The effect of Ag+ on arginine kinase: inhibition kinetics.

We studied the effects of Ag(+) on arginine kinase (AK) from Fenneropenaeus chinensis. Ag(+) inactivated the activity of AK in a dose dependent manner (IC(50) = 15 microM). Kinetic studies showed that the inactivation of AK by Ag(+) was reversible and occurred in a noncompetitive inhibition manner (K(i) = 2.8 microM). Spectroflurorimetry results showed that Ag(+) did not induce conspicuous tertiary structural changes in AK at the corresponding concentration ranges of inactivation studies. However, the secondary structure measured by circular dichroism was slightly changed by Ag(+). Taken together, these data suggest that the active site of AK is flexible, with the complete loss of activity occurring prior to significant changes in overall structures. Our study provides important insight into the inhibitory mechanism of Ag(+) on AK and increases our understanding of the influence of Ag+ on the mechanism of this metabolic enzyme.

Cultivation and modelling of encapsulated Saccharomyces cerevisiae in NaCS-PDMDAAC polyelectrolyte complexes.

The cultivation of encapsulated S. cerevisiae in NaCS-PDMDAAC polyelectrolyte complexes was studied. The results showed that the encapsulated microorganisms had the same growth trends as in its free cell culture and, thus, NaCS-PDMDAAC microcapsules were suitable for the encapsulation of these biological substances. The encapsulated S. cerevisiae cells were fermented sequentially for 16 batches. The highest cell density in the capsules reached 2.64 x 10(10) cells mL(-1) and the ethanol concentration was 47.0 g L(-1). A model of the cultivation of the encapsulated S. cerevisiae was developed.

An improved procedure for production of human epidermal growth factor from recombinant E. coli.

An improved procedure for the fermentation and purification of human epidermal growth factor (hEGF) was developed. Recombinant Escherichia coli HB-101 [lacUV5omp08hEGF] harboring plasmid lacUV5omp08hEGF encoding hEGF was used in fermentation to increase levels of hEGF. Medium composition, and the levels of inoculum, inducer (isopropyl-beta-D-thiogalactoside) and ampicillin were optimized with respect to volumetric fermentation of hEGF. As a result, the hEGF concentration reached a high value of 242 mg l(-1) and the amount of heterogeneous protein decreased by 62% compared with that before optimization in batch fermentation. High-quality hEGF was purified from the fermentation culture by centrifugation, salting-out, resuspension, recentrifugation and finally gel chromatography on a Grad-iFrac System using Sephadex G-50 superfine. The purity of hEGF and the total yield were more than 94% and higher than 36%, respectively, and SDS-PAGE of the purified hEGF demonstrated a single band corresponding to an hEGF standard. In particular, a very important phenomenon was found, i.e. that the amount of heterogenous protein in fermentation broths cultured in media with high concentrations of lactose is far less than that cultured in media with high concentrations of glucose.

[Genetical effect of different rye chromosomes on the acid phosphatase (Acph) secretion of common wheat roots under phosphorus starvation conditions].

The effects of different rye chromosomes on Acph secretion of common wheat roots under P starvation conditions were studied by using a set of Chinese Spring-Imperial (CS-IMP) alien addition lines as materials. The Acph activity measurement results showed that P starvation is an induction factor for Acph gene expression; Different chromosomes of Imperial rye in Chinese Spring background has different effects on the secretion of Acph by corresponding addition line roots. Among them, chromosome 1R had the strongest promoting effect; The IEF diagram of Acph isozymes clearly demonstrated that chromosome 1R in rye genome carries P starvation inducible Acph gene(s).

A micro carbon electrode for nitric oxide monitoring.

A nitric oxide (NO) probe, consisting of a micro carbon fiber working electrode, 10 microns diameter, a platinum counter electrode, and a silver/silver chloride (Ag/AgCl) reference electrode, has been developed. The carbon fiber working electrode is covered with a Nafion cation exchange membrane. Using differential pulse voltammetry (DPV), we found the NO to N2O reduction current peak at approximately -1.35 V versus Ag/AgCl. This has been reported by others. The DPV current outputs are linearly related to dissolved NO concentrations [NO] in the 2-10 microM range. Catecholamines were found not to interfere with the reduction signal. The Nafion membrane also prevents interference by NO2-, NO3-, and amino acids at normal physiologic pH (pH 7.4). The effects of O2 are accounted for through sampling and subtracting background currents from the peak current. To increase sensitivity and shorten response time, a method of integrated pulse amperometry (IPA) was used for the study. The IPA charge outputs (delta C) are linear to the dissolved [NO] in the 50-350 nM range. The carbon fiber electrode has the potential of being miniaturized to a smaller electrode, allowing detection of NO released from the subendothelial space.

