The proton-translocating nicotinamide–dinucleotide (phosphate) transhydrogenase of rat liver mitochondria

1. The NAD(P) transhydrogenase activity of the soluble fraction of sonicated rat liver mitochondrial preparations was greater than the NAD-linked isocitrate dehydrogenase activity, and the NAD-linked and NADP-linked isocitrate dehydrogenase activities were not additive. The NAD-linked isocitrate dehydrogenase activity was destroyed by an endogenous autolytic system or by added nucleotide pyrophosphatase, and was restored by a catalytic amount of NADP. 2. We concluded that the isocitrate dehydrogenase of rat liver mitochondria was exclusively NADP-specific, and that the oxoglutarate/isocitrate couple could therefore be used unequivocally as redox reactant for NADP in experiments designed to operate only the NAD(P) transhydrogenase (or loop 0) segment of the respiratory chain in intact mitochondria. 3. During oxidation of isocitrate by acetoacetate in intact, anaerobic, mitochondria via the rhein-sensitive, but rotenone- and arsenite-insensitive, NAD(P) transhydrogenase, measurements of the rates of carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive pH change in the presence of various oxoglutarate/isocitrate concentration ratios gave an -->H(+)/2e(-) quotient of 1.94+/-0.12 for outward proton translocation by the NAD(P) transhydrogenase. 4. Measurements with a K(+)-sensitive electrode confirmed that the electrogenicity of the NAD(P) transhydrogenase reaction corresponded to the translocation of one positive charge per acid equivalent. 5. Sluggish reversal of the NAD(P) transhydrogenase reaction resulted in a significant inward proton translocation. 6. The possibility that isocitrate might normally be oxidized via loop 0 at a redox potential of -450mV, or even more negative, is discussed, and implies that a P/O quotient of 4 for isocitrate oxidation might be expected.     

Impact of coenzyme regeneration on the performance of an enzyme-based optical biosensor: a computational study

A mathematical model of a reagent-less optical sensing scheme composed of an enzymatic reaction coupled to light-controlled photochemical coenzyme regeneration is described. The model is based on previous experimental work describing the regeneration of NADPH from NADP(+) by excited state thionine coupled to the oxidation of isocitrate by isocitrate dehydrogenase. The system is capable of repeated isocitrate measurements without the addition of exogenous coenzyme. The model is simulated using numerical integration to determine the effect of regeneration on the sensor sensitivity, response time and maximum sample throughput rate. Prediction of these effects without a model is difficult due to activation and inhibition of the dehydrogenase by both forms of the coenzyme. The regeneration parameters, including thionine concentration and thionine excitation pattern, are varied to determine optimal sensor conditions to maximize performance. A periodic regeneration approach is found to be superior to a continuous regeneration approach as the former maximizes sensitivity and minimizes response time in most cases. In addition periodic regeneration results in a maximum sample throughput frequency that is achieved at a single optimal thionine level and is independent of the analyte concentration. In contrast the optimal thionine concentration during continuous regeneration varies with the sample analyte concentration. These findings highlight the importance of designing controllable regeneration for dehydrogenase-based optical biosensors.     

A plug-in minielectrode for measurements in stirred photometric cuvettes

A device is described to perform potentiometric measurements with ion-sensitive electrodes in stirred photometric cuvettes. Its design allows to make additions to the reaction medium with microliter syringes during measurements. Originally, this plug-in electrode was designed to register the free Ca²⁺ concentrations in the incubation medium of mitochondrial suspensions during measurements of the free matrix Ca²⁺ concentration with a permeant fluorescent Ca²⁺ indicator. However, numerous other applications can be easily realized, such as the combination of mitochondrial light-scattering measurements and ion-transport measurements, the combination of the permeabilized cell technique with fluorescence measurements of intracellular organelles or simply the calibration of the fluorescence of Ca²⁺ indicators with a Ca²⁺ ion-sensitive minielectrode. Compared with the use of a second fluorescent indicator the use of an electrode has the advantage that the signal can be transformed into ion concentrations already during the measurements.     

Determination of the concentration of free Ca2+ in the presence of magnesium (or manganese) and chelating effectors of the NAD+-linked isocitrate dehydrogenase.

An equation was formulated that enabled the calculation of the concentration of free Ca²⁺ in a reaction medium containing calcium, magnesium (or manganese), and up to five metal chelators. The concentrations of all ionic and chelating species present in the incubation mixture could also be estimated. The equation, a polynomial to the sixth power with respect to free Ca²⁺, is expressed in terms of known equilibrium (formation) constants and concentrations of reactants. The calculated values of free Ca²⁺ were compared with those determined experimentally, using a calcium ion-specific electrode, and the two were found to be in excellent agreement. To illustrate the usefulness of the technique, we examined the inhibition by calcium of NAD⁺-linked isocitrate dehydrogenase, in the presence of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA), ADP, ATP, isocitrate, citrate, and magnesium. The effect of manganese on the concentration of free Ca²⁺ in solutions of calcium and EGTA was also deseribed. It was found that manganese, but not magnesium, displaced calcium from the Ca-EGTA cholate resulting in a much higher than expected concentration of free Ca²⁺. This raises questions about the inhibition of the isocitrate dehydrogenase by physiological concentrations of calcium previously reported.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

A rapid controller of temperature for use in determining Arrhenius profiles in biomembrane systems

To minimize artifacts in temperature-velocity (Arrhenius) profiles due to aging of preparations of biological membranes, a rapid controller of temperature was developed for spectrophotometric or polarographic (O₂ electrode) measurements. The reaction mixture is cooled or heated through contact with Peltier elements. One Pt temperature sensor in the cuvette or electrode holder controls current flow into the Peltier units, and another Pt temperature sensor in the reaction mixture is used to read out the sample temperature on a meter or recorder, and to provide feedback control. The sample temperature can be reproducibly set to within 0.1°C, with a noise level of 0.04°C or less; a change of 4°C takes 1 min.On leave at the Arrhenius Laboratory, University of Stockholm.     

Electrochemical analysis of copper ion using a Gly–Gly–His tripeptide modified poly(3-thiopheneacetic acid) biosensor

A novel biosensor harnessing a conducting polymer functionalized with a copper ion specific peptide proved to be highly effective for electrochemical analysis of copper ions. The developed sensor comprised a transducer based on a conducting polymer (poly(3-thiopheneacetic acid)) electrode and a probe (tripeptide, Gly–Gly–His) selectively cognitive of copper ions. For functionalization of the electrode, the carboxylic group of the polymer was covalently coupled with the amine group of the tripeptide, and its structural features were confirmed by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection infrared (ATR-IR) spectroscopy. The peptide modified polythiophene biosensor was used for the electrochemical analysis of various trace metal ions by square wave voltammetry. The electrode was found to be highly sensitive and selective to Cu²⁺ in the range of 0.02–20 μM with almost no cross binding to other metal ions such as Ni²⁺ and Pb²⁺. Furthermore, the developed sensor exhibited a high stability and reproducibility despite the repeated use of the sensor electrode and probe. With the advent of more diverse affinity bioprobes specific towards a broad range of analytes, the demonstrated strategy harnessing peptide modified polythiophene biosensor is likely to provide an excellent platform for the selective determination of trace amount of analytes whose detection is otherwise cumbersome.     

Proteinases and the instability of isocitrate lyase in extracts of developing flax seedlings

In extracts of flax seedlings 4 days after imbibition, isocitrate lyase activity is unstable in comparison to that in extracts from 2.5-day seedlings or to malate syntheses analysed at several stages of development. This instability in extracts of 4-day seedlings is especially pronounced when a large number of seedlings is homogenized per unit volume of Tris-Mg²⁺-EDTA-dithioerythritol buffer. However, isocitrate lyase can be stabilized when the resultant homogenate is diluted soon after seedling breakage. The pronounced instability in more concentrated extracts is not due to inadequate buffering by the homogenization medium, nor can it be due to polyphenols because added polyvinylpyrrolidone has no effect. Mixing of a heated supernatant from concentrated extract with dilute unheated extract yields the units of stable isocitrate lyase expected in the dilute extract, ruling out stoichiometric inactivation by a heat-stable component. The pronounced instability is attributed to the action of proteinases. A theoretical model assuming a decay process that is first order in isocitrate lyase and first-order in one or more proteinases is in reasonable agreement with the results. Malate synthase and NADP⁺-isocitrate dehydrogenase are much more stable in concentrated extracts prepared from 4-day flax seedlings. Isocitrate lyase is stable in concentrated extracts of 5-day watermelon seedlings, which is a developmental stage analogous to that for 4-day flax seedlings.     

Amperometric estimation of BOD by using living immobilized yeasts

Summary A microbial electrode consisting of immobilized living whole cells of yeasts, porous membrane and an oxygen electrode was prepared for continuous estimation of biochemical oxygen demand (BOD). Immobilized Trichosporon cutaneum was employed for the microbial electrode sensor for BOD. When a sample solution containing the equivalent amount of glucose and glutamic acid was injected into the sensor system, the current of the electrode decreased markedly with time until steady state was reached. The response time was within 18 min. A linear relationship was observed between the current decrease and the concentration below 41 mg l ^– of glucose and 41 mg l ^– glutamic acid (5-day BOD 60 mg l ^–). The current decrease was reproducible within ± 6% of the relative error when a sample solution containing 27 mg l ^– of glucose and 27 mg l ^– of glutamic acid (5-day BOD 40 mg l ^–) was employed. The microbial electrode sensor was applied to untreated waste waters from a fermentation factory. Good comparative results were obtained between BOD estimated by the microbial electrode and that determined by the conventional 5-day method (regression coefficient was 1.2). Furthermore, the effect of various compounds on BOD estimation was also examined. The current output of the microbial electrode sensor was almost constant for 17 d and 400 tests.     

Molecularly imprinted electrochemiluminescence sensor based on ZIF-8 doped with CdSe quantum dots for the detection of trace estriol

A composite of the metal–organic framework compound ZIF-8 doped with CdSe quantum dots (QDs) with sensitive and stable luminescence was synthesized, and a molecularly imprinted electrochemiluminescence (ECL) sensor was constructed based on this composite. The ZIF-8@CdSe molecularly imprinted ECL sensor combines the high sensitivity of ECL and the high selectivity of molecular imprinting to realize the sensitive and specific detection of estriol. CdSe QDs and gold nanoparticles were encapsulated within ZIF-8 to obtain the ZIF-8@CdSe QDs/GNP (ZIF@CdSe/GNP) composite. Subsequently, the GNPs were further loaded on the surface of this composite to obtain the GNP/ZIF@CdSe/GNP composite. l -Cysteine was used to immobilize the GNP/ZIF@CdSe/GNP composite on the surface of a gold electrode to obtain the GNP/ZIF@CdSe/GNP-modified gold electrode. A molecularly imprinted polymer (MIP) film was prepared on the surface of the modified electrode by electropolymerization with o-phenylenediamine as the functional monomer and estriol as the template molecule. After elution, estriol could be specifically recognized by the cavities. The readsorption of estriol by the MIP can prevent the coreactant from reaching the electrode surface through the cavities, thereby weakening ECL. A good linear relationship existed between the ∆ECL and lg C of estriol concentrations of 1 × 10⁻¹⁴ to 1 × 10⁻⁹ mol·L⁻¹. The detection limit was as low as 8.9 × 10⁻¹⁶ mol·L⁻¹. The sensor was applied in the determination of estriol in serum samples with a recovery of 97.0–102%.     

13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate – oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.     

New and Fast Method To Quantify Respiration Rates of Bacterial and Plankton Communities in Freshwater Ecosystems by Using Optical Oxygen Sensor Spots

