Chemical decomposition of glutamine in cell culture media: effect of media type, pH, and serum concentration.

The chemical decomposition of glutamine to ammonia and pyrrolidonecarboxylic acid was studied at 37 degrees C in a pH range of 6.8-7.8 in different media preparations containing various amounts of fetal bovine serum. The media type influenced the decomposition rate, and the first-order rate constants increased with increasing pH values. The serum concentration had little or no effect on the decomposition rate. The importance of chemical decomposition of glutamine on the analysis of glutamine and ammonia metabolism was illustrated by an example of batch cultivation of a hybridoma cell line. The difference between the apparent uptake rate of glutamine and the actual uptake rate (which is corrected for the chemical decomposition) is shown to be as high as 200%. Similar discrepancy between the apparent and actual ammonia production rate is observed. Mathematical analysis was carried out to develop the relationship between the apparent and actual glutamine uptake and ammonia production rates. The analysis reveals that there are three important dimensionless parameter ratios that govern the difference between the apparent and actual glutamine uptake and ammonia production rates.     

Surfactant-lactoperoxidase complex catalytically active in organic media

A surfactant-lactoperoxidase (LPO) complex catalytically active in organic solvents was developed by the emulsion coating method. The oxidation of 2,6-dimethoxyphenol (2,6-DMP) was conducted by the surfactant-LPO complex in organic media. The LPO complex efficiently catalyzed the oxidation of 2,6-DMP in various organic solvents, although lyophilized LPO did not display the catalytic activity at all. To optimize the preparation and reaction conditions for the surfactant-LPO complex, we examined the effects of pH value in the water pools of W/O emulsions, kinds of oxidants, and the nature of organic solvents on the oxidation reaction. Its optimum activity was obtained when the pH value of the aqueous enzyme solution was adjusted to ca. 8 at the preparation stage. The LPO complex exhibited the highest catalytic activity in chloroform when H(2)O(2) was employed as the oxidant. Furthermore, the storage stability of the surfactant-LPO complex was far better than that of the surfactant-horseradish peroxidase complex. This high storage stability of the LPO complex will be a benefit for industrial usage of peroxidases.     

Freezing of isolated thylakoid membranes in complex media

Chloroplast thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw treatment in a buffered medium containing 70 mM KCl, 30 mM NaNO₃ and 20 mM K₂SO₄ in different combinations. In the presence of the three predominant inorganic electrolytes, inactivation of photophosphorylation was mainly caused by a decrease in the capacity of the photosynthetic electron transport; release of proteins from the membranes was not manifest and light-induced H⁺ gradient and proton permeability were largely unaffected. Omission of nitrate from the medium had little effect. When either sulfate or chloride or both were omitted prior to freezing, inactivation of photophosphorylation was correlated with stimulation of the phosphorylating electron flow, marked increase in H⁺ permeability and loss of the ability of the thylakoids to accumulate protons in the light. In the absence of sulfate, uncoupling was mainly a consequence of the dissociation of chloroplast coupling factor (CF₁). Partial restoration of proton impermeability and pH gradient occurred upon the addition of N,N-dicyclohexylcarbodiimide (DCCD). When sulfate was present but chloride omitted, CF₁ remained attached to the membranes and the addition of DCCD had no effect, indicating that the increase in proton efflux was caused by a different mechanism. It is concluded that sulfate stabilizes the CF₁ and prevents its release from the membranes, but KCl is also necessary for maintaining the low permeability of the membranes to protons. The importance of complex media for investigations on isolated biomembrane systems is stressed.Abbreviations CF₁ chloroplast coupling factor - DCCD N,N-dicyclohexylcarbodiimide - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid I=Santarius 1986 b     