A microchip glucose sensor.

A major problem in development of a glucose sensor for use in an implantable artificial pancreas is the lack of reproducibility in signals from sensor to sensor. Each glucose sensor fabricated with currently used methods has a unique response to varying levels of glucose concentration and thus needs to be individually calibrated before use. We have adapted microchip manufacturing techniques for the fabrication of electrochemically based glucose sensors with standardized and reproducible function. Scanning electron microscopic study of the resulting electrode surfaces shows them to be smooth and featureless at all levels of magnification. X-ray diffraction analysis of the electrodes indicates preferential exposure of the [1,1,1] crystal interface. Cyclic voltammetry evaluation of initial sensor response to varying glucose concentrations shows excellent sensor to sensor reproducibility for all sensors made with the same underlayment. Sensors made with titanium underlayment appear to be more differentiated and thus more sensitive to variations in glucose concentration than are sensors with chromium underlayment. Although the initial response of microchip glucose sensors appears to be standardized and reproducible, additional development of an appropriate electrical insulation material is required before long-term study of signal stability is feasible.

A nitric oxide sensor using reduction current.

Nitric oxide (NO) has a wide range of biologic activity. Methods commonly used for the detection of biologically derived NO are indirect and measure only the amount of NO released during an interval of time. An electrochemical method available is capable of being direct and continuous but is subject to interference. The recent explosion of scientific research into NO activity requires better methods of NO detection. This article reports a new NO electrochemical sensing method and sensor design. The tip of the sensor is covered with a hydrophobic membrane and contains an internal electrolyte. Platinum is used for the working and counter electrodes and silver/silver bromide (Ag/AgBr) for the reference electrode. The components of the internal electrolyte are potassium bromide and sulfuric acid. The NO that diffuses to the working electrode is first oxidized to NO+; the NO+ is reduced to NO; and the reduction current is determined. An integrated pulsed amperometric method is used to achieve the redox of NO and the measurement and integration of the reduction current. The results show that the NO sensor is sensitive and has a rapid response and less interference.

Zeolitic ammonium ion exchange for portable hemodialysis dialysate regeneration.

Ammonia removal from a recirculating dialysate stream is a major challenge in developing a truly portable, regenerable hemodialysis system. Three zeolites, type F, type W, and clinoptilolite, were found to have good ammonia ion exchange capacity with linear equilibrium ion exchange coefficients of 0.908, 0.488, and 0.075 L/g, respectively. The linear equilibrium ion exchange coefficient relates dialysate ammonia concentration (mumol/L) to the amount of ammonia absorbed by zeolite (mumol/g) at equilibrium. Ammonia uptake by zeolite powders was fast, with equilibrium reached within 15 sec. Zeolite ammonia ion exchange and regeneration through multiple cycles was studied using an ion exchange column containing clinoptilolite pellets. Zeolite ion exchange capability was regenerated by flushing the column with 2 mol/L sodium chloride after an ion exchange run. The column maintained ammonia ion exchange capacity through six ion exchange/regeneration cycles, demonstrating multiple dialysis use possibilities. Atomic absorption spectroscopy of the column effluent showed no detectible (< 1 part per million) Si or Al leached from the zeolite.

Ammonia transport across hydrophobic membranes. Application to dialysate regeneration.

Removal of ammonia from a recirculating dialysate buffer in a portable hemodialysis application can be achieved by countercurrent, gas phase ammonia transfer across a hydrophobic membrane into an acid solution. Ammonia transfer fluxes as high as 0.076 mumol/sec/m2 have been achieved using a Sarns Turbo Membrane Oxygenator (Sarns-3M, Ann Arbor, MI) with a 1.9 m2 membrane surface area (0.145 mumol/sec actual rate). A simple physical model based upon ammonia desorption at the gas-dialysate buffer interface in a membrane pore, ammonia diffusion through the gas filled pore, and subsequent ammonia absorption at the gas/acid interface side of the pore quantitatively describes the experimental data. The ammonia transfer rate is most dependent upon dialysate buffer pH (higher pH promoting transfer rate) and ammonia concentration in the dialysate buffer (higher concentrations promoting transfer rate). A 500 fold improvement in transfer rate, however, will be required for clinical application.

