bluuebunny/crossref_metadata_2025 huggingface dataset:
Rare right ventricular myxoma detected incidentally on a FAST examination
Authors: Brestas Paraskevas S. , Malagari Katerina S. , Kelekis Dimitrios A.
Published: March 2005

A young man underwent Focused Assessment with Sonography in Trauma (FAST) scan for blunt abdominal trauma. An incidental finding on the pericardial view was the presence of a tumor in the right ventricle. Detailed cardiac imaging and subsequent excision revealed the presence of a myxoma. The patient had no obvious cardiac symptoms, but the awareness and experience of the examiner during the FAST examination detected this rare heart tumor at a latent stage, preventing serious, irreversible complications. This case demonstrates the potential importance of looking above the diaphragm during routine abdominal sonography, especially in patients who are critically ill. © 2005 Wiley Periodicals, Inc. J Clin Ultrasound 33:143–145, 2005
On restricted connectivities of permutation graphs
Authors: Balbuena C. , Marcote X. , García‐Vázquez P.
Published: March 2005

A permutation graph (or generalized prism) Gπ of a graph G is obtained by taking two disjoint copies of G and adding an arbitrary matching between the two copies. Permutation graphs can be seen as suitable models for building larger interconnection networks from smaller ones without increasing significantly their maximum transmission delays, in such a way that these larger networks are highly fault‐tolerant. For permutations graphs, in this article we provide conditions that guarantee optimal values for two parameters of connectivity, λ′ and κ′. For a connected graph G the restricted edge‐connectivity λ′(G) is defined as the minimum cardinality of a restricted edge‐cut; that is, the minimum cardinality of a set S of edges such that G − S is not connected and S does not contain the set of incident edges of any vertex of the graph. A graph G is said to be λ′‐optimal if λ′(G) = ξ(G), where ξ(G) is the minimum edge‐degree in G defined as ξ(G) = min{d(u) + d(v) − 2 : uv ∈ E(G)}, and d(u) denotes the degree of vertex u. Among other things, we prove that permutation graphs satisfy: min{λ′(G) + δ(G), 2λ′(G), ξ(Gπ)} ≤ λ′(Gπ) ≤ ξ(Gπ) if |V(G) | ≥ ξ(G) + 2. Furthermore, min{ 2λ′(G), ξ(Gπ)} ≤ λ′(Gπ) ≤ ξ(Gπ) if G is triangle‐free. We also study the vertex case considering the restricted connectivity κ′(G) and relating it to the superconnectivity κ1(G); the latter is defined as the minimum cardinality of a set of vertices, if any, whose deletion disconnects G in such a way that every remaining component has at least two vertices. For instance, we prove that 2κ(G) ≤ κ1(G) ≤ κ′(Gπ) ≤ ξ(Gπ) if G is triangle‐free and the permutation graph has no cycles of length five. © 2005 Wiley Periodicals, Inc. NETWORKS, Vol. 45(3), 113–118 2005
Anatomic Reconstruction of the Distal Radioulnar Ligaments: Long-Term Results
Authors: TEOH L. C. , YAM A. K. T.
Published: March 2005

Chronic post-traumatic dynamic distal radioulnar joint instability following rupture of the distal radioulnar ligaments may cause disabling and progressive ulnar-sided wrist symptoms. We use a free tendon graft to anatomically reconstruct both dorsal and palmar distal radioulnar ligaments simultaneously. Nine patients who underwent this procedure between 1990 and 1997 were assessed using a modified Mayo Wrist Score. Following surgery, average wrist scores improved from 66 (95% CI, ±3.0) to 92 (95% CI, 92±6.9) and were maintained in the long term (average score, 87 at 9 years). Joint stability was restored and maintained in seven of the nine patients, but two developed recurrent instability. Pain, grip strength and function were better following surgery in the short and long term. Range of supination and pronation decreased slightly from an average of 169° pre-operatively to 155° (90% of normal) in the long term. Long-term radiographs show no distal radioulnar or ulnocarpal joint osteoarthritis.
Long-Term Outcome of Radial Shortening with or Without Ulnar Shortening for Treatment of Kienböck’s Disease: A 13–25 Year Follow-Up
Authors: ZENZAI K. , SHIBATA M. , ENDO N.
Published: March 2005

Fourteen of 35 patients who underwent radial shortening with or without ulnar shortening for the treatment of Kienböck’s disease were followed up for a median of 19 (range, 13–25) years. Radial shortening was performed for patients with ulnar negative or neutral variance, and combined shortening of radius and ulna for those with ulnar positive variance. Overall the clinical situation was significantly improved at the final follow-up. There was no significant advanced collapse of the wrists and Lichtman’s stage of disease increased in only three cases. Although osteoarthritic changes in the distal radio–ulnar joint progressed in five patients, this may have little influence on clinical outcome. Radial shortening osteotomy is a reliable method for treatment of Kienböck’s disease.
Expression of the lactotransferrin receptor during the differentiation process of the megakaryocyte Dami cell line
Authors: Nillesse Nathalie , Pierce Annick , Lecocq Myriam , Benaissa Monique
Published: March 2005