Modeling of the TCR-MHC-peptide complex

The methodology for generating a homology model of the T1 TCR-PbCS-K(d) class I major histocompatibility complex (MHC) class I complex is presented. The resulting model provides a qualitative explanation of the effect of over 50 different mutations in the region of the complementarity determining region (CDR) loops of the T cell receptor (TCR), the peptide and the MHC's alpha(1)/alpha(2) helices. The peptide is modified by an azido benzoic acid photoreactive group, which is part of the epitope recognized by the TCR. The construction of the model makes use of closely related homologs (the A6 TCR-Tax-HLA A2 complex, the 2C TCR, the 14.3.d TCR Vbeta chain, the 1934.4 TCR Valpha chain, and the H-2 K(b)-ovalbumine peptide), ab initio sampling of CDR loops conformations and experimental data to select from the set of possibilities. The model shows a complex arrangement of the CDR3alpha, CDR1beta, CDR2beta and CDR3beta loops that leads to the highly specific recognition of the photoreactive group. The protocol can be applied systematically to a series of related sequences, permitting the analysis at the structural level of the large TCR repertoire specific for a given peptide-MHC complex.     

The flocculation of yeast with chitosan in complex fermentation media: The effect of biomass concentration and mode of flocculant addition

Summary The minimum effective dose of chitosan necessary to flocculate a suspension of yeast in a complex medium is not proportional to the yeast concentration in the medium and is dependent upon the mode of addition of the flocculant to the suspension.     

Whole cell biocatalysis in nonconventional media

Summary In this paper biocatalytic reactions carried out by whole cells in nonconventional media are reviewed. Similar relationships are observed between solvent hydrophobicity and catalytic activity in reactions carried out by isolated enzymes and whole cells. In addition to the effect of organic solvent on biocatalyst stability, microbial cells are susceptible to damaging effects caused by the organic phase. In general, more hydrophobic solvents manifest lower toxicity towards the cells. Whole cell biocatalysts require more water than isolated enzymes and two-phase systems have been most widely used to study whole cell biocatalysis. Immobilization makes cell biocatalysts more resistant to organic solvents and helps achieve homogeneous biocatalyst dispersion. Cell entrapment methods have been widely used with organic solvent systems and mixtures of natural and/or synthetic polymers allow adjustment of the hydrophobicity-hydrophilicity balance of the support matrix. Some examples of stereoselective catalysis using microbial cells in organic solvent media are presented.     

Modeling of growth, lactate consumption, and volatile fatty acid production by Megasphaera elsdenii cultivated in minimal and complex media

Use of the Pirt and Luedeking-Piret equations permits the determination of the effect of medium composition on the metabolic patterns of Megasphaera elsdenii grown in minimal and complex media with lactate as the major carbon source. To establish the significance of the parameters involved in the Pirt and Luedeking-Piret equations, a quantitative statistical criterion was proposed. In the complex medium, lactate was completely used for growth and product formation, whereas in the minimal medium a fraction of the energy obtained from lactate was used for maintenance purposes. Modeling of VFA production by the Luedeking-Piret equation showed that, independent of the type of medium, acetate and propionate are growth-associated products, while butyrate and valerate are only partially growth-associated. The growth-associated products are related to energy-yielding metabolism and the non-growth-associated products are related to the consumption of reducing equivalents.     

Thermal stress of Pseudomonas fluorescens in complex media.

Pseudomonas fluorescens (P7) cells were stressed by incubation at 43 degrees C for 2 h. The stress induced a 9-h lag in replication after the return of the temperature of the culture to 25 degrees C. Stressed cells demonstrated a sensitivity to diluents and plating media during the recovery period. Data from utilization of selective inhibitors suggested that ribonucleic acid and protein, but not deoxyribonucleic acid, syntheses were required for recovery by the cells. The cells lost uracil- and leucine-labeled material as a result of the stress, further suggesting that ribonucleic acid and protein damage had occurred. Membrane damage was indicated by sensitivity to sodium dodecyl sulfate near the end of the lag period. Membrane damage was also suggested by the failure of cells to incorporate labeled material from the recovery medium. The lesions induced in this foodlike system are compared with those previously reported for a minimal media model system (Gray et al., Appl. Microbiol. 26:78-85, 1973; Gray et al., Appl. Environ. Microbiol. 33:1074-1078, 1977).     

Thermal stress of Pseudomonas fluorescens in complex media.