Interference of glucose sensing by amino acids.

Interference by membrane permeable substances on nonspecific electrodes is a major problem in glucose sensing. Alanine, lysine, phenylalanine, and cystine were chosen for study to gain insight into this problem. These compounds represent the classes of mono-amino aliphatic, di-amino aliphatic, aromatic, and sulfur containing amino acids, respectively. Cyclic voltammetry experiments were performed using a Pt electrode (1.77 mm2). The reductive current of glucose at -0.750 V versus Ag/AgCl was measured with increasing concentrations of interfering substances in Krebs-Ringer phosphate buffer (pH 7.4) at 37 degrees C. Experimental results have shown that these amino acids have an inhibitory effect on the glucose signal. An important finding was that the interferences from phenylalanine and cystine were more pronounced than those of lysine and alanine. An initial drop in the glucose signal was seen at less than 2.0 mg/dl of alanine or lysine and at less than 0.5 mg/dl of phenylalanine or cystine. Additional increase in the concentrations of interfering substance did not cause further appreciable signal reduction. The results confirm that glucose sensing using a non-specific electrode is possible in fluids containing interfering substances such as amino acids.

Voltage polarity relay--optimal control of electrochemical urea oxidation.

Voltage polarity relay (VPR) is shown to optimize the urea oxidation rate and urea current utilization under constant current conditions in direct electrochemical urea oxidation. Direct electrochemical urea oxidation is characterized by reversible deactivation of the working electrode due to oxidation products remaining on the surface and the requirement that the working electrode potential remain below about 1.1 V relative to Ag/AgCl in order to prevent undesirable secondary electrochemical oxidations. The VPR method monitors the potential of the working electrode relative to a suitable reference and changes system polarity when the upper potential set limit is reached. Thus, what was the working electrode becomes the counter electrode and vice versa. Since urea oxidation products are desorbed from the counter electrode when its potential drops below about -0.6 V relative to Ag/AgCl, alternating electrode functions between working and counter provides cyclic electrode regeneration and continuous urea oxidation. VPR is believed to optimize constant current control for any electrochemical system that exhibits behavior similar to direct electrochemical urea oxidation.

A thin-film glucose electrode system with compensation for drift.

Thin-film platinum electrodes have been prepared for use in a system for measurement of glucose. Cyclic voltammetry was used to test the electrodes in phosphate and bicarbonate solutions, and in the dialysate of human serum. The peak current output for glucose concentrations between 50-300 mg/dl in a phosphate buffer, and 100-250 mg/dl in the dialysate of human serum, were linear. Two of the three inflection points occurring in the voltammograms are invariant with changes in scan rate, pH, CO2, O2, and glucose. A method for using these points to compensate for voltage drift and current shift is discussed.

Electrochemical glucose sensing at low potentials.

An electrochemical sensing method was studied, employing cyclic voltammetry at smooth Pt-electrodes in the low potential region. It has certain advantages over methods that employ higher potentials or constant voltage amperometry. There are also advantages over methods employing active enzymes or other unstable elements. In addition to a specific scanning potential we have studied voltage-delay-pulsing techniques for electrode rejuvenation and innovative methods of creating analyte-selective covering membrane. At potentials below -0.6 V vs Ag/AgCl, peaks of adsorbed glucose species have been observed in cyclic voltammograms. These peaks at about -0.7 V (oxidation peak) and -0.8 V (reduction peak) have yielded reproducible current-glucose concentration relationships, linear through the region of clinically important glucose concentrations (50-300 mg/dl) and stable over time. Since these potentials are not in the redox range of many potentially interfering substances, selectivity is enhanced.

Orbital reconstruction with proplast.