Summary— In order to determine whether the human lactotransferrin receptor recently described on platelets was also present onhematopoietic precursors, we investigated its presence and characteristics on the megakaryocytic Dami cell line. The reversible binding of human 5‐([2‐(carbo(hydrazino)methyl]thio)acetyl)aminofluorescein‐labeled lactotransferrin showed that such a receptor was only present on the subpopulation of the largest cells. The increase in numbers of large cells during culture was paralleled by a concurrent increase in lactotransferrin receptor positive cells. Scatchard analysis of the binding of [125I]‐labeled lactotransferrin showed that a single affinity class of binding site was present (Kd = 446 ± 40 nM) and that there were 52 ± 3 × 105 sites per cell. The mouse monoclonal antibody DP5B3G10, specific for the human lactotransferrin receptor, allowed its characterization as a 105 kDa protein on Western blots. The same monoclonal antibody was used to separate the small and large cell subpopulations of Dami cells by panning. Separate culture of the small cells showed that the receptor appeared prior to and independent from endomitosis. In contrast, GPIb was expressed only by large megakaryocytes. The use of conditioned medium from cultures of whole Darni cell populations indicated that a soluble factor is involved in differentiation, but not in the appearance of the lactotransferrin receptor.
THE EFFECT OF HEAT TREATMENT OF WATERMELON SEED ON GERMINATION, HYPOCOTYL EMERGENCE AND ABSCISIC ACID CONTENT
Authors: DEMIR I. , VAN DE VENTER H. A.
Published: March 2005

Seeds of watermelon (Citrullus lanatus) with a moisture content of 7.2% were heat treated at 60°C for 15 or 20 h, and their performances compared with that of untreated seeds. Radicle emergence of treated seeds was only slightly lower than that of the control when germinated at 25°C. To ascertain the effect of the treatments on hypocotyl growth (emergence) per se, radicle emergence was synchronized in all treatments and emergence from deep plantings in sand determined. Small differences in emergence were observed when seeds were germinated at 25°C, but seed treatment resulted in large reductions in emergence at 15°C. This demonstrates that the hypocotyls developing from heat-treated, germinated seeds are less able to withstand low germination temperatures, which could be an important factor in emergence failure. Levels of free abscisic acid (ABA) were higher at 15°C than at 25°C, with hypocotyls of treated seeds containing more free ABA and its breakdown products than the control at the lower temperature.
Opinion Polls in 2004
Authors: KABASHIMA IKUO
Published: March 2005

When the last opinion polls were published one year ago, Prime Minister Junichiro Koizumi was enjoying a high approval rate despite Japan's poor economic performance. The poll conducted in June 2003 showed that 48.2% of the public supported the Koizumi administration, which is an exceptionally high number in comparison with the approval rates of historical LDP prime ministers and their cabinets. Only 29.5% of those polled expressed dissatisfaction with the administration. Today, however, public support for Koizumi is on a sudden decline. Just as Koizumi's managed to sustain public support throughout 2003, his loss of support in 2004 manifests a peculiar quality; he is now losing popularity despite Japan's significant economic improvement.
Continuous Counteracting Chromatographic Electrophoresis
Authors: Ivory Cornelius F. , Gobie William A.
Published: March 2005

Counteracting chromatographic electrophoresis (CACE) uses opposing electric and convective fields to focus proteins at the boundary separating distinct granular gel phases. In batch operation, CACE can purify, stack, and concentrate gram quantities of proteins. This technique has been extended to continuous operation by modifying the batch column. Experiments were then used to demonstrate operation of the column. The principles underlying focusing and separation are elucidated by linear chromatography theory.
Two‐Barrel Bile‐Acids‐Sensitive Microelectrodes Based on Liquid Ion Exchanger
Authors: Bi Yu , Wen‐Bin Hong
Published: March 2005

Several liquid membrane microelectrodes sensitive to bile acids (two barrel, tip diameter about 0.5 μm) are described. The results of different liquid ion exchangers such as Aliquat 336/decanol, trioctylmethylammonium/decanol, hexadecyltrimethylammonium/ decanol, benzyldimethylhexadecylammonium/ decanol, hexadecyltributylammonium/5% hexachlorobenzene + 0.5% bromoacetanilide in o‐dichlorobenzene are compared with each other, and the better one among them is the mixture of benzyldimethylhexadecylammonium cholate/decanol with hexadecyltributylammonium taurocholate/5% hexachlorobenzene + 0.5% bromoacetanilide in o‐dichlorobenzene because of its quicker response time and low drift. The calibration curves, slopes, test limits, selective coefficients, drifts, and response times of the various bile‐acids‐sensitive microelectrodes in different calibration solutions were demonstrated and compared with each other.
Continuous Operation of a Pressure‐Cycled Membrane Bioreactor
Authors: Steinmeyer D. E. , Shuler M. L.
Published: March 2005

A membrane bioreactor system has been used to continuously ferment glucose to eth‐anol with Saccharomyces cerevisiae and extract the product into a solvent, tributyl phosphate. The reactor cycles gas pressure to force a convective flux of nutrient into the cell chamber and product out. The reactor has been run for over 3000 h with minimal deterioration of operating characteristics after the first hours of operation. A corn starch hydrolysate was also fermented, yielding similar results. Zymomonas mobilis was tested as an alternative catalyst, but it formed filaments that reduce the effectiveness of the pressure cycle. Z. mobilis appears to be less attractive than S. cerevisiae for such a reactor.
Immobilization of Microbial Cells in a Mixed Matrix of Silicone Polymer and Calcium Alginate Gel: Epoxidation of 1‐Octene by Nocardia corallina B‐276 in Organic Media
Authors: Kawakami Koei , Tsuruda Satoshi , Miyagi Katsuyoshi
Published: March 2005