A new method of respiration rate measurement based on oxygen luminescence quenching in sensor spots was evaluated for the first time for aquatic bacterial communities. The commonly used Winkler and Clark electrode methods to quantify oxygen concentration both require long incubation times, and the latter additionally causes signal drift due to oxygen consumption at the cathode. The sensor spots proved to be advantageous over those methods in terms of precise and quick oxygen measurements in natural bacterial communities, guaranteeing a respiration rate estimate during a time interval short enough to neglect variations in organism composition, abundance, and activity. Furthermore, no signal drift occurs during measurements, and respiration rate measurements are reliable even at low temperatures and low oxygen consumption rates. Both a natural bacterioplankton sample and a bacterial isolate from a eutrophic river were evaluated in order to optimize the new method for aquatic microorganisms. A minimum abundance of 2.2 × 10⁶ respiring cells ml⁻¹ of a bacterial isolate was sufficient to obtain a distinct oxygen depletion signal within 20 min at 20°C with the new oxygen sensor spot method. Thus, a culture of a bacterial isolate from a eutrophic river (OW 144; 20 × 10⁶ respiring bacteria ml⁻¹) decreased the oxygen saturation about 8% within 20 min. The natural bacterioplankton sample respired 2.8% from initially 94% oxygen-saturated water in 30 min. During the growth season in 2005, the planktonic community of a eutrophic river consumed between 0.7 and 15.6 μmol O₂ liter⁻¹ h⁻¹. The contribution of bacterial respiration to the total plankton community oxygen consumption varied seasonally between 11 and 100%.     

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10⁻⁹ M to 2.0 × 10⁻⁸ M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10⁻¹⁰ M. Additionally, the specificity and hybridization response of this simple sensor were investigated.     

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP⁺. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at 90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups. Received: 6 February 1997 / Accepted: 12 June 1997     

Rechargeable glucose electrodes for long-term implantation

A unique enzyme electrode was designed using glucose oxidase immobilized on fine graphite powder. The graphite-enzyme is in a fluid state which enables recharging of the system when the enzyme activity decreases, therefore allowing the system to have a long lifetime in a diabetic patient. The sensor was tested using glucose concentration in the range of 20–300 mg d⁻¹. The electrode construction includes a hydrophobic membrane and a platinum electrode for detection of hydrogen peroxide. The signal output is large with minimal noise when tested in buffer. The sensor has been performing at ambient temperature for 4 months when stored overnight at 4° C.     

Voltammetric detection of anti-HIV replication drug based on novel nanocomposite gold-nanoparticle–CaCO<Subscript>3</Subscript> hybrid material

A novel bionanocomposite, horse radish peroxidase- gold-nanoparticle–Calcium carbonate (HRP-AuNPs-CaCO₃), hybrid material was encapsulated by silica sol on a glassy carbon electrode (GCE). The fabricated modified electrode was used as a novel voltammetric sensor for electrochemical sensing of anti-HIV replication drug i.e. deferiprone. The surface morphology of the modified electrode was characterized by scanning electron microscopy (SEM). Results obtained from the voltammetric measurements show that HRP-AuNPs-CaCO₃ modified GCE offers a selective and sensitive electrochemical sensor for the determination of deferiprone. Under experimental conditions, the proposed voltammetric sensor has a linear response range from 0.01 to 10,000 μM with a detection limit of 0.01 μM. Furthermore, the fabricated sensor was successfully applied to determine deferiprone level in spiked urine and serum samples.     

Excretion of Glycolate, Mesotartrate and Isocitrate Lactone by Synchronized Cultures of Ankistrodesmus braunii

Fixation of ¹⁴CO₂ by synchronized cultures of Ankistrodesmus braunii was highest for young growing cells, low for mature cells, and lowest for dividing cells. The amount of ¹⁴C excreted during photosynthesis followed the same trend. Cells at the end of the growing phase, after 10 hours of a 16-hour light phase, excreted nearly 35% of the total ¹⁴C fixed as one product, glycolate. Dividing cells from the dark phase, when tested in the light, excreted only 4% as much glycolate-¹⁴C as the young growing cells. Dividing cells also excreted as much mesotartrate as glycolate and also some isocitrate lactone and an unidentified acid. None of these excreted acids were found inside the cells in significant amounts. Methods for isolation and identification of the excreted acids are present. With ¹⁴C-labeled algae, it was shown that the excretion of glycolate was light-dependent and inhibited by 1,1-dimethyl-3-(p-chlorophenyl) urea. The excretion of labeled mesotartrate, isocitrate lactone, and an unknown acid, but not glycolate, also occurred in the dark. The excreted mesotartrate was predominantly carboxyl-labeled even after long periods of ¹⁴CO₂ fixation. Since glycolate is known to be uniformly labeled, glycolate could not be the precursor of the carboxyl-labeled mesotartrate. The reason for the specific excretion of glycolate, mesotartrate, and isocitrate lactone is not known, but the metabolism of all three acids by the algae may be limited and each can form dilactides or lactones by dehydration. In this context isocitrate lactone was excreted rather than the free acid.     

Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen ^1-7. The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion². Among the relatively few in situ surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation^8-12. It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition^{8, 10, 13, 14} ("coking") and sulfur poisoning^{11, 15} and the manner in which surface modifications stave off this degradation¹⁶. The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM and STM, other properties such as local electronic states, ion diffusion coefficient and surface potential can also be investigated^17-22. In this work, electrochemical measurements, Raman spectroscopy, and SPM were used in conjunction with a novel test electrode platform that consists of a Ni mesh electrode embedded in an yttria-stabilized zirconia (YSZ) electrolyte. Cell performance testing and impedance spectroscopy under fuel containing H₂S was characterized, and Raman mapping was used to further elucidate the nature of sulfur poisoning. In situ Raman monitoring was used to investigate coking behavior. Finally, atomic force microscopy (AFM) and electrostatic force microscopy (EFM) were used to further visualize carbon deposition on the nanoscale. From this research, we desire to produce a more complete picture of the SOFC anode.     

Detection and Monitoring of Toxic Chemical at Ultra Trace Level by Utilizing Doped Nanomaterial

Composite nanoparticles were synthesized by eco-friendly hydrothermal process and characterized by different spectroscopic techniques. All the spectroscopic techniques suggested the synthesis of well crystalline optically active composite nanoparticles with average diameter of ∼30 nm. The synthesized nanoparticles were applied for the development of chemical sensor which was fabricated by coating the nanoparticles on silver electrode for the recognition of phthalimide using simple I–V technique. The developed sensor exhibited high sensitivity (1.7361 µA.mM⁻¹.cm⁻²), lower detection limit (8.0 µM) and long range of detection (77.0 µM to 0.38 M). Further the resistances of composite nanoparticles based sensor was found to be 2.7 MΩ which change from 2.7 to 1.7 with change in phthalimide concentration. The major advantages of the designed sensor over existing sensors are its simple technique, low cost, lower detection limit, high sensitivity and long range of detection. It can detect phthalimide even at trace level and sense over wide range of concentrations. Therefore the composite nanoparticals would be a better choice for the fabrication of phthalimide chemical sensor and would be time and cost substituted implement for environmental safety.     

The control of isocitrate oxidation by rat liver mitochondria

1. The factors capable of affecting the rate of isocitrate oxidation in intact mitochondria include the rate of isocitrate penetration, the activity of the NAD-specific and NADP-specific isocitrate dehydrogenases, the activity of the transhydrogenase acting from NADPH to NAD(+), the rate of NADPH oxidation by the reductive synthesis of glutamate and the activity of the respiratory chain. A quantitative assessment of these factors was made in intact mitochondria. 2. The kinetic properties of the NAD-specific and NADP-specific isocitrate dehydrogenases extracted from rat liver mitochondria were examined. 3. The rate of isocitrate oxidation through the respiratory chain in mitochondria with coupled phosphorylation is approximately equal to the maximal of the NAD-specific isocitrate dehydrogenase but at least ten times as great as the transhydrogenase activity from NADPH to NAD(+). 4. It is concluded that the energy-dependent inhibition of isocitrate oxidation by palmitoylcarnitine oxidation is due to an inhibition of the NAD-specific isocitrate dehydrogenase. 5. Kinetic studies of NAD-specific isocitrate dehydrogenase demonstrated that its activity could be inhibited by one or more of the following: an increased reduction of mitochondrial NAD, an increased phosphorylation of mitochondrial adenine nucleotides or a fall in the mitochondrial isocitrate concentration. 6. Uncoupling agents stimulate isocitrate oxidation by an extent equal to the associated stimulation of transhydrogenation from NADPH to NAD(+). 7. A technique is described for continuously measuring with a carbon dioxide electrode the synthesis of glutamate from isocitrate and ammonia.