminophenol, which is detected amperometrically at +350 mV. The high binding capacity and very short response time of the working electrode allow within 20 min the quantification of FABP in the measuring range 10-350 ng/ml, covering the pathological range of FABP release into the circulation. Measurements of plasma samples from a patient with acute myocardial infarction show an excellent correlation of the results obtained with the biosensor and those obtained with the respective reference ELISA. Owing to the long stability of the electrodes with immobilized capture antibody (> 3 months) a quick application without the need of labour-intensive electrode preparation is possible.     

Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to sublethal ischemia-like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum- and glucose-free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Concomitant transient increases (2-3 times above control levels) of both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 4-24 h of hypoxia, 4 h of glucose deprivation, and 2-4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K-ATPase activity. The observed increases in Na,K-ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia-like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply.