摘要:

AbstractThe reconstruction of functioning human tissues ex vivo is becoming an important part of biotechnology. There are compelling scientific, clinical, and biotechnological reasons for fully or partially reconstituting human tissues such as skin, bone marrow, and liver ex vivo. In particular, bone marrow is a tissue of much importance, and there are significant societal and health benefits derived from a successfully constructed ex vivo hematopoietic system. In this article, we review the current status of this effort. The topics covered include the current understanding of the biology of human hematopoiesis, the motivation for reconstructing it ex vivo, the current state of ex vivo human hematopoietic cultures, the development of important metrics to judge culture performance, and an approach based on in vivo mimetics to accomplish this goal. We discuss some applications of functional ex vivo hematopoietic cultures and the biological and engineering challenges that face research in this area. © 1993 John Wiley&Sons, Inc

ISSN:0006-3592    

DOI:10.1002/bit.260420802

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe kinetics of the esterification of lauric acid by (‐)menthol, catalyzed byPenicillium simplicissimumlipase, was studied in water/bis‐(2‐ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsions. Due to their low water content, microemulsions assist in reversing the direction of lipase activity, favoring synthetic reactions. The kinetics of this synthesis follows a Ping‐Pong BiBi mechanism. The values of all apparent kinetic parameters were determined. The theoretical model for the expression of enzymic activity in reverse micelles, proposed by Verhaert et al. (Verhaert, R., Hilhorst, R., Vermüe, M., Schaafsma, T. J., Veeger, C. 1990. Eur. J. Biochem. 187: 59–72) was extended to express the lipase activity in an esterification reaction involving two hydrophobic substrates in microemulsion systems. The model takes into account the partitioning of the substrates between the various phases and allows the calculation of the intrinsic kinetic constants. The experimental results showing the dependence of the initial velocity on the hydration ratio,Wo= [H2O]/[AOT], of the reverse micelles, were in accordance with the theoretically predicted pattern. © 1993 John Wi

ISSN:0006-3592    

DOI:10.1002/bit.260420803

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractA kinetic study of the activity of mushroom polyphenol oxidase in an organic system was carried out to obtain detailed enzyme kinetic data in relation to optimization of reaction conditions and substrate specificity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobilized polyphenol oxidase and a hydrated solution of the substrate in chloroform) was designed. The aqueous content of the system was optimized usingp‐cresol as the substrate. With this system, a crude extract ofAgaricus bisporuswas used to hydroxylate and oxidize a range of selectedp‐substituted phenolic substrates, yieldingo‐quinone products. Michaelis–Menten kinetics were used to obtain apparentKMandVmaxvalues with respect to each of these substrates. Results from this analysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could be rationalized in terms of the restricted flexibility of the enzyme when it is in chloroform and also in terms of substrate and solvent hydrophobicity. In the course of the investigation UV molar absorption coefficients of severalo‐quinones were measured by a novel method:1H nuclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the transformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding the necessity to isolate the water‐sensitive, unstableo‐quinones. © 1993 John W

ISSN:0006-3592    

DOI:10.1002/bit.260420804

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractEscherichia coli, genetically engineered with a mercury(II)‐sensitive promoter and theluxgenes fromVibrio fischeri, were used as microbial bioluminescent sensors for the detection of mercury. Evaluation of this genetic construction was carried out by determining the effects of various parameters on cell suspensions maintained at constant conditions in a small 100‐mL vessel. The strongest light intensities and quickest induction times occurred with cells in the midexponential growth phase maintained at 28°C, concentrated to 1 × 109cells/mL, mixed at very fast speeds, and aerated at 2 vvm (volume of air per volume of culture per minute) during light measurement in the small vessel. The cells were sensitive to the mercuric ion in the range of 20 nMto 4 μM(4 to 800 ppb), and the total response time was on the order of 1 hour, depending on the above parameters. The cells exhibited great specificity for mercury. The cells had almost equal specificity for organic and inorganic forms of the mercuric ion and responded more weakly to the mercurous ion. A simple, inexpensive, durable miniature probe (3 mL) was constructed and operated using the optimum parameters found in the small vessel as a guide. The range of sensitivity to the mercuric ion detected in the probe was 10 nMto 4 μMwhen aeration was provided. © 1993 John Wiley&S

ISSN:0006-3592    

DOI:10.1002/bit.260420805

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe lipase‐catalyzed acylglycerol synthesis with fatty acids of different chain length is studied. Measured ester mole fractions at equilibrium are compared with calculated mole fractions. For these calculations the computer program TREP (Two‐phase Reaction Equilibrium Prediction) is used. This program is based on the UNIFAC group contribution method and is developed for nondilute two‐phase reaction systems.With one set of equilibrium constants, namely 1.3, 0.8, and 0.6 for monoester, diester, and triester synthesis, respectively, the equilibrium position of the reaction between glycerol and all saturated fatty acids with a chain length from 6 to 18 and oleic acid (cis‐9‐octadecenoic acid) can be calculated. Deviations, expressed as the ratio between calculated and measured ester mole fractions, usually were between 0.7 and 1.2. In the presence of solvents, the deviations of the monoester mole fractions were higher and rose up to 3. Without addition of a solvent, the ester mole fractions at equilibrium are dependent on the fatty acid chain length. With the short‐chain hexanoic acid, the monoester mole fraction is the highest ester mole fraction, while for the long‐chain oleic acid, the diester mole fraction is the highest one. The ester mole fractions become independent on the chain length of the fatty acid with a solvent added in a sufficient high concentration. Both reactions, with saturated and unsaturated C18fatty acids, lead to the same equilibrium position. The program TREP is found to make good predictions of the equilibrium amounts of ester and fatty acid. However, systematic deviations arise between measured and calculated amounts of water and glycerol in the organic phase. The calculated water and glycerol amounts are always lower than the measured ones. These deviations seem to be highest in nonpolar media and are probably due to deficiencies in the UNIFAC calculation method. Some preliminary experiments show the effect of the choice of solvent on the reaction rates. In polar solvents, the monoester production rate is enhances by a factor of 1.5 as compared to the reaction rate in a system without solvent. © 1993 John W

