bluuebunny/crossref_metadata_2025_split huggingface dataset:
The effect of ammonia on the O‐linked glycosylation of granulocyte colony‐stimulating factor produced by chinese hamster ovary cells
Authors: Andersen Dana C. , Goochee Charles F. 
Published: December 2004

AbstractAmmonium ion concentrations ranging from 0 to 10 mM are shown to significantly reduce the sialylation of granuiocyte colony‐stimulating factor (G‐CSF) produced by recombinant Chinese hamster ovary cells. Specifically, the degree of completion of the final reaction in the O‐linked glycosylation pathway, the addition of sialic acid in an α(2,6) linkage to N‐acetylgalactosamine, is reduced by NH concentrations of as low as 2 mM. The effect of ammonia on sialylation is rapid, sustained, and does not affect the secretion rate of G‐CSF. Additionally, the effect can be mimicked using the weak base chloroquine, suggesting that the effect is related to the weak base characteristics of ammonia. In support of this hypothesis, experiments using brefeldin A suggest that the addition of sialic acid in an α(2,6) linkage to N‐acetylgalactosamine occurs in the trans‐Golgi compartment prior to the trans‐Golgi network, which would be expected under normal conditions to have a slightly acidic pH in the range from 6.5 to 6.75. Ammonium ion concentrations of 10 mM would be expected to reduce significantly the differences in pH between acidic intracellular compartments and the cytoplasm. The pH‐activity profile for the CHO O‐linked α(2,6) sialytransferase using monosialylated G‐CSF as a substrate reveals a twofold decrease in enzymatic activity across the pH range from 6.75 to 7.0.Mathematical modeling of this sialylation reaction supports the hypothesis that this twofold decrease in sialyltransferase activity resulting from an ammoniainduced increase in trans‐Golgi pH could produce the observed decrease in G‐CSF sialylation. © 1995 John Wiley & Sons, Inc.
Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation
Authors: Ohno Akihiro , Ano Takashi , Shoda Makoto 
Published: December 2004

AbstractProduction of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa‐14, a gene related to surfactin production cloned at our laboratory from a wild‐type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37°C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30°C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4–5 times more efficient than in SMF. © 1995 John Wiley & Sons, Inc.
Studies of transcriptional state heterogeneity in sporulating cultures of bacillus subtilis
Authors: Chung John D. , Stephanopoulos Gregory 
Published: December 2004

AbstractUsing a flow cytometry‐based technique for detecting p‐galactosidase. lacZ fusions have been used to study the pattern of gene expression exhibited by stationary phase cultures of Bacillus subtilis that have initiated the developmental pathway of spore formation. We have found that within such cultures there exist two distinct cell types: one that has induced the developmental program of gene expression and one that has not. This heterogeneity among transcriptional states is shown to be established early during the stationary phase and plays a significant role in influencing later stationary phase events. Additionally, when this technique is used to study gene expression in mutants that display altered patterns of gene expression, we are able to conclude that the cellular apparatus responsible for sensing and transducing stationary phase developmental signals represents a bottleneck that controls the expression of certain early stationary phase genes. These findings provide a demonstration of the utility of single cell measurements of gene expression and indicate that unless culture heterogeneity is properly taken into account, standard measurements of gene expression may not provide information suitable for the analysis of events occurring at the cellular level. © 1995 John Wiley & Sons, Inc.
Nutrient enrichment and in‐situ waste removal through electrical means for hybridoma cultures
Authors: Chang Yu‐Hsiang David , Grodzinsky Alan J. , Wang Daniel I. C. 
Published: December 2004

AbstractIn‐situ dc electric fields were applied to remove ammonium and lactate from suspension hybridoma cultures (ATCC‐CRL‐1606) which used enriched media. Nutrient concentration was increased fourfold above the normal concentration of DMEM to study enhanced protein product formation in a dc electric field. In the presence of the electric field, hybridoma growth and antibody production were increased 1.5‐fold (from 3.7 × 106 to 9.1 × 106 viable cells/mL) and twofold (from 170 to 505 mg IgG/L), respectively, compared with the control. The effective removal of ammonium and lactate and increased concentrations of the various nutrients accounted for this enhancement. The enriched media caused the overflow metabolism of glucose, glutamine, and various essential amino acids. The overconsumption of glucose also produced substantial amounts of lactate, which in turn greatly increased the medium osmolarity. The increase in medium osmolarity is believed to be one of the causes of cell death in these culture systems.© 1995 John Wiley & Sons, Inc
Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein‐free medium
Authors: Renner Wolfgang A. , Lee Kelvin H. , Hatzimanikatis Vassily , Bailey James E. , Eppenberger Hans M. 
Published: December 2004

AbstractExogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell‐cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein‐free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell–cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell‐cell contact and a transformed and rounded‐up morphology. Compared with insulin‐stimulated cells, bFGF‐stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild‐type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein‐free basal medium and had similar cyclin b levels, distributions of nuclear cell‐cycle times, and cell morphologies as bFGF‐stimutated CHO K1 culture. Metabolic engineering of cell‐cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. © 1995 John Wiley & Sons Inc.
The role of glucose in ajmalicine production by catharanthus roseus cell cultures
Authors: Schlatmann J. E. , Koolhaas C. M. A , Vinke J. L. , ten Hoopen H. J. G. , Heijnen J. J. 
Published: December 2004

AbstractThe role of glucose in ajmalicine production by Catharanthus roseus was investigated in the second stage of a two‐stage batch process. Activities of tryptophan decar‐boxylate (TDC) and anthranilate synthase (AS), two enzymes In the pathway leading to ajmalicine, were higher after induction with 40 g/L glucose than after induction with 60 or 80 g/L glucose. Experiments with different media containing mixtures of glucose and the nonpermeating osmotic agent xylose, and using an already induced culture as inoculum, revealed that a minimum amount of glucose is required to support ajmalicine production after enzyme induction. This requirement was not an osmotic effect. The relation between the glucose concentration and the specific ajmalicine production rate, qp, was investigated in seven (fed‐)batch cultures with constant glucose concentrations: 23, 29, 35, 53, 57, 75, and 98 g/L. In the cultures with a low glucose concentration (23, 29, and 35 g/L) the qp was 2.7‐times higher than the cultures with 53 and 57 g/L, and almost six times higher than the cultures with a high glucose concentration (75 and 98 g/L). A glucose perturbation experiment (from 53 to 32 g/L) demonstrated that the ajmalicine production rate was adjusted without much delay. A kinetic equation is proposed for the relationship between the glucose concentration and qp. Differences in enzyme induction and ajmalicine production at different glucose levels could not be explained by the intracellular concentrations of glucose, fructose, sucrose, or starch. © 1995 John Wiley & Sons Inc.
Surface IgG content of murine hybridomas: Direct evidence for variation of antibody secretion rates during the cell cycle
Authors: Charlet Marc , Kromenaker Sandra J. , Srienc Friedrich 
Published: December 2004

AbstractPrevious experiments have shown that population average surface lgG content is correlated with the specific antibody production rates of batch hybridoma cultures. Therefore, surface associated lgG content of single hybridoma cells might indicate antibody secretion rates of individual cells. Moreover, the surface lgG content should reflect the pattern of secretion rates during the cell cycle. To probe for lgG secretion rates during the cellcycle, a double staining procedure has been developed allowing simultaneousflow cytometric analysis of surface lgG content and DNA content of murine hybridoma cells. Crosslinking of the surface associated immunofluorescence with the cell by paraformaldehyde fixation permits subsequent DNA staining without loss of immunofluorescence. The optimized protocol has been used to determine the pattern of the surface lgG fluorescence as a function of the cell cycle position. It is highest during the G2+M cell cycle phase and the experimental data are in excellent agreement with the previously predicted secretion pattern during the cell cycle. © 1995 John Wiley & Sons Inc.
Kinetic model of disaccharide oxidation by Agrobacterium tumefaciens
Authors: Walter Jan , Jördening Hans‐Joachim 
Published: December 2004

AbstractDisaccharides were microbaially transformed to their corresponding 3‐keto‐derivatives by resting cells of Agrobacterium tumefaciens NCPPB 396. The kinetics and yield of this highly specific oxidation depend on several factors. The oxygen concentration especially has a major influence on the production of 3‐keto‐derivatives and was investigated kinetically with respect to low stationary oxygen concentrations in solution. Experiments showed unconventional results that conflicted with normal Michaelis‐Menten kinetics. A kinetic model was developed and the kinetic constants were calculated. The model and experimental data for sucrose, maltose, iso‐maltulose (palatinose), and leucrose are in good agreement with each other. Initial reaction rates with different sugars using constant oxygen concentrations resulted in a Michaelis–Mentent type function. The complete kinetics, including the effect of disaccharide and oxygen concentrations, are presented. © 1995 John Wiley & Sons, Inc.
Characterization of an oligonucleotide‐binding fluorescent ligand for application in affinity purification of dsDNA
Authors: Haynes Charles A. , Sherwood Christopher S. , Turner Robin F. B. 
Published: December 2004