Proplast was implanted in 15 cases of orbital reconstruction. Seven cases were orbital floor fractures, four cases were phthisical enophthalmos, and four cases were anophthalmic enophthalmos. In orbital floor fracture cases, thin (2-mm) Proplast was placed onto the fractured orbital floor after the incarcerated orbital tissue was released. To correct the enophthalmic socket, Proplast was shaped into small pieces (10 x 5 x 2 mm), then inserted into the subperiosteal space from all directions to fill up the orbital cavity. The results of these 15 cases were satisfactory. The average follow-up period is 13 months. The increased orbital volume seems less than the volume of the implanted Proplast; therefore, we suggest an 80-90% overcorrection rate in accordance with the computer measurements. In sum, Proplast serves as a very good material for orbital floor reconstruction and volume replacement.

Immobilized glucose oxidase in the potentiometric detection of glucose.

Previous work has shown that glucose oxidase can be immobilized on platinum to give an electrode that responds potentiometrically to glucose over the clinically useful range of about 10-250 mg glucose/100 mL. The present studies were carried out with electrochemically pretreated platinum and with gold or porous graphite substituted for the platinum support. The presence of the enzyme gave a significantly enhanced potentiometric response over that obtained with the bare support for both the pretreated platinum and the porous graphite, but not with gold. However, with platinum the potentiometric response became more negative with increasing glucose concentration. With porous graphite, the potential changed in the positive direction as the glucose concentration was increased. Hysteresis was demonstrated for the platinum-enzyme electrode. Mass transfer measurements with a rotating ring-disc electrode (RRDE) showed measurable diffusional resistances to the transport of a model electroactive compound (potassium ferrocyanide) through a matrix of immobilized enzyme attached to the disc of the RRDE. These results are part of a larger study to define the source of the potentiometric response by examining the roles of the support and the mass transfer resistances through the immobilized enzyme matrix.

Glucose concentration at possible sensor tissue implant sites.

It is generally acknowledged that the ideal automatic insulin infusion system would be a closed loop that metered insulin delivery in response to a feedback sensor such as an implantable glucose detector. Most current efforts are aimed at a transducer located within the blood vascular tree. We believe that the blood constitutes an especially hostile environment for such a device. The possibility of placing the sensor with a reasonably rapid response to dynamic alterations in glucose metabolism in a space containing fluid outside the bloodstream was studied. The subcutaneous extracellular space, peritoneum, pleura, and pericardium were included. Generally, the concentration of glucose ranged between 50 and 115 mg/dl under steady-state conditions. The experimental design did not permit monitoring rapid responses to artificially induced dynamic changes. There were several situations where lower values were recorded, suggesting that a wide range of concentrations might occur. The authors have concluded that these experimental results are compatible with the possibility of a suitable locus for the glucose sensor in the extracellular, extravascular space.

Potentiometric measurement of glucose concentration with an immobilized glucose oxidase/catalase electrode.

A series of enzyme electrodes for measurement of glucose have been constructed. The electrodes contain glucose oxidase immobilized on platinum, either with or without co-immobilization of catalase. When placed in buffered glucose, the enzyme electrodes show a potentiometric response to glucose with respect to a Ag/AgCl reference electrode. This response is reproducible in the physiologic range of glucose concentrations. The immobilization technique, some of the environmental variables such as oxygen concentration and pH, and several compounds that might interfere with the selectivity of the enzyme electrodes for glucose have received preliminary study. This direct potentiometric approach is undergoing further evaluation to determine the basic electrochemical mechanism responsible for the potentiometric signal and whether it can be adapted for continuous in vivo monitoring of the glucose concentration in body fluids.

Immobilized enzyme electrodes for the potentiometric measurement of glucose concentration: immobilization techniques and materials.

Glucose oxidase, catalase, and bovine serum albumin were co-immobilized with glutaraldehyde around a platinum screen or around a single platinum-iridium wire. The potential difference between this dual enzyme electrode and a Ag/AgCL reference electrode was proportional to the logarithm of the glucose concentration over the range from 10 to about 150 mg glucose per 100 ml in buffered solution at pH 7.4 and 37 degrees C. The enzyme electrode responded in serum only if coated with a semipermeable film, such as cellulose acetate, to exclude serum macromolecules. The potentiometric results were similar to those obtained with the two enzymes co-immobilized in polyacrylamide gel around a platinum screen or with only one of the enzymes, glucose oxidase, covalently coupled to a platinum screen. The results so far suggest that glucose for development of a continuous in vivo glucose sensor.