Continuous Buffer Research Articles

Textured buffer layers for YBCO coated conductors by continuous sol-gel processing

In order to develop alternative buffer layers for YBCO coated conductors, RE/sub 2/O/sub 3/ (RE=La, Pr, Sm, Eu, Gd, Tb, Ho, Yb, and Lu) thin films were studied as candidate materials. Buffer layers were grown on Ni tapes using organometallic compounds that are 2,4-pentanedionates, isopropoxides, and acetates by a continuous, non-vacuum sol-gel process. Films were annealed at 900/spl deg/C-1200/spl deg/C under 4%H/sub 2/-Ar gas mixture. The buffer layers were characterized by means of ESEM, SEM, EDS, XRD and pole figure analysis. Textured, pinhole free crack free, continuous buffer layers were obtained by using 2,4-pentanedionates.

Review article: the control of gastric acid and Helicobacter pylori eradication.

This review focuses on the gastric acid pump as a therapeutic target for the control of acid secretion in peptic ulcer and gastro-oesophageal reflux disease. The mechanism of the proton pump inhibitors is discussed as well as their clinical use. The biology of Helicobacter pylori as a gastric denizen is then discussed, with special regard to its mechanisms of acid resistance. Here the properties of the products of the urease gene clusters, ureA, B and ureI, E, F, G and H are explored in order to explain the unique location of this pathogen. The dominant requirement for acid resistance is the presence of a proton gated urea transporter, UreI, which increases access of gastric juice urea to the intrabacterial urease 300-fold. This enables rapid and continuous buffering of the bacterial periplasm to approximately pH 6.0, allowing acid resistance and growth at acidic pH in the presence of 1 mM urea. A hypothesis for the basis of combination therapy for eradication is also presented.

Enzymatic resolution of (S)-(+)-naproxen in a trapped aqueous-organic solvent biphase continuous reactor.

A trapped aqueous-organic biphase system for the continuous production of (S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen methyl ester by Candida rugosa lipase in a trapped aqueous-organic biphase system. The reaction has been carried out in a laboratory-scale continuous-flow stirred tank reactor (CSTR). The staring material has been supplied in and remaining substrate recovered by organic phase. YWG-C(6)H(5), a poorly polar synthetic support, has been employed to immobilize the lipase and to restrict the aqueous phase. Lipase immobilized on YWG-C(6)H(5) containing aqueous phase has been added into the CSTR to catalyze the hydrolysis. A dialysis membrane tube containing a continuous flow closed-loop buffer has been applied in the CSTR for the extraction of product and recruiting of the aqueous part consumed. Various reaction conditions have been studied. The activity of immobilized enzyme was effected by the polarity of support, the substrate concentration, logP value of organic phase and the product inhibition. At steady-state operating conditions, an initial conversion of 35% has been obtained. The CSTR was allowed to operate continuously for 60 days at 30 degrees C with a 30% loss of activity. The hydrolysis reaction yielded (S)-(+)-Naproxen with >90% enantiomeric excess and overall conversion of 30%.

Fanout optimization using bipolar LT-trees

In this paper we present a new fanout optimization algorithm which is particularly suitable for digital circuits designed with submicron CMOS technologies. Restricting the class of fanout trees to the so-called bipolar LT-trees, the topology of the optimal fanout tree is found by means of a dynamic programming algorithm. The buffer selection is in turn performed by using a continuous buffer sizing technique based on a very accurate delay model especially developed for submicron CMOS processes. The fanout trees can distribute a signal with arbitrary polarity from the root of the tree to a set of sinks with arbitrary required time, required minimum signal slope, polarity and capacitive load. These trees can be constructed to maximize the required time at the root or to minimize the total buffer area under a required time constraint at the root. The performance of the algorithm shows several improvements with respect to conventional fanout optimization methods. More precisely, the average improvements in area and delay of the optimized circuits, using a standard library which contains tapered and nontapered buffers with different strengths, are 30% and 7%, respectively.