Pseudomonas fluorescens (P7) cells were stressed by incubation at 43 degrees C for 2 h. The stress induced a 9-h lag in replication after the return of the temperature of the culture to 25 degrees C. Stressed cells demonstrated a sensitivity to diluents and plating media during the recovery period. Data from utilization of selective inhibitors suggested that ribonucleic acid and protein, but not deoxyribonucleic acid, syntheses were required for recovery by the cells. The cells lost uracil- and leucine-labeled material as a result of the stress, further suggesting that ribonucleic acid and protein damage had occurred. Membrane damage was indicated by sensitivity to sodium dodecyl sulfate near the end of the lag period. Membrane damage was also suggested by the failure of cells to incorporate labeled material from the recovery medium. The lesions induced in this foodlike system are compared with those previously reported for a minimal media model system (Gray et al., Appl. Microbiol. 26:78-85, 1973; Gray et al., Appl. Environ. Microbiol. 33:1074-1078, 1977).     

Modeling TCR signaling complex formation in positive selection

T cell receptor signaling in the thymus can result in positive selection, and hence progressive maturation to the CD4(+)8(-) or CD4(-)8(+) stage, or induction of apoptosis by negative selection. Although it is poorly understood how TCR ligation at the CD4(+)8(+) stage can lead to such different cell fates, it is thought that the strength of signal may play a role in determining the outcome of TCR signaling. In this study, we have characterized the formation of an active signaling complex in thymocytes undergoing positive selection as a result of interaction with thymic epithelial cells. Although this signaling complex involves redistribution of cell surface and intracellular molecules, reminiscent of that observed in T cell activation, accumulation of GM1-containing lipid rafts was not observed. However, enforced expression of the costimulatory molecule CD80 on thymic epithelium induced GM1 polarization in thymocytes, and was accompanied by reduced positive selection and increased apoptosis. We suggest that the presence or absence of CD80 costimulation influences the outcome of TCR signaling in CD4(+)8(+) thymocytes through differential lipid raft recruitment, thus determining overall signal strength and influencing developmental cell fate.     

Modeling complex neutrophil dynamics in the grey collie

We have developed a mathematical model for the peripheral regulation of neutrophil production mediated by granulocyte colony-stimulating factor. We have used that model to show that the pattern of neutrophil oscillations in nine grey collies is consistent with the hypothesis that cyclical neutropenia is due to an oscillatory stem cell input to the neutrophil regulatory system, and not due to autonomous oscillations in the peripheral neutrophil regulatory system. In the process of interfacing our model with the laboratory data, we have estimated parameters for the peripheral neutrophil control system consistent with higher than normal apoptotic cell loss within the recognizable neutrophil precursors. This is in agreement with other experimental data. Our estimated model parameters also predict that the peripheral neutrophil production system is globally stable in the grey collies we studied. This further supports our hypothesis that the origin of the oscillatory behavior in cyclical neutropenia is in the stem cell population, consistent with other clinical and experimental evidence.     

Rapid characterization and quality control of complex cell culture media solutions using raman spectroscopy and chemometrics

The use of Raman spectroscopy coupled with chemometrics for the rapid identification, characterization, and quality assessment of complex cell culture media components used for industrial mammalian cell culture was investigated. Raman spectroscopy offers significant advantages for the analysis of complex, aqueous‐based materials used in biotechnology because there is no need for sample preparation and water is a weak Raman scatterer. We demonstrate the efficacy of the method for the routine analysis of dilute aqueous solution of five different chemically defined (CD) commercial media components used in a Chinese Hamster Ovary (CHO) cell manufacturing process for recombinant proteins.The chemometric processing of the Raman spectral data is the key factor in developing robust methods. Here, we discuss the optimum methods for eliminating baseline drift, background fluctuations, and other instrumentation artifacts to generate reproducible spectral data. Principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were then employed in the development of a robust routine for both identification and quality evaluation of the five different media components. These methods have the potential to be extremely useful in an industrial context for “in‐house” sample handling, tracking, and quality control. Biotechnol. Bioeng. 2010;107: 290–301. © 2010 Wiley Periodicals, Inc.