Immobilization of Nocardia corallina B‐276 was examined for production of 1,2‐epoxyoctane from 1‐octene in the presence of n‐hexadecane as the organic solvent. Hydrophobic silicone polymer was the most suitable material for entrapment of the cells. Coentrapment of aqueous reaction medium into the silicone polymer matrix improved the epoxide productivity. It was also effective to immobilize the cells in a mixed matrix composed of silicone polymer and calcium alginate gel involving the reaction medium. In the organic monophase, the amount of epoxide accumulated with the cells immobilized in an almost equivolumetric composite of both materials was 2 and 7 times the amounts in the silicone and alginate single matrices, respectively, and it became larger than with the free cells in the aqueous—organic two‐liquid phase after a longer period of batch operation. The use of such an optimized composite matrix enabled us to perform a relatively simple operation of the continuous three‐phase bioreactor.
Fed‐Batch Culture of Saccharomyces cerevisiae in an Airlift Reactor with Net Draft Tube
Authors: Wu Jiumn‐Yih , Wu Wen‐Teng
Published: March 2005

A modified airlift reactor has been developed for a fed‐batch culture of Saccharomyces cerevisiae. The draft tube of the proposed reactor is a net one. The reactor was coupled with a computer control system for substrate addition. Since the oxygen transfer of the new reactor is much higher than that of a conventional airlift reactor and bubble column, experimental study showed that higher cell mass was obtained by using the new reactor.
Lactic Acid Formation by Free and Immobilized Cells of an Obligate Methylotroph
Authors: Dinarieva Tatiana , Netrusov Alexander
Published: March 2005

Cells of the Gram‐negative obligate methylotrophic bacterium Methylobacillus flagellation KT, which can only use methanol and methylamine as sole carbon and energy source, oxidize 1,2‐propanediol to lactaldehyde and lactate by using a pyrroloquinoline quinone (PQQ) containing cytochrome c dependent methanol dehydrogenase and an NAD+‐dependent aldehyde dehydrogenase, both of which have a broad substrate specificity. The kinetics of 1,2‐propanediol oxidation and lactic acid appearance in the buffered medium were followed, and Km for substrate, pH, and temperature optima were determined. M. flagellatum KT cells immobilized in polyacrylamide, agarose, or carrageenan gels maintain the ability to oxidize 1,2‐propanediol to lactic acid. The half‐life of the process with the cells in carrageenan was increased 5–6 times compared with free cell suspensions.
Model for Calculating the Compressive Deformability of Double‐Layered Curdlan Gels
Authors: Nussinovitch A. , Lee S. J. , Kaletunc G. , Peleg M.
Published: March 2005

The compressive force‐deformation relationships of double‐layered curdlan gels (made of 2.5% and 3.5% gels) were calculated from those of the components. On the basis of the assumptions that the normal force in the layers is the same, that their deformations are additive, and that the effects of lateral stresses and viscoelasticity can be neglected, the calculation was done by using a mathematical model the constants of which were determined from the behavior of the individual layers. The calculated relationships were in agreement with experimental relationships over a considerable range of strain. Failure of the arrays, however, preceded that of the layers and was accompanied by their separation, a phenomenon that is unrelated to the model but limits the strain range of its applicability.
Measurement of Heat Transfer Coefficients in Rotating Liquid/Particulate Systems
Authors: Stoforos Nikolaos G. , Merson Richard L.
Published: March 2005

A new method, involving the use of liquid crystals as temperature sensors, was developed to measure surface temperatures on moving particles. The method involves coating the monitored particles with aqueous solutions of encapsulated liquid crystals and recording the color changes on the particle surface as a function of temperature. Experimentally measured particle surface and liquid temperatures were used to calculate heat transfer coefficients for water‐heated, axially rotating, cylindrical acrylic vessels containing liquid and solid spherical particles. The theoretical analysis associated with calculating the heat transfer coefficients from the experimental data is presented. The method is illustrated by measuring overall (heating medium/container wall/internal liquid) and liquid‐particle film heat transfer coefficients for Teflon spheres in deion‐ized water and aluminum spheres in 1.5 cSt silicone fluid.
Adsorption Characteristics of an Immobilized Metal Affinity Membrane
Authors: Iwata Hideki , Saito Kyoichi , Furusaki Shintaro , Sugo Takanobu , Okamoto Jiro
Published: March 2005