Electrophoretic focusing preconcentration technique using a continuous buffer system for capillary electrophoresis

Detection and analysis of dilute small volume samples can be achieved by preconcentration techniques. Published techniques use specialized discontinuous buffer systems (sample stacking, field-amplification, and isotachophoresis) or incorporation of a chromatographic preconcentration chamber–capillary. By carefully exploiting flow and electric fields, preconcentration of analytes can be achieved without the need for discontinuous buffer systems or physical chromatographic devices. This focusing is achieved by independently controlling pressure flow and electrophoretic migration of analytes. Initiation of a voltage field at the immediate entrance of the capillary combined with adjusting bulk flow equal and opposite to the electrophoretic migration of the analytes results in preconcentration. Data are presented indicating an increase in local concentration of 200 nm carboxylate modified latex spheres within the immediate volume of the capillary entrance (specifically ∼15 pL) using laser-induced fluorescence detection. ©2000 John Wiley & Sons, Inc. J Micro Sep 12: 98–106, 2000

Use of nonvolatile buffers in liquid chromatography/mass spectrometry: advantages of capillary-scale particle beam interfacing.

The use of nonvolatile buffers like phosphate and citrate is usually incompatible with mass spectrometric detection or may heavily interfere with ion generation. In the case of conventional HPLC flow rates, the continuous buffer deposition produces fouling of the mass spectrometer ion source and may worsen the performance of any LC/MS interface as well. Our research group demonstrated that reducing the mobile phase flow rate in a particle beam interface improves the nebulization and enhances the overall performance. We have also assumed that such a modification may better handle potentially instrument harmful solvents or nonvolatile HPLC buffers. Since the presence of salts does not interfere with the electron ionization process, the particle beam interface can be considered, in principle, particularly suitable for those chromatographic separations that benefit from nonvolatile buffers. The microscale flow rate interface, developed in our laboratory, generates an aerosol from 1 microL/min of mobile phase flow rate. Under these conditions, the absolute amount of a nonvolatile buffer actually introduced into the system is approximately 1/1000 of that carried by a conventional HPLC column, slowing its deposition. This assumption was verified with a real-world application requiring the use of a phosphate buffer at a maximum concentration of 10 mM. It involved the determination of dexamethasone, an anti-inflammatory drug, in human blood after its administration to a patient. Several crucial mass spectral and chromatographic parameters were monitored during a 35-day period of intense use. Neither appreciable signal modification nor evident buffer deposition was observed during the test period, with only a normal and limited run-by-run and day-by-day variation of the instrument response.

Mnium orientale sp. nov. from Japan Is Morphologically and Genetically Distinct from M. hornum in Europe and North America

Mnium orientale sp. nov. from Japan Is Morphologically and Genetically Distinct from M. hornum in Europe and North America

Fast simulation of Markov fluid models

In this paper we study continuous flow finite buffer systems with input rates modulated by Markov chains. Discrete event simulations are applied for estimating loss probabilities. The simulations are executed under a twisted version of the original probability measure (importance sampling). We present a simple rule for determining a new measure, then show that the new measure matches the ‘most likely' empirical measure that we expect from large deviations arguments, and finally prove optimality of the new measure.

Fast simulation of Markov fluid models

In this paper we study continuous flow finite buffer systems with input rates modulated by Markov chains. Discrete event simulations are applied for estimating loss probabilities. The simulations are executed under a twisted version of the original probability measure (importance sampling). We present a simple rule for determining a new measure, then show that the new measure matches the ‘most likely' empirical measure that we expect from large deviations arguments, and finally prove optimality of the new measure.

Upside-Down Stopped-Flow Electrofractionation of Complex Protein Mixtures

The excellent resolution of SDS–PAGE in protein analysis stimulated the creation of various preparative devices. The main approach used in these devices is the construction of a elution chamber in the lower end of the polyacrylamide gel cylinder or plate. Although this continuous lower buffer flow electrofractionation system serves as an acceptable preparative electrophoresis, some limitations to this approach exist. There is strong dilution of protein zones by the eluting buffer, which drastically restricts the sensitivity of the determination of minor proteins, and the restricted current flow caused by electric resistance arising from the column holder prevents application to purification of complex protein mixtures. To overcome these problems, the upside-down stopped-flow electrofractionation system (UDSFE) was designed. The necessary quantity of fraction is drawn with a pipet in a small volume from just above the gel cylinder. This invention improves the possibility of electrofractionation of deluted complex protein mixtures. The efficiency of this technique is demonstrated by purification a protein kinase from rat liver. The method has also been successfully used for purification of error-correcting 3′–5′ exonuclease.

Reproducibility of mobility in gel electrophoresis.

