摘要:

AbstractA numerical model based on the convective‐diffusive transport of reacting and adsorbing proteolytic enzymes within erodible fibrous biopolymers was used to predict lysis fronts moving across biogels such as fibrin or collagen. The fiber structure and the transport properties of solutes in fibrin (or collagen) were related to the local extent of dissolution within the dissolving structure. An accounting for solubilization of adsorbed species into solution from the eroding fiber phase provided for complete conservation of mass in reacting systems containing over 10 species. At conditions of fibrinolysis typical of clinical situations, the model accurately predicted the dynamic rate of lysis front movement for plasmin, urokinase, and tissue plasminogen activator (tPA)‐mediated lysis of fibrin gels measured in vitro. However, under conditions of extremely fast fibrinolysis using high enzyme concentrations, fibrinolytic fronts moved very rapidly (>0.1 mm/mm)—faster than predicted for diffusionlimited reactions—at nearly constant velocity for over 2 h, indicating non‐Fickian behavior. This was due to proteolysis‐mediated retraction of dissolving fibrin fibers that resulted in fiber convection and front‐sharpening within 3 μm of the reaction front, as observed by digitally enhanced microscopy. In comparing the model to fibrinolysis measurements using human lys77‐plasmin, the average first order rate constant for non‐crosslinked fibrin bond cleavage by fibrin‐bound plasmin was calculated to be 5s−1assuming that 10 cleavages per fibrin monomer were required to solubilize each monomer. The model accurately predicted lysis front movement using pressure‐driven permeation of plasmin or urokinase into fibrin as well as literature data obtained under well‐ mixed conditions for tPA‐mediated fibrinolysis. This numerical formulation provides predictive capability for optimization of proteolytic systems which include thrombolytic therapy, wound healing, controlled drug release, and tissue engineering applications.

ISSN:0006-3592    

DOI:10.1002/bit.260480203

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe hydraulic resistance and membrane fouling effects ofCandida utilisin fermentation broth were investigated using Millipore PVDF 0.22‐μm membranes (GVWP and GVHP) in a stirred‐cell system at 50 kPa and 700 rpm. With the various components of broth, spent medium, which contains colloidal particles and macromolecules having sizes (0.32 to 2.67 μm) comparable with the membrane pores (actual range 0.26 to 0.63 μm), was found to be the major contributing factor to the membrane fouling by broth through pore plugging. This led the spent medium to exhibit the highest hydraulic resistance (Rsmof 5.8E+ 12 m−1) and percentage flux loss (81.0%) when compared with either intact cells alone in buffer or to whole broth. Intact cells appeared to physically block and protect the pores without significant adhesion, because of the relatively hydrophilic nature of their cell walls (hydrophobicity of 5.9% at hour 36), resulting in the lowest hydraulic resistance (Rsbcof 7.5E+ 11m−1) and percentage flux loss (19.3%).However, the hydraulic resistance and percentage flux loss of broth increased as cells aged. This was attributed to the increase in particle loading (intact cells by 15.37%, released cell contents and cell fragments) and in the hydrophobicity of cell walls. Autoclaved broth, lysed broth and aged broth, which contained a larger portion of colloidal particles and released cell contents caused a more pronounced fouling effect. This was revealed by the absence of flux recovery after depressurization with continuous stirring, even when a hydrophilic membrane was used. Furthermore, the hydrophobicity ofC. utiliswas found to increase with yeast extract present in medium, and use of hydrophobic membranes helped enhance the fouling effect. Overall, the degree of irreversible membrane fouling could be revealed by the value ofRsm/Rt′and the hydraulic resistance, which resulted from concentration polarrzation, could be revealed by the value ofRc/Rt′whereRt=Rm+Rsm+Rc′andRmis the clean membrane resistance. © 1995 Joh

ISSN:0006-3592    

DOI:10.1002/bit.260480204

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

年代:1995

数据来源: WILEY

摘要:

AbstractHuman bcl‐2 DNA was introduced into mouse hybridoma 2E3 cells and expressed at a high level by using BCMGSneo vector, which reportedly amplifies as multiple copies in the cells independently of their chromosomes. The high expression of bcl‐2 in BCMGSneo‐bcl‐2 transfectants was confirmed by western blotting. In batch cultures, the overexpression of bcl‐2 raised the maximum viable cell density by 45%, delayed the initiation of apoptosis by 2 days, and prolonged the viable culture period by 4 days. The delayed initiation of apoptosis was detected by emergence of the ladder pattern on DNA electrophoresis and increase of the dead cell number. The bcl‐2 transfectants produced lgG1fourfold per batch culture in comparison with 2E3 cells transfected with BCMGSneo but not with bcl‐2: a little less than twofold due to the improved survival of the cells and more than twofold due to the enhanced lgG1production rate per cell of the bcl‐2 transfectants. The method to engineer hybridoma cells genetically with bcl‐2 using BCMGSneo vector for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures. © 1995 Jo

ISSN:0006-3592    

DOI:10.1002/bit.260480205

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe effect of different concentrations and combinations of oxygen, carbon dioxide, and ethylene on cell growth and taxol production in suspension cultures ofTaxus cuspidatawas investigated using several factorial design experiments. Low head space oxygen concentration (10% v/v) promoted early production oftaxol. High carbon dioxide concentration (10% v/v) inhibited taxol production. The most effective gas mixture composition in terms of taxol production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Cultures grown underambient concentration of oxygen had a delayed uptake of glucose and fructose compared to cultures grown under 10% (v/v) oxygen. Average calcium uptake rates into the cultured cells decreased and average phosphate uptake rates increased as ethylene was increased from 0 to 10 ppm. These results may indicate that gas composition alters partitioning of nutrients, which in turn affects secondary metabolite production. © 1995 John Wiley&Sons, Inc