An immobilized metal affinity (IMA) hollow‐fiber membrane was prepared by radiationinduced graft polymerization of glycidyl methacrylate (GMA) onto a porous polyethylene hollow fiber, followed by chemical conversion of the produced epoxide group into an iminodiacetate (IDA) group and its chelation with copper (II) ion. The IDA hollow fiber, whose degree of GMA grafting was 120%, was found to retain 0.42 mol of Cu ion/kg of dry weight of the resulting IMA hollow fiber. The pure water flux of the affinity membrane was 0.90 m/h at a filtration pressure of 1 × 105 Pa. The 0.1 g/L L‐histidyl‐L‐leucine (His‐Leu) solution permeated across the IMA hollow fiber, whose inner diameter and thickness were 0.78 and 0.365 mm, respectively, at a prescribed filtration pressure ranging from 0.2 × 105 to 1.0 × 105 Pa. The adsorption of His‐Leu during permeation of the solution showed that the overall adsorption rate was independent of the filtration pressure, i.e., the residence time, because of the negligible diffusional resistance of His‐Leu to the pseudobioaffinity ligand located on the pore surface of the membrane. No deterioration in the adsorption capacity was observed after five cycles of His‐Leu adsorption, its elution, and reimmobilization of copper. The adsorption isotherm of bovine serum albumin (BSA) on the IMA hollow fiber was measured and compared with that for the conventional agarose‐based bead containing the IDA‐Cu ligand. The diol group formed by H2SO4 treatment of the epoxide group remaining after conversion of epoxide into IDA functionality significantly reduced the nonselective adsorptivity of His‐Leu and BSA on the membrane.
Kinetics and Mechanism of the Adsorption of Sulfolobus acidocaldarius on Coal Surfaces
Authors: Vitaya Virote B , Toda Kiyoshi
Published: March 2005

The adsorption of Sulfolobus acidocaldarius on five types of coals (with different sulfur contents) was investigated to define the kinetics of the bacterial cell adsorption and to clarify the mechanism of the adsorption involved in biological desulfurization of coals. A kinetic model, in which two stages of adsorption are considered, is proposed on the basis of the experimental results. The estimated values of the maximum cell numbers adsorbed per unit area of coal surface (including internal surface area of pores larger than 0.5 μm), [Sm], showed a good correlation with the pyritic sulfur content of the coals. The mechanism of the adsorption was studied by investigating the effects of pH, ζ potentials of coal particles and the bacteria, a specific inhibitor of membrane‐bound ATPase (20 mM NaF), a surfactant (Tween 80, 0.05%), and heat treatment (121 °C, 15 min) of the bacteria. From these observations, it was concluded that the rapid adsorption in the first stage was related with the physical force of hydrophobicity and the adsorption in the second stage was strongly related with the oxidation of pyrite. An electromicrographic study on the erosion of pyrite surfaces by the bacterial cells was also conducted. The micrographs of the pyrite particles after 5 days of the incubation showed that surfaces of pyrite minerals were eroded by adsorbed cells.
α‐Amylase Fermentation with Bacillus amyloliquefaciens in an Aqueous Two‐Phase System
Authors: Park Kyung‐Moon , Wang Nam Sun
Published: March 2005

α‐Amylase production from Bacillus amyloliquefaciens in an aqueous two‐phase system of poly(ethylene glycol) and dextran was investigated. In both batch and continuous fermentations, cells preferentially partitioned to the dextran‐rich bottom phase, glucose distributed almost evenly in both phases, and α‐amylase generally preferred the bottom phase by a small margin. A continuous fermentor with bottom‐phase recycle is proposed to achieve a significantly improved volumetric productivity of α‐amylase. Continuous α‐amylase fermentation with simultaneous product removal was demonstrated in 84 g/L poly(ethylene glycol) (MW 8000) and 84 g/L dextran (MW 71 500) at various dilution rates. Low overall cell densities economized glucose consumption. Unlike the polymer‐free fermentation, α‐amylase synthesis was not repressed even at high glucose concentrations. A properly designed continuous system with cell recycle can be considered as a nonconventional, but highly effective, method of cell immobilization.
Growth, Metabolic, and Antibody Production Kinetics of Hybridoma Cell Culture: 2. Effects of Serum Concentration, Dissolved Oxygen Concentration, and Medium pH in a Batch Reactor
Authors: Ozturk Sadettin S , Palsson Bernhard O
Published: March 2005

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/γ2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monodtype model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20–50 % air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell‐specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2‐fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.
Cell Death in the Thin Films of Bursting Bubbles
Authors: Cherry Robert S. , Hulle Carl T.
Published: March 2005

A sparged gas bubble floating at the liquid interface has a liquid film which drains and thins until the film spontaneously ruptures at a point. This causes rapid retraction of the film, forming a rim of collected fluid. This rim moves at a constant velocity of about 3 m/s and any cells in the bubble film are rapidly accelerated to this velocity in the moving rim. Half of the surface energy originally in the thin film is converted to kinetic energy of the rim, while the rest is dissipated in this rim. The rate of energy dissipation per mass of rim fluid is approximately 9000 m2/s3, which corresponds to a Kolmogorov eddy size of 3.2 μm in fully developed turbulence or a shear stress of 95 N/m2 in laminar flow. Either of these limiting cases presents an environment in which rapid cell death would be expected. Experiments with'Sf‐9 insect cells suggest that the cell concentration in these thin films is 0.6 times the bulk liquid concentration and that about 20 % of these cells are killed when the film ruptures. An equation based on this mechanism accurately predicts the death rate.
Integrated Ozonation‐Biotreatment of Pulp Bleaching Effluents Containing Chlorinated Phenolic Compounds
Authors: Heinzle E. , Geiger F. , Fahmy M. , Kut O. M.
Published: March 2005