The quantitative exploitation of gel electrophoresis to yield molecular and gel fiber properties rests on the assumption that mobility is characteristic of the macromolecule migrating as a band and is a physical constant for any system defined by pH, ionic strength and temperature. This assumption has not been tested intra-experimentally in previous literature. With the commercial introduction of automated gel electrophoresis apparatus, the collection of multiple mobility data during a single run without additional expense of labor has made it possible to test the assumption. As a start, we undertook that test for three proteins and their sodium dodecyl sulfate (SDS) derivatives, in agarose and polyacrylamide gel electrophoresis, various field strengths, continuous and discontinuous buffers, as well as intra- and interexperimentally. It was found that in agarose gel electrophoresis conducted in a single buffer, the standard deviation of mobility over a wide concentration range ranges intra-experimentally from 0.2 to 1.3% for two globular proteins and 1.4 to 5.3% for the same proteins derivatized with SDS. Interexperimentally, it was 3% in the single case tested to date. The standard deviation in polyacrylamide appears to be higher, varies in inverse relation to the mobility value, i.e. increases with gel concentration in the range of 11 to 19%T, and varies substantially between the two SDS-proteins investigated. Mobility in a discontinuous buffer system decreases continuously due to the decreasing leading phase/trailing phase ratio along the migration path. The decrease is sharpest in the "nonrestrictive" stacking gel.

Infusion of the GABAergic antagonist bicuculline into the medial septal area does not block the impairing effects of systemically administered midazolam on inhibitory avoidance retention

This experiment investigated the effect of intraseptal administration of the GABAergic antagonist bicuculline methiodide on benzodiazepine-induced amnesia. Male Sprague-Dawley rats were implanted with cannula aimed at the medial septal area and allowed to recover for 1 week. Ten minutes prior to training in a continuous multiple trial inhibitory avoidance task, buffer solution or bicuculline methiodide (56 or 100 pmol/0.5 microliter) was injected into the medial septal area. This infusion was immediately followed by systemic (ip) administration of saline or midazolam (1.5 or 3.0 mg/kg). In comparison with saline controls, animals given the higher dose of midazolam (3.0 mg/kg), required more trials to reach acquisition criterion (remaining in the starting chamber for 100 s). This midazolam-induced acquisition deficit was blocked by an intraseptal infusion of bicuculline methiodide (100 pmol). On a 48-h retention test the performance of animals given either dose of midazolam was significantly impaired relative to vehicle controls. Furthermore, although intraseptal infusion of bicuculline methiodide prior to systemic injection of midazolam blocked the midazolam-induced acquisition impairment, bicuculline did not block the midazolam-induced retention impairment. These results suggest that although the medial septal area may be involved in midazolam-induced acquisition deficits, this area is not a critical site of action for benzodiazepine-induced effects on inhibitory avoidance retention.

Stopped Lower Buffer Flow Electrofractionation: Simple Electrofractionation for Complex Protein Mixture

The standard electrofractionation technique simulated the liquid chromatography to create a continuous elution system. Though this continuous lower buffer flow electrofractionation system served as an excellent model for preparative electrophoresis, the restricted current flow caused by electric resistance arising from the column holder prevented the application to the purification of complex protein mixtures. To overcome the electric current problem, the stopped lower buffer flow electrofractionation (SFE) system is designed to work without using a column holder and use prestained markers as the detection tool of protein position during the electrofractionation process. Through this improvement, the SFE system widens the availability of electrofractionation to the purification of complex and low-concentration protein mixtures. In this report, the efficiency of this technique is shown through the partial purification of a 32P-labeled protein in the rat liver microsome.

Looking at Bands from Another Side

In high-percentage gels run in the submerged electrophoresis mode separated DNA bands were bent, regardless of whether the gel was prepared from polyacrylamide, poly-N-acryloyl-tris(hydroxymethyl)aminomethane, or hydroxyethylated agarose. This declination from the vertical axis is the major reason for the appearance of diffuse bands after recording by a camera positioned vertically above the gel. Five major factors which could cause the bending, including an inhomogeneous electric field, electroendosmosis, nonuniform heat dissipation, different effective porosity across the gel thickness, and different conductivity in the gel and electrophoresis buffer, were investigated. The results indicate that the major cause is the difference in the ratio of current density and specific conductivity between the gel and the running buffer. When that ratio was adjusted by empirically optimizing gel ionic composition, the bending could be prevented or greatly diminished. Other results described in this work suggest that electrophoretic systems in which gels are polymerized and run in the same buffer, frequently referred to as "continuous buffer systems," may not provide a continuous, constant electric field. Furthermore, the pH change regularly observed in circulated running buffers could be related to difference in the amount of current transported by buffer anions and cations. A buffer in which current was equally transported by anions and cations showed no detectable pH change after prolonged electrophoresis.

Fluid-melt-rock interaction in mafic eclogites and coesite-bearing metasediments: Constraints on volatile recycling during subduction

The question as to whether the metasomatizing-slab agent needed to form island-arc magmas is an aqueous fluid or a hydrous melt requires a knowledge of fluid-melt-rock interaction during high-pressure low-temperature metamorphism. In the Western Alps, typical “anhydrous” mafic eclogites (Monviso Massif) and coesite-bearing metasediments (Dora-Maira Massif) have experienced a prograde P- T path characteristic of a mature (cold) subduction zone. Microstructural, petrologic, geochemical and fluid inclusion studies show that fluid flow was limited during eclogite and high-grade blueschist facies metamorphism. Most of the fluids were driven off the rocks at pressures lower than 1 and 1.6 GPa, respectively. With increasing pressure and temperature, the remaining fluid phase was released during crystal plastic flow processes or devolatilisation reactions but retain the host rocks to form veins, partial melts or dense hydrous silicates. Tectonic erosion at modern convergent margins delivers large amounts of terrigenous sediments (KMASH system) in subduction zones. In contrast to the basalt and peridotite systems, the KMASH system is characterized by a wide range of pressure-sensitive reactions which allow the reconstruction of dehydration/hydration depths in the subducted slab. Independently of the thermal structure of subduction zones, terrigenous sediments will begin to melt at pressures < 2 GPa. The melt phase can absorb tremendeous quantities of water and represent major sinks for devolatilisation fluids. Dense hydrous silicates will form in any subducted sediments at all depths. These silicates can store significant amount of water in their structure. In the subducted oceanic crust and upper mantle, heterogeneous distribution of fluids inherited from hydrothermal alteration at mid-ocean ridges will cause contemporaneous dehydration and hydration reactions on a local scale rather than large-scale fluid infiltration in the overlying mantle wedge. The situation where a cold, competent and fluid-rich subducted slab is bounded above by a hot, relatively weak and anhydrous material is likely to act against buoyant rise of devolatilisation fluids by forcing the fluids to flow downwards. At sub-arc depths (50 ± 10 to 80 150 km ) and for any geothermal gradient, continuous internal buffering of volatile activities will retain the fluid phase in the slab. This is in contrast with shallow, sub-forearc depths (20 to 50 ± 10 km), where tectonic vein arrays, localized thrust faults, serpentine diapirs, mud volcanoes and boninite magmas attest to the release of large volume of fluids into the overriding mantle wedge.

Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties.

Using a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel ("sea-level electrophoresis"), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris-boric-acid-EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA system, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer.

Horizontal semi-dry electroblotting for the detection of the low density lipoprotein receptor in solubilized liver membranes.

A high efficiency transfer of the low density lipoprotein (LDL) receptor proteins from polyacrylamide slab gel onto immobilizing nitrocellulose membranes using the horizontal semi-dry electrophoretic system is described. The transfer of the LDL receptors from solubilized rat liver microsomes was performed between two graphite plate electrodes in a continuous buffer system containing methanol and sodium dodecyl sulfate. The protein transfer was achieved in only 150 min at a constant current of 0.8 mA/cm2 at room temperature with very low Joule heat development. The homogeneous electric field yield between the two electrode plates produced a satisfactory transfer of the LDL-receptor protein band in spite of its high molecular weight, and only few protein traces remained in the polyacrylamide gel after blotting. This improved method allows a rapid and quantitative transfer of the LDL receptors without protein denaturation, since the specific binding activity of the blotted receptor is retained as demonstrated by ligand-blotting and immunoblotting.

Production of an enzymatic active protein using a continuous flow cell-free translation system

We have examined the characteristics of protein synthesis in an improved continuous flow cell-free translation system prepared from wheat germ extract with dihydrofolate reductase (dhfr) mRNA as the translated message. Continuous buffer flow and separation of product from the reaction mixture were accomplished by the use of a modified Amicon ultrafiltration chamber as reaction vessel. The system produced protein for more than 20 h, and the product had an activity of dhfr comparable to that of authentic enzyme from E. coli. Analysis of RNA recovered from the filtrate supports the notion that a functionally active protein-synthesizing machinery is superorganized in a dynamic complex.

Historical Relationships Between Research and Resource Management in the Apalachicola River Estuary.