AbstractThe fluorescent probe PO‐PRO‐3 was investigated as a potential ligand for the affinity immobilization and purification of genomic or plasmid DNA fragments. Affinities and mechanisms for PO‐PRO‐3 binding to superhelical and linearized pUC 18 plasmid DNA were examined through measurement of binding isotherms, continuous‐variation analysis, and DNA titrations. In addition, the effects of DNA conformation, protein and RNA contaminants, solvent polarity, and ionic strength are examined with the aim of optimizing binding and elution conditions and of assisgning limits to the range of applicability of the affinity purification. © 1995 John Wiley & Sons, Inc.
Transesterification catalyzed by polystyrene‐supported chymotrypsin in toluene: The effect of neutralization of basic or acidic groups attaching to polystyrene resins
Authors: Ohtani Noritaka , Inoue Yukihiko , Kobayashi Atsushi , Sugawara Toshiki 
Published: December 2004

AbstractCrosslinked polystyrene resins containing a low level of either basic or acidic groups were used for supports of α‐chymotrypsin (CT), which catalyzed the transesterification of N‐acetyl‐L‐phenylalanine ethyl ester (AcPheOEt) with propanol in toluene. With a minimal amount of water, CT was sorbed to the resins, basic or acidic groups of which were partly or fully neutralized by several soluble acids or bases. With an increasing degree of neutralization of basic resins by free acids, the rate of disappearance of AcPheOEt was decreased, whereas the by‐product formation of AcPheOH, due to hydrolysis, was considerably suppressed, compared with the ester‐exchange product, AcPheOPr. The pKa value of the neutralizing acid was also important for both CT activity and reaction selectivity. AcPheOPr was selectively produced at a certain range of pKa values. On the other hand, the neutralization of acidic resins with free amines enhanced the CT activity but a strong base promoted the formation of hydrolysis product. © 1995 John Wiley & Sons, Inc.
A combined cell‐cycle and metabolic model for the growth of hybridoma cells in steady‐state continuous culture
Authors: Martens D. E. , Sipkema E. M. , de Gooijer C. D. , Beuvery E. C. , Tramper J. 
Published: December 2004

AbstractThe Model presented in this work demonstrates the combination of cell‐cycle model with a model describing the growth and conversion kinetics of hybridoma cells in a steady‐state continuous culture. The cell‐cycle model is based upon a population balance model as described by Cazzador et al. and assumes the existence of a cycling‐and apoptotic‐cell population, which together form the viable‐cell population. In this part the fraction of apoptotic cells, the age distribution of the cycling and apoptotic‐cell population, the mean volume and biomass content per cell of the cycling, apoptotic, and viable cells, and the specific growth and death rates of the cells are calculated. The metabolic part consists of a Monod‐type growth equation, four elemental balances, an equation assuming a constant yield of ammonia on glutamine, an equation for product formation, and the relation of Glacken for energy production. Furthermore, a maintenance‐energy model for the consumption of glucose and glutamine is introduced, which combines the approaches of Herbert and Pirt into one model in a way similar to Beeftink et al. For energy consumption a Pirt model is assumed. The model is capable of predicting trends in steady‐state vaues of a large number of variables of interest like specific growth rate, specific death rate, viability, cell numbers, mean viable‐cell volume, and concentrations and conversion rates of product, glucose, glutamine, lactate, and ammonia. Also the concentrations and conversion rates of oxygen and carbon dioxide are qualitatively predicted. The values of the model predictions are generally close to experimental data obtained from literature. © 1995 John Wiley & Sons, Inc.
Studies on the synthesis of βhCG hormone in vero cells by recombinant vaccinia virus
Authors: Mukhopadhyay Asok , Mukhopadhyay S. N. , Talwar G. P. 
Published: December 2004

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. mm2 βhCG/106 cells. h and 1.55 AU. mm2 βhCG/106 cells. 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 with 125I‐hCG for radioreceptors and stimulated testosterone synthesis in Leydig cells. © 1995 John Wiley & Sons, Inc.
Sporulation: An alternative way to recover recombinant proteins from Bacillus subtilis
Authors: Maurizi Giovanni , Bossù Paola , Boraschi Diana , Ulisse Edy , Tagliabue Aldo , Ruggiero Paolo 
Published: December 2004

AbstractA recombinant strain of Bacillus subtilis engineered for endocellular expression of human interleukin‐1 receptor antagonist (IL‐Ira) was subjected to sporulation. The recombinant protein was recovered from the sporulation supernatant in quantities, purity, and activity comparable with those obtained from a traditional cell lysate. Thus, exploitation of this natural mechanism of autolysis could overcome problems of intact protein recovery related to the cell disruption step. © 1995 John Wiley & Sons, Inc.
Real‐time monitoring of protein secretion in mammalian cell fermentation: Measurement of monoclonal antibodies using a computer‐controlled HPLC system (BioCad/RPM)
Authors: Ozturk S. S. , Thrift J. C. , Blackie J. D. , Naveh D. 
Published: December 2004

AbstractOn‐line, “real‐time” monitoring of product concentration is important for mammalian cell culture fermentation. The continuous measurement of monoclonal antibodies allows for instantaneous determination of cell productivity and effective manipulation of the fermentor operating conditions for optimal production. This article will present the evaluation and application of a BioCad/RPM system (Per Septive Biosystems) for rapid analysis of lgG concentration for hybridoma cell cultivation. Several commercial crossflow filtration devices are tested for low protein retention and fouling properties. A protein G column is used successfully for analyzing about 400 samples of lgG1, without significant loss in separation efficiency. The Immuno Detection system is integrated into a computer‐controlled 15‐L fermentor. This fermentor could be operated in batch and perfusion modes with cell densities up to 20 million cells/mL. A continuous cell‐free sample stream obtained by a hollow fiber filter system is introduced to the BioCad/RPM for analysis. The speed of this system allows for real‐time monitoring even at high densities with fast dynamics. A murine hybridoma cell (A10G10) is cultivated in batch and continuous reactors and antibody concentration is measured continuously with complete sterility. The results are compared to offline measurements with good agreement. © 1995 John Wiley & Sons, Inc.
Fed‐batch culture of Bacillus thuringiensis for thuringiensin production in a tower type bioreactor
Authors: Jong Jian‐Zhong , Hsiun Ding‐Yu , Wu Wen‐Teng , Tzeng Yew‐Min 
Published: December 2004

AbstractFed‐batch culture of Bacillus thuringiensis in a modified airlift reactor has been developed by using adaptive control of glucose concentration in the reactor. The glucose concentration was estimated via a correlation equation between carbon dioxide production rate and glucose consumption rate. The estimated glucose concentration as the output variable was fed back to computer for calculation of substrate addition. The modified reactor was an airlift reactor with a net draft tube. The airlift reactor had high oxygen transfer rate and low shear stress which were important factors for production of thuringiensin. Fed‐batch culture of Bacillus thuringiensis in the modified airlift reactor provided significant improvement of thuringiensin production. © 1995 John Wiley & Sons, Inc.
The effect of particle morphology and concentration on the directly measured yield stress in filamentous suspensions
Authors: Mohseni M. , Allen D. G. 
Published: December 2004

AbstractThe influence of concentration (mass and volume fraction) and particle morphology on the yielding properties of filamentous broths ofAspergillus niger and Strepto‐myces levoris was investigated using the rotating vane technique and compared with those of pulp suspensions. Two methods were applied to determine the volume fraction of the cells growing in filamentous form: the measurement of interstitial volume using a high molecular weight dye or dextran, and the measurement of dewatered broth filter cake volume by displacement using a pycnometer. The latter method provided the most reliable results. Cell concentrations ranging from 3 to 20 g dw/L, with corresponding volume fractions between 0.005 and 0.05, were obtained with broths generated in stirred tank and shake flask fermentations. The yield stress values obtained using the vane technique (0.1 ≤ τy ≤ 37 Pa) correlated with biomass concentration (X, grams dry weight per liter) using the equation τy = aXb with “b” ranging between 2 and 3. Increasing the length of organisms significantly increased the yield stress at a given cell concentration; this morphology effect was described using a “morphology factor” (range 0.016 to 0.11 Pa/[g dw/L]2.5), determined by setting the value of “b” to 2.5. The vane yield strain, which is the critical strain or deformation at the yield point, was independent of cell concentration, but increased with increasing length of organisms. Filamentous fermentation broths with highly branched organisms had a greater yield stress than pulp suspensions. © 1995 John Wiley & Sons, Inc.