Bleaching effluents from pulp and paper industry are problematic because they contain toxic chlorinated phenolic compounds and recalcitrant chlorinated lignin fragments of higher molecular weight. The chlorinated phenolic compounds have been shown to be rather easily removable in fluidized bed bioreactors. The total amount of adsorbable organic halogens (AOX), however, was only reduced by 20%‐30%. It is generally possible to oxidize all unsaturated organic compounds in the bleaching effluents by ozonation. However, ozone is still too expensive to allow complete treatment. Nevertheless, the increase of biodegradability of recalcitrant components by dechlorination and degradation of high molecular weight fractions by ozone can lead to increased efficiency in a subsequent biotreatment. In this study the simple ozonation of bleaching effluents with and without biological pretreatment was compared with sequential or cyclic ozonation—biotreatment schemes. By integrated ozonation—biotreatment, the degradation efficiency relative to the amount of ozone consumed was markedly increased. The overall efficiency of the treatment process in terms of fractional removals of global parameters COD, TOC, and AOX increased. More than 70% of AOX were eliminated. No toxic effect was observed in the cyclic reactor, where the liquid passed was cycling between an ozonation and a biotreatment zone. In the liquid phase no dissolved ozone could be detected in this reactor.
An Improved Method for Disruption of Microbial Cells with Pressurized Carbon Dioxide
Authors: Lin Ho‐Mu , Yang Zhiying , Chen Li‐Fu
Published: March 2005

Disruption of microbial cells by pressurized carbon dioxide at both subcritical and supercritical temperatures has been previously investigated. This method differs in principle from other disruption techniques and was found to have potential applications for rupture of a variety of microorganisms. However, it is not as effective for some of the microbial cells, including yeast, of which the cell walls are extremely robust and rigid. This work suggests an alternative operation to improve the disruption rates of cells by repeatedly releasing the applied fluid pressure within the cells in the midst of a disruption process. The improvement is substantial at all the experimental conditions studied.
Techniques for the Estimation of Cell Concentration in the Presence of Suspended Solids
Authors: Kennedy M. J. , Thakur M. S. , Wang D. I. C. , Stephanopoulos Gregory N.
Published: March 2005

Measuring cell concentration is of fundamental importance in many biochemical processes. However, this measurement is very difficult to make when solid particles are present along with the cells. This review examines strategies that have been used to estimate cell concentration in the presence of solid particles.
Recovery of Propionic and Acetic Acids from Fermentation Broth by Electrodialysis
Authors: Weier Anthony J. , Glatz Bonita A. , Glatz Charles E.
Published: March 2005

Acetic and propionic acids can be produced by fermentation. In this process, acid recovery is important both for producing a concentrated product from the broth and for removing acid from the broth to reduce product inhibition during the fermentation. The purpose of this work was to characterize the performance of an electrodialysis unit for carrying out both tasks. Current efficiencies, water transport, and product concentrations were determined both for model solutions of the acids and for fermentation broths to determine the limiting factors in using this separation method. In addition, observations were made of fouling behavior of the membranes in this use. The highest concentrations of product were obtained by contacting with the most concentrated feed and doing so in the absence of competing ions. For the range studied here, a maximum concentration of 13.8% (w/v) sodium propionate was obtained. In the batch recycle mode, final concentrations in the concentrate were reduced, but acid recoveries were improved by the presence of other ions because the low‐current shut‐off was delayed. The use of electrodialysis for producing a concentrated acid product is not promising unless salt levels in the medium can be reduced and acid levels increased. However, electrodialysis can be used to maintain low acid levels in the fermentor during extractive fermentation.
The Use of Pressure to Modify Enzyme Activity in Reversed Micelles
Authors: Rao A. Madhusudhan , Kommareddi N. , John V. T.
Published: March 2005

Pressurization of enzyme‐containing AOT‐water‐isooctane reversed micelles with low molecular weight gases leads to markedly different responses in activity characteristics. Microbial lipases exhibit a total cutoff in activity with as low a pressure as 2 MPa and a remarkable activity regain with depressurization. The observation also holds for reaction in monophasic organic solvents. The protease, α‐chymotrypsin, is unaffected by pressurization until a critical pressure wherein micellar instability occurs. The use of pressure as a switch for lipase reaction in nonaqueous media is discussed.
Dextran Retention in the Rat Brain Following Release from a Polymer Implant
Authors: Dang Wenbin , Saltzman W. Mark
Published: March 2005

Intracranial controlled release polymers may improve drug administration to the brain, where therapy is frequently limited due to the low permeability of brain capillaries to therapeutic agents. On the basis of drug transport and elimination rates, we proposed that high molecular weight, water‐soluble molecules would be retained in the brain space following release from an intracranial implant. To test this hypothesis, solid particles of different molecular weight fractions of fluorescein isothiocyanate labeled dextran (FITC‐dextran; 4 × 103 Da (4 kDa) < weight‐averaged molecular weight (Mw) < 150 kDa) or fluorescein were uniformly dispersed in matrices of a polyanhydride copolymer synthesized from a fatty acid dimer and sebacic acid in a 50:50 ratio, P(FAD: SA). When incubated in buffered saline, FITC‐dextran fractions of 70 kDa Mw were released from the polymer within 48 h; 4 kDa Mw FITC‐dextran and fluorescein were released more slowly. Following implantation of P(FAD:SA) matrices containing either 70 kDa Mw FITC‐dextran, 4 kDa Mw FITC‐dextran, or fluorescein into the brains of normal rats, fluorescent tracers were continuously released into the brain tissue for 30 days. Tracer concentrations within the brain were significantly higher for large molecular weight tracers (70 kDa Mw FITC‐dextran ≫ 4 kDa Mw FITC‐dextran > fluorescein). The rate of elimination, 𝔨app, of each tracer from the brain was determined by comparing experimental data with a model describing tracer diffusion/elimination in the brain extracellular space; 𝔨app decreased with increasing molecular weight (fluorescein > 4 kDa Mw FITC‐dextran > 70 kDa Mw FITC‐dextran).
Application of an On‐Line Turbidimeter for the Automation of Fed‐Batch Cultures
Authors: Yamane Tsuneo
Published: March 2005

Recent advances in the technology of continuous in situ turbidity measurements in microbial cultivations are reviewed. Some characteristics of commercially available on‐line laser turbidimeters are estimated in relation to requirements of sensors for microbial cultures. A system for the automation of fed‐batch cultures using an on‐line turbidimeter is proposed. Experimental data are presented concerning fed‐batch cultures grown on synthetic and natural complex media. In situ turbidity data were found to be particularly useful for the automation of the feeding of natural complex nutrient solutions by converting the data into dry cell mass concentration and at the same time utilizing the instantaneous data on the culture broth volume through a computer.
Fermentation of Crystalline Cellulose to Ethanol by Klebsiella oxytoca Containing Chromosomally Integrated Zymomonas mobilis Genes
Authors: Doran Joy B. , Ingram L. O.
Published: March 2005

Complete enzymatic hydrolysis of cellulose to glucose is generally required for efficient fermentation to ethanol. This hydrolysis requires endoglucanase, exoglucanase, and cellobiase. The Gram‐negative bacterium, Klebsiella oxytoca, contains the native ability to transport and metabolize cellobiose, minimizing the need for extracellular cellobiase. Strain P2 is a recombinant derivative in which the Zymomonas mobilis pdc and adhB genes have been integrated into the chromosome and expressed, directing the metabolism of pyruvate to ethanol. This organism has been evaluated in simultaneous sacchari‐fication and fermentation (SSF) experiments to determine optimal conditions and limits of performance. The temperature was varied between 32 and 40 °C over a pH range of 5.0–5.8 with 100 g/L crystalline cellulose (Sigmacell 50, Sigma Chemical Company, St. Louis, MO) as the substrate and commercial cellulase (Spezyme CE, South San Francisco, CA). A broad optimum for SSF was observed, with a pH of 5.2–5.5 and temperatures of 32–35 °C, which allowed the production of over 44 g of ethanol/L (81–86% of the maximum theoretical yield). Although the rate of ethanol production increased with cellulase, diminishing improvements were observed at enzyme loadings above 10 filter paper units/g of cellulose. Over 40 g of ethanol/L was produced with relatively low enzyme loadings: 7–10 filter paper units/g of cellulose. Two optimal SSF conditions were identified for fermentation yield with strain P2: pH 5.2 at 35 °C and pH 5.5 at 32 °C. Under these conditions, 47 g of ethanol/L was produced in 144 h (0.48 g of ethanol/g of cellulose). Maximal rates of ethanol production were observed at 37 °C and pH 5.0 and produced over 40 g of ethanol/L in 72 h (final yield of 0.432 g of ethanol/g of cellulose after 96 h). All fermentations except those conducted at 40 °C and low pH (pH 5.0–5.5) exceeded 70% of theoretical ethanol yields. Tight process controls may not be required using this organism since temperatures between 32 and 37 °C at pH ranges between 5.0 and 5.8 still produced good yields.
Comparison of Trypan Blue Dye Exclusion and Fluorometric Assays for Mammalian Cell Viability Determinations
Authors: Altman Steven A. , Randers Lisa , Rao Govind
Published: March 2005

A hemocytometer‐based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide. Viable and nonviable cell densities were measured, and culture viability was calculated both during the normal growth cycle of a murine hybridoma and in response to the application of millimolar concentrations of either tert‐butyl hydroperoxide or ferrous iron. During the early phase of rapid hybridoma cell growth, assay‐based differences in viable cell density were not significant. As the culture aged, the trypan blue dye exclusion assay significantly overestimated cell viability, thereby underestimating nonviable cell density and yielding an erroneous estimation of the overall viability of the culture. Because of its lack of ambiguity in the identification of stained, nonviable cells and its resulting increased accuracy in the estimation of culture viability, the fluorometric assay was considered a better choice for the evaluation of cell viability.
Gene Structural Analysis and Expression of Human Renal Dipeptidase
Authors: Satoh Susumu , Ohtsuka Kazuyuki , Keida Yuriko , Kusunoki Chihiro , Konta Yoshiyuki , Niwa Mineo , Kohsaka Masanobu
Published: March 2005

Human renal dipeptidase cDNA and genomic DNA were isolated from human kidney cDNA and genomic libraries, respectively. The human renal dipeptidase gene has a total length of approximately 6 kb and consists of ten exons and nine introns. The exons and cDNA each encode the 411 amino acid residues of the precursor protein, including 16 amino acid residues of signal sequence and a hydrophobic carboxyl terminal sequence for the attachment of a phosphatidylinositol glycan. Although the cDNA was slightly different from the cDNA reported by Adachi et al. (1990), the differences observed suggest, by comparison with human genomic DNA, that it may not represent an allelic variant but a cloning artifact. The recombinant human renal dipeptidase was produced on the surface of transfected L929 cells and had the same character as native renal dipeptidase. Northern blotting hybridization analysis showed that renal dipeptidase mRNA is only transcribed in kidney.
Production of Oryzacystatins I and II in Escherichia coli Using the Glutathione S‐Transferase Gene Fusion System
Authors: Michaud Dominique , Nguyen‐Quoc Binh , Yelle Serge
Published: March 2005

In recent years, several studies have demonstrated the potential of proteinase inhibitors for the biocontrol of insect pests. For such a control approach, however, strategies must be developed for the simple and efficient production of active inhibitors. In this study, oryzacystatins I (OCI) and II (OCII) were produced in Escherichia coli using the glutathione S‐transferase (GST) gene fusion system. Both inhibitors were produced in large amounts as fusion products (∼100 mg/L E. coli culture) and were apparently stable when accumulated in bacterial cells. Enzyme specificities and inhibition constants of the fusion proteins were similar to those determined for free inhibitors and those reported for naturally occurring inhibitors isolated from rice, preventing the necessity of using an expensive and time‐consuming cleavage step for the obtention of active OCs. The GST system thus appears appropriate for simple and efficient large‐scale production of the two cysteine proteinase inhibitors.
Stability of Producer Hybridoma Cell Lines after Cell Sorting: A Case Study
Authors: Kromenaker Sandra J. , Srienc Friedrich
Published: March 2005

Flow cytometry was used in combination with immunofluorescent staining for intracellular and surface‐associated antibody contents to identify a significant nonproducer cell fraction in a murine hybridoma cell line that had shown a decline in monoclonal antibody productivity with passaging. Viable producer cells stained for surface‐associated antibody content were isolated by cell sorting on the basis of surface fluorescence intensity. The fraction of nonproducers was initially reduced from 85% to 20%. Sorting a second time, after these cells were cultivated for 2 weeks, further reduced the fraction of nonproducers to less than 4%. The stability of this purified producer hybridoma cell line during passaging and after a freeze‐thaw cycle was investigated. This cell line was found to be highly unstable. A simple batch‐growth model simulation was used as follows: (i) to demonstrate that nonproducers appear in hybridoma cell lines after a rare, random mutation event that results in the loss of the heavy‐chain gene and/or light‐chain gene expression; (ii) to show that a high rate of conversion of producer hybridomas to nonproducer hybridomas cannot entirely explain the population dynamics; (iii) to estimate the rate of conversion of producers to nonproducers to be 8.7 ± 10−5 h−1; (iv) to show that, for this conversion rate, the nonproducer cells' specific growth rates need only be 9% higher than those of the producer cells to dominate the hybridoma culture after only 25 passages; and (v) to predict that producer cells are preferentially lost during cell freezing and thawing procedures. The data suggest that particularly unstable cell lines should be analyzed and purified frequently to prevent overgrowth by nonproducers.
Rapid Quantitation of Recombinant Retroviruses
Authors: Morgan Jeffrey R. , Lee Jongwon , Tompkins Ronald G. , Yarmush Martin L.
Published: March 2005

A method for measuring the concentration of recombinant retrovirus encoding the Escherichia coli LacZ gene is described. The assay is based on the quantitative measurement of β‐galactosidase activity in extracts of cells infected with the LacZ‐encoding retrovirus. LacZ‐encoded β‐galactosidase activity in transduced cells is measured using the colorimetric substrate, O;‐nitrophenyl β‐D‐galactopyranoside (ONPG), and the results are read using an ELISA plate‐reader. Because the entire assay is performed in a 96‐well plate, large numbers of samples are easily measured, and the assay has the advantages of rapidity, precision, and ease of data collection. The assay was used to determine the optimal concentration of polybrene, a polycation known to enhance the infectivity of retroviruses, and can be used to evaluate other factors that affect infection, as well as the optimal conditions for production of the recombinant retrovirus.
New Materials for Biotechnology: Chromatographic Stationary Phases Based on Zirconia
Authors: Nawrocki Jacek , Dunlap Christopher J. , Carr Peter W. , Blackwell John A.
Published: March 2005

This review explores the usefulness of zirconia‐based materials in separations in biotechnology. The physical and chemical properties of zirconia are discussed briefly to familiarize the reader with the advantages of zirconia. The use of native zirconia is then examined, with a study of the Lewis acid/base chemistry that defines chromatography with zirconia. Modification of the zirconia surface with small molecules is then discussed. Finally, polymer‐coated zirconia materials are examined. Examples of separations using these various materials are shown. The advantages and disadvantages of each of these materials are presented.
Suboptimal Control of Batch Fermentation Processes Based on Their Classification by the Peculiarity Index
Authors: Kazakov Alexandr V. , Ganipol'skiy Sergey L.
Published: March 2005

A new method of suboptimal control for batch and fed‐batch fermentation processes is proposed. It combines the properties of program and adaptive control methods. The main idea of the method is to divide the controlled fermentation processes into classes using the integral characteristic of their individual properties‐the peculiarity index (PI). It is assumed that any process that belongs to a class is identical to the class' standard process (CSP). The optimal control programs for the CSP are derived off‐line. The value of PI for the current process is determined on‐line, and the control program that is used is the optimal one for the corresponding CSP. Industrial scale lysine fermentation processes are taken as an example. It is shown that the efficiency of the method is close to the efficiency of adaptive control, but that it is much simpler and its performance characteristics are better.
Stimulatory Effect of a Sulfonylurea Analogue and Its Polymer Conjugate on Insulin Secretion from Rat Islets
Authors: Kikuchi Akihiko , Bae You Han , Kim Sung Wan
Published: March 2005

A sulfonylurea with a polymerizable end group (a glyburide analogue) was synthesized. This monomer was copolymerized with N, N‐dimethylacrylamide (DMAAm). In vitro bioactivity was evaluated by adding these sulfonylurea monomers and copolymers into normal rat islet cultures and measuring the insulin concentration in the supernatant. The sulfonylurea monomer showed a stimulatory effect on rat islets at glucose concentrations of 50 and 100 mg/dL, with equivalent stimulation indices for glyburide, the potent sulfonylurea drug, while no effect was observed at a glucose concentration of 200 mg/dL. The sulfonylurea polymer stimulated Langerhans islets to secrete insulin at a low glucose concentration of 50 mg/dL, with 80% bioactivity of its monomeric compound. However, at 100 and 200 mg/dL glucose concentrations, there was no significant stimulation in insulin secretion. Because it is suggested that the sulfonylurea receptors are located in the lipid phase on the ATP‐sensitive K+ channels, the solubilization of sulfonylurea into, and its diffusion through, the lipid phase in the cell membrane may affect the interactions of sulfonylurea with its receptors. In the case of the sulfonylurea polymer, the sulfonylurea unit in the polymer can interact with its receptor as can be seen by the increased insulin secretion at a glucose concentration of 50 mg/dL, but its interaction would be interfered with to some extent by a large hydrophilic polymer backbone. Thus, less change in insulin secretion was observed at higher glucose concentrations by the addition of this particular sulfonylurea polymer.
Isolation of Human Blood Cells, Platelets, and Plasma Proteins by Centrifugal SPLITT Fractionation
Authors: Fuh C. Bor , Giddings J. Calvin
Published: March 2005

Centrifugal SPLITT fractionation, a technique designed for the continuous high‐resolution separation of colloids and low‐density particles, is applied here to fresh human blood, producing six purified fractions consisting of proteins and lipoproteins, platelets, red blood cells, lymphocytes, monocytes, and neutrophils. Production of the six fractions requires five steps, each yielding two fractions. These five steps are carried out in sequence using a single apparatus, with conditions varying from step to step in accordance with theoretical guidelines in order to achieve the desired cut points. In the first step, a stream of diluted blood is separated into one fraction consisting of platelets and plasma and another containing blood cells. The throughput of diluted blood is 162 mL/h and that of whole blood is about 2 mL/h or ∼1010 cells/h; guidelines are given for significantly increasing throughput. The purity of the blood cell fractions ranged from 92 to 98%, and the viability fell in the range 97–99%.
Flow Injection Analysis of Glucose, Fructose, and Sucrose Using a Biosensor Constructed with Permeabilized Zymomonas mobilis and Invertase
Authors: Park Je‐Kyun , Shin Min‐Chol , Lee Seung‐Goo , Kim Hak‐Sung
Published: March 2005

Cells of Zymomonas mobilis possessing glucose‐fructose oxidoreductase and glucono‐lactonase were permeabilized with toluene and co‐immobilized with invertase within a gelatin membrane. This membrane was coated over a pH electrode, and the resulting biosensor was placed in a flow‐through cell to develop a flow injection analysis (FIA) system for the specific determination of glucose, fructose, and sucrose. Peak height resulting from the production of hydrogen ion was correlated with sugar concentration, and the effect of operating variables on the response characteristics of the FIA system was investigated on the basis of theoretical and experimental analyses. Under the optimized conditions, the calibration curves for glucose, fructose, and sucrose were linear up to 8, 80, and 60 g/L, respectively. The FIA system was applied to the online monitoring of glucose production in the enzymatic hydrolysis of cellulose, and the glucose concentrations determined using the FIA system coincided well with those determined by the conventional enzymatic method.
Thermal Deactivation of Free and Immobilized β‐Glucosidase from Penicillium funiculosum
Authors: Aguado José , Romero M. Dolores , Rodríguez Lourdes , Calles José A.
Published: March 2005

Thermal deactivation of free and immobilized β‐glucosidase from Penicillium funiculosum has been studied in the temperature range 30–70 °C. Immobilization results in a remarkable increase in β‐glucosidase thermal stability. A two‐step series deactivation mechanism has been used to describe the experimental results for both free and immobilized β‐glucosidases. The kinetic equations resulting from this model fit our experimental results with a mean deviation of less than 10%.
Rapid, High‐Yield Recovery of a Recombinant Digoxin Binding Single Chain Fv from Escherichia coli
Authors: Burks Elizabeth A. , Iverson Brent L.
Published: March 2005

We have isolated milligram quantities of active single chain antibody from the insoluble fraction of Escherichia coli cultures. The system relies on high‐level expression from a T7 RNA polymerase‐directed gene construct, 8 M urea to dissolve the desired protein out of the insoluble fraction, presumably inclusion bodies, isolation and concentration of the desired protein by nickel chelate [IDA‐Ni(II)] immobilized metal‐ion affinity chromatography (IMAC), and removal of urea from column fractions by dialysis directly into storage buffer. Routinely, about 50% of the protein loaded onto an IMAC column is recovered as single chain Fv at a concentration of approximately 0.7 mg/mL. As little as 3 days are required to obtain 10 mg of final product when starting with an overnight inoculum.