A continuous field research effort has been carried out in the Apalachicola River estuary since March 1972. The information generated from this interdisciplinary study has been directly applied to the management of the Apalachicola resource by means of close associations among local, state, and federal officials and university scientists. During the early years, scientific data were instrumental in the prevention of the impoundment of the Apalachicola River. A series of regional studies was carried out to evaluate various forms of effects due to forestry activities, pesticides, and stormwater runoff from urban areas. A review was made of fisheries problems associated with dredging, overfishing, and marine pollution. Results of such studies were directly applied to local management questions. Research that linked the river wetlands with the estuary, in terms of the input of fresh water, nutrients, and organic matter, served as the basis for the purchase of extensive bottomland tracts. Other initiatives were carried out that were designed to protect the naturally high productivity of the river estuary. Further purchases of estuarine wetlands and barrier island properties were made that formed an almost continuous buffer of publicly held lands between upland developments and critical habitats and important populations of the bay system. A regional management plan was adopted that was designed to limit local municipal development in the estuarine region. Analyses of the long-term scientific data indicated that dominant, commercially important estuarine populations are associated with river flow, local salinity characteristics, and biological (predation, competition) interactions with the salinity regime and food web structure. Such interactions are not straight forward, however; they reflect complex interactions of the freshwater influxes and biological response in the estuary that are not well understood. Species-specific responses to the principal driving factors further complicate the biological relationships of the Apalachicola system. The management of a river-dominated estuary should be based on protection and control of freshwater sources, nutrients, and organic matter with a minimization of physical alterations that often lead to increased salinity stratification and the associated loss of the nursery function of the estuary. There is growing evidence that changes in upland characteristics and within-system habitat alterations are associated with changes in nutrient distributions and salinity relationships and that such changes can have serious impacts on estuarine systems. Issues involving basic changes in estuarine productivity and associated food webs are far more important than those involving species diversity in the management of such resources. Processes such as nutrient flow and salinity alteration underlie the very basis of estuarine productivity and usefulness. Over the past decade, there has been a gradual reduction in the importance of research as an integral part of the planning and management process in the Apalachicola system. This regional trend follows a national pattern. Dredging effects on the river and bay have gone on without effective challenge even though such activities are damaging productive habitats of the system. The once influential Apalachicola oyster fishery remains in disarray following a series of natural disasters and poor management practices. There is a growing problem with the deliberate obstruction of the generation and use of scientific data to determine management policies by various state and federal agencies. The substitution of public relations activities for the development of needed scientific information concerning factors such as the importance of sustained freshwater input to estuaries is further evidence that even the most elaborate and well-conceived management plans can be reversed by political manipulations and short-sighted bureaucratic policies.

Binding of lysozyme on the surface of entrapped phosphatidylserine—phosphatidylcholine vesicles and an example of high-performance lipid vesicle surface chromatography

Phospholipid vesicles (liposomes) composed of phosphatidylserine (PS) and phosphatidylcholine (PC) in five different molar ratios were formed and entrapped in agarose gel beads (Sepharose 6B and Superose 6). For this purpose a previous dialysis—entrapment procedure was improved by use of an apparatus with continuous buffer flow and rotating dialysis cells. By rotary dialysis combined with a method of vesicles fusion with the aid of Ca 2+ ions, the capacity of entrapped vesicles composed of 80–85% phosphatidylserine from bovine brain [denoted “PS (brain extract)”] was increased. The entrapment of PSPC vesicles increased as the content of PS was increased. Lysozyme was applied to a PSPC vesicle—Sepharose 6B column. The amount of bound lysozyme was about 9 nmol per μmole of lipids for entrapped PSPC vesicles with 20 and 45 mol-% PS contents. The amounts of bound lysozyme increased to 29, 30 and 40 nmol per μmole of lipids as the fraction of PS in the vesicles was increased to 65, 80–85 and 100 mol-% PS, respectively. The lysozyme molecules thus closely packed on the surface of the vesicles with the higher PS contents. The losses of the entrapped vesicles in the first two chromatographic lysozyme-binding experiments were 5% for 80–85% PS (brain extract) vesicles, 10–12% for vesicles with 20 to 65 mol-% PS content and 23% for pure PS vesicles. PS (brain extract) vesicles were also formed and entrapped in Superose 6 gel beads (diameter 13 μm). Ribonuclease A, lysozyme and cytochrome c were well separated on this PS-vesicle—Superose column at an ionic strength of 0.05–0.15 M.