ISSN:0006-3592    

DOI:10.1002/bit.260480206

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe carminomycin 4‐O‐methyltransferase enzyme fromStreptomyces peucetiuswas covalently immobilized on 3M Emphaze ABI‐activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S‐adenosyl‐L‐methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4°C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparentKm(K′m) for carminomycin while theK′mforS‐adenosyl‐L‐methionine was approximately doubled. A 57% decrease in theVmaxvalue occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. © 19

ISSN:0006-3592    

DOI:10.1002/bit.260480207

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

年代:1995

数据来源: WILEY

摘要:

AbstractIncreased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6‐aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding forEscherichia colipenicillin acylase was subjected to oligonucleotide‐directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned inE. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431‐Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half‐life of the mutant enzyme when stored at pH 8.5 as compared with the wild‐type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH‐stability profile. © 1995 John Wil

ISSN:0006-3592    

DOI:10.1002/bit.260480208

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

年代:1995

数据来源: WILEY

摘要:

AbstractNutrient depletion and inhibitory end‐product accumulation are the major problems for hydrogel‐entrapment cell cultures. An electrokinetic technique was developed to enhance intrahydrogel mass transfer to overcome these problems.Escherichia colicells (ATCC 15224) were entrapped in 3.2‐mm‐thick potassium‐K‐carrageenan and agarose hydrogel slabs. With a electric current density of 180A/m2the cell densities were increased by 140%(from 3.9 to 9.6 dry cell weight [DCW] g/L) in potassium‐K‐carrageenan and by 80% (from 3.9 to 7.0 DCW g/L) in agarose. A mathematical model taking into account nutrient depletion, inhibitory end‐product formation, and cell growth kinetics under facultatively anaerobic conditions was developed to rationalize the overall transport and biological behaviors in the hydrogel. The cell growth in hydrogel was successfully simulated. It is concluded that the augmented transports for glucose and inhibitory end‐products accounted for these increases in cell growth. The increase in cell density in potassium‐K‐carrageenan was due to the enhanced removal of inhibitory end‐products (through electroosmosis and electro‐phoresis: 80%) and due to the augmented glucose transport (through electroosmosis: 20%).

ISSN:0006-3592    

DOI:10.1002/bit.260480209

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

年代:1995

数据来源: WILEY

摘要:

AbstractSynthesis of the β‐subunit of human chorionic gonado‐tropin (βhCG) in Vero cells by the recombinant vaccinia virus has been studied. The yield of βhCG was a function of the multiplicity of infection (MOI), and was highest at 25 MOI. The kinetics of synthesis and initial secretion of βhCG, deduced from the pulse‐chase experiments were “zero order.” At 30 h postinfection, the relative values of net synthesis and secretion rates were 4.0 AU. mm2βhCG/106cells. h and 1.55 AU. mm2βhCG/106cells. h, respectively. The time required to secrete 50% of intracellular βhCG was 210 min. Pulse‐chase data also showed that 24% of βhCG was degraded intracelluiarly within 10 h, of which 17% was detected in the autoradiogram. Along with 30 kD βhCG, a satellite band of 28 kD was evident among the peptide synthesized in Vero cells. The molecular weight of vaccinia‐derived βhCG was 13 kD more that its nonglycosylated form, indicating extensive glycosylation in Vero cells. The mRNA levels in infected Vero cells at different postinfection times were quantified by excess DNA dot‐blot hybridization. It appears that the Vero cell possesses some host cell‐associated factor(s), which prevented the transcription of early βhCG–mRNA promoted by the early signal of the vaccinia P 7.5 promoter. The half‐life of βhCG–mRNA, as determined by follow‐up of decay after blocking transcription initiation, was found to be 6.4 h. The synthesized βhCG was immunoreactive as it reacted with monoclonal and polyclonal monospecific antibodies. The subunit was also biologically active, as it combined with native βhCG to form heterodimer βhCG, which competed with125I‐hCG for radioreceptors and stimulated testosterone synthesis in

ISSN:0006-3592    

DOI:10.1002/bit.260480210

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

年代:1995

数据来源: WILEY

摘要:

AbstractChemical treatments similar to those routinely used to extract cellulose from plant biomass caused significant increases in the relative crystallinity index (RCI) of Sig‐macell 100 (a commercial cellulose of moderate crystallinity), as measured by x‐ray powder diffraction in both the reflectance and transmittance modes. In general, the largest increases in RCI were observed following higher (rather than lower) temperature treatments. Substantial increases in crystalliity were also observed upon resuspension in water prior to drying, with higher temperatures again resulting in the greatest increases in RCI. Measurement of the RCIs of wetted Sigmacell 100 samples by acid hydrolysis kinetics revealed that most of the increased crystallinity occurred rapidly upon contact with water. In contrast to Sigmacell 100, a cellulose of higher initial crystallinity (the microcrystalline cellulose Sigmacell 50) showed little change in crystallinity following the above treatments. The results provide a partial explanation for the inconsistent relationships reported between cellulose crystallinity and cellulose biodegradation. © 1995 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260480211

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

年代:1995

数据来源: WILEY

ISSN:0006-3592    

DOI:10.1002/bit.260480202

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

年代:1995

数据来源: WILEY