ISSN:0006-3592    

DOI:10.1002/bit.260420806

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractExposure of spreading anchorage‐dependent cells to laminar flow is a common technique to measure the strength of cell adhesion. Since cells protrude into the flow stream, the force exerted by the fluid on the cells is a function of cell shape. To assess the relationship between cell shape and the hydrodynamic force on adherent cells, we obtained numerical solutions of the velocity and stress fields around bovine aortic endothelial cells during various stages of spreading and calculated the force required to detach the cells. Morphometric parameters were obtained from light and scanning electron microscopy measurements. Cells were assumed to have a constant volume, but the surface area increased during spreading until the membrane was stretched taut. Two‐dimensional models of steady flow were generated using the software packages ANSYS (mesh generation) and FIDAP (problem solution). The validity of the numerical results was tested by comparison with published results for a semicircle in contact with the surface. The drag force and torque were greatest for round cells making initial contact with the surface. During spreading, the drag force and torque declined by factors of 2 and 20, respectively. The calculated forces and moments were used in adhesion models to predict the wall shear stress at which the cells detached. Based upon published values for the bond force and receptor number, round cells should detach at shear stresses between 2.5 and 6 dyn/cm2, whereas substantially higher stresses are needed to detach spreading and fully spread cells. Results from the simulations indicate that (1) the drag force varies little with cell shape whereas the torque is very sensitive to cell shape, and (2) the increase in the strength of adhesion during spreading is due to increased contact area and receptor densities within the contact area. © 1993 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260420807

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous‐flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)–polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous‐flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h−1(experiment 1). However, at a lower dilution rate of 0.015 h−1(experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensisagglutinin,Galanthus nivalisagglutinin, andDatura stramoniumagglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme‐linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA–280 nm ratio of 1.02–1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03–2.94 and 2.53–4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA–A280nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS–polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein‐ and carbohydrate‐rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long‐term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth

ISSN:0006-3592    

DOI:10.1002/bit.260420808

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractDisruption of animal cells in turbulent capillary flows has been predicted from a model of cell–hydrodynamic interactions using cell mechanical properties determined by micromanipulation. Eddies of sizes similar to or smaller than the cells are presumed to interact with those cells, causing local surface deformations. The proposed mechanism of cell damage is that such deformations result in an increase in membrane tension and surface energy and that a cell disrupts when its bursting membrane tension and bursting surface energy are exceeded. The surface energy of the cells is estimated from the kinetic energy of appropriately sized eddies. To test the model, cells were disrupted in turbulent flows in capillaries at mean energy dissipation rates up to 2 × 104m2/s3. In all cases the model underestimated the cell disruption by about 15%. Such good agreement implies that the approach of the model to the complicated phenomena of cell turbulence interactions is reasonable. © 1993 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260420809

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractA new bioreactor, in which a series of air‐lift reactors with an internal loop is incorporated into one vessel, is introduced. With this multiple air‐lift loop reactor (MAL) and approximation of an aerated plug‐flow fermentor is strived for. Mixing, liquid velocity, and gas holdup were measured as a function of the gas flow rate in this new internal‐loop reactor geometry. As a reference, hydrodynamics were also investigated in a conventional internal‐loop reactor. A model description of the hydrodynamics in the second compartment of the MAL is given. This model is based on a two‐phase, drift‐flux model and a friction coefficient. Frictional losses were independent of the reactor bottom geometry, and were observed to increase with the gas flow rate as a result of the presence of stationary gas bubbles in the downcomer. The hydrodynamics and mixing of the second MAL compartment were comparable with those of conventional internal‐loop reactors. © 1993 John

ISSN:0006-3592    

DOI:10.1002/bit.260420810

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe activities of six purifiedThermomonospora fuscacellulases andTrichoderma reeseiCBHI and CBHII were determined on filter paper, swollen cellulose, and CMC. A simple method to measure the soluble and insoluble reducing sugar products from the hydrolysis of filter paper was found to effectively distinguish between exocellulases and endocellulases. Endocellulases produced 34% to 50% insoluble reducing sugar and exocellulases produced less than 8% insoluble reducing sugar. The ability of a wide variety of mixtures of these cellulases to digest 5.2% of a filter paper disc in 16 h was measured quantitatively. The specific activities of the mixtures varied from 0.41 to 16.31 μmol cellobiose per minute per micromole enzyme. The degree of synergism ranged from 0.4 to 7.8.T. reeseiCBHII andT. fuscaE3 were found to be functionally equivalent in mixtures. The catalytic domains (cd) ofT. fuscaendocellulases E2 and E5 were purified and found to retain 93% and 100% of their CMC activity, respectively, but neither cd protein could digest filter paper to 5.2%. When E2cd and E5cd were substituted in synergistic mixtures for the native proteins, the mixtures containing E2cd retained 60%, and those containing E5cd retained 94% of the original activity. Addition of a β‐glucosidase was found to double the activity of the best synergistic mixture. Addition of CBHI toT. fuscacrude cellulase increased its activity on filter paper 1.7‐fold. © 1993 John Wiley&Son

ISSN:0006-3592    

DOI:10.1002/bit.260420811

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY