Kegg Pathway: Amino Acid Metabolism

Biosci Rep. 2008 Jul 17;
Ross-Inta C, Tsai CY, Giulivi C

Mitochondria can synthesize a limited number of proteins encoded by mtDNA by using its own biosynthetic machinery, whereas most of the proteins are imported from the cytosol. It could be hypothesized that the mitochondrial pool of Amino Acids follows the frequency of Amino Acids in mtDNA-encoded proteins or alternatively, the profile is the result of the participation of Amino Acids in pathways other than protein synthesis (e.g. heme biosynthesis, Aminotransferase reactions). These hypotheses were tested by evaluating the pool of free Amino Acids and derivatives in highly-coupled, purified liver mitochondria obtained from rats fed a nutritionally adequate diet for growth. Our results indicated that the pool mainly reflects the Amino Acid composition of mtDNA-encoded proteins suggesting that there is a posttranslational control of protein synthesis. This conclusion was supported by the following findings: one, correlation between the concentration of free Amino Acids in the matrix and the frequency of abundance of Amino Acids in mtDNA-encoded proteins; two, the similar ratios of essential-to-nonessential Amino Acids in mtDNA-encoded proteins and mitochondrial pool of Amino Acids; three, lack of a correlation between codon usage or tRNA levels and Amino Acid concentrations. Quantitative information on the mammalian mitochondrial content of Amino Acids, such as that presented in this report, along with functional studies will help us better understand the pathogenesis of mitochondrial diseases or the biochemical implications in mitochondrial Metabolism.

Biotechnol Bioeng. 1997 Oct 20; 56(2): 168-80
Marx A, Striegel K, de Graaf AA, Sahm H, Eggeling L

To evaluate the importance of reactions within the central Metabolism under different flux burdens the fluxes within the pentose phosphate pathway (PPP), as well as the other reactions of the central Metabolism, were intensively analyzed and quantitated. For this purpose, Corynebacterium glutamicum was grown with [1-(13)C]glucose to metabolic and isotopic steady state and the fractional enrichments in precursor metabolites (e.g., pentose 5-phosphate) were quantified. Matrix calculus was used to express these data together with metabolite mass data. The detailed analysis of the dependence of (13)C enrichments on exchange fluxes enabled the transketolase-catalyzed exchange rate (2 pentose 5-phosphate <--> sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate) to be quantified as 74.3% (molar metabolite flux) at a net flux of 10.3% and the exchange rate (pentose 5-phosphate + erythrose 4-phosphate <--> fructose 6-phosphate + glyceraldehyde 3-phosphate) to be quantified as 5.6% at a net flux of 8.1%. The flux entering the tricarboxylic Acid cycle was 93.3%. The same comprehensive flux analysis as performed for the nonexcreting condition was done with the identical strain that had been forced to excrete L-glutamate. Because we had already quantified the fluxes for L-lysine excretion with an isogenic strain, three directly comparable flux situations are thus available. Consequently, this comparison permits a direct cause-and-effect relationship to be specified. In response to the different flux burdens of the cell, the PPP flux decreased from a maximum of 67% to 26%, with the glycolytic flux increasing accordingly. The carbon flux through isocitrate dehydrogenase increased from 20% to 36%. The bidirectional carbon flux between pyruvate and oxaloacetate decreased from 36% to 9%. Since the cause of the three different flux states was the allelic exchange in the final L-lysine assembling pathway or the glutamate export activity, respectively, the flexible response is the effect. This shows conclusively the enormous flexibility within the central Metabolism of C. glutamicum to supply precursors upon their withdrawal for the synthesis of Amino Acids. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 168-180, 1997.

Biotechnol Bioeng. 1997 Sep 20; 55(6): 909-20
Rhiel M, Mitchell-Logean CM, Murhammer DW

Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Fivetrade mark) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell Metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and Amino Acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Fivetrade mark cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Fivetrade mark cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Fivetrade mark cell cultures, but not in Sf-9 cell cultures. In addition, High Fivetrade mark cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Fivetrade mark cell cultures. It was also found that the medium had a significant effect on High Fivetrade mark cell Metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.

Cell Cycle. 2008 May 12; 7(14):
Mullany LK, White P, Hanse EA, Nelsen CJ, Goggin MM, Mullany JE, Anttila CK, Greenbaum LE, Kaestner KH, Albrecht JH

The D-type cyclins (D1, D2 and D3) are components of the cell cycle machinery and govern progression through G(1) phase in response to extracellular signals. Although these proteins are highly homologous and conserved in evolution, they contain distinct structural motifs and are differentially regulated in various cell types. Cyclin D1 appears to play a role in many different types of cancer, whereas cyclins D2 and D3 are less frequently associated with malignancy. In this study, we transiently expressed cyclin D1, D2 or D3 in hepatocytes and analyzed transcriptional networks regulated by each. All three D-type cyclins promoted robust hepatocyte proliferation and marked liver growth, although cyclin D3 stimulated less DNA synthesis than D1 or D2. Accordingly, the three D-type cyclins similarly activated genes associated with cell division. Cyclin D1 regulated transcriptional pathways involved in the Metabolism of carbohydrates, lipids, Amino Acids, and other substrates, whereas cyclin D2 did not regulate these pathways despite having an equivalent effect on proliferation. Comparison of transcriptional profiles following 70% partial hepatectomy and cyclin D1 transduction revealed a highly significant overlap, suggesting that cyclin D1 may regulate diverse cellular processes in the regenerating liver. In summary, these studies provide the first comparative analysis of the transcriptional networks regulated by the D-type cyclins and provide insight into novel functions of these key cell cycle proteins. Further study of the unique targets of cyclin D1 should provide further insight into its prominent role in proliferation, growth and cancer.

Rapid Commun Mass Spectrom. 2008 Jul 15; 22(16): 2436-2446
Law WS, Huang PY, Ong ES, Ong CN, Li SF, Pasikanti KK, Chan EC

A method using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and (1)H NMR with pattern recognition tools such as principle components analysis (PCA) was used to study the human urinary metabolic profiles after the intake of green tea. From the normalized peak areas obtained from GC/MS and LC/MS and peak heights from (1)H NMR, statistical analyses were used in the identification of potential biomarkers. Metabolic profiling by GC/MS provided a different set of quantitative signatures of metabolites that can be used to characterize the molecular changes in human urine samples. A comparison of normalized metabonomics data for selected metabolites in human urine samples in the presence of potential overlapping peaks after tea ingestion from LC/MS and (1)H NMR showed the reliability of the current approach and method of normalization. The close agreements of LC/MS with (1)H NMR data showed that the effects of ion suppression in LC/MS for early eluting metabolites were not significant. Concurrently, the specificity of detecting the stated metabolites by (1)H NMR and LC/MS was demonstrated. Our data showed that a number of metabolites involved in glucose Metabolism, citric Acid cycle and Amino Acid Metabolism were affected immediately after the intake of green tea. The proposed approach provided a more comprehensive picture of the metabolic changes after intake of green tea in human urine. The multiple analytical approach together with pattern recognition tools is a useful platform to study metabolic profiles after ingestion of botanicals and medicinal plants. Copyright (c) 2008 John Wiley & Sons, Ltd.

ISME J. 2008 Jul 17;
Sharif DI, Gallon J, Smith CJ, Dudley E

Quorum sensing involving acyl homoserine lactones (AHLs) is a density-dependent form of intercellular communication that occurs in many different members of the group Proteobacteria. However, to date, there have been few investigations of its occurrence in cyanobacteria. Here, using both a bioreporter Agrobacterium tumefaciens NTL4 (PZLR4) and mass spectrometry, we provide evidence of N-octanoyl homoserine lactone (C8-AHL) production in axenic cultures of the cyanobacterium Gloeothece PCC6909 and its sheathless mutant PCC6909/1. Accumulation of C8-AHL in the culture medium of laboratory cultures of Gloeothece followed a pattern characteristic of the phenomenon of autoinduction, a common feature of functional AHL-based quorum-sensing systems. Analysis by two-dimensional gel electrophoresis showed that, in response to treatment with C8-AHL, early growth-stage cells of PCC6909/1 showed changes in expression of 43 proteins compared with untreated cells. Among the 15 proteins that showed more than a twofold change in expression were RuBisCo, glutamate synthase, chorismate synthase, a member of the LysR family of transcriptional regulators (all upregulated), and enolase and aldolase, both of which were downregulated. The significance of such changes in response to C8-AHL is discussed in relation to carbohydrate and Amino-Acid Metabolism and involvement of Gloeothece in biofilms.The ISME Journal advance online publication, 17 July 2008; doi:10.1038/ismej.2008.68.

J Microbiol Biotechnol. 2008 May; 18(5): 968-74
Oh MH, Jeong HG, Choi SH

Proteomic analysis led to identification of the proteins of Vibrio vulnificus that were induced upon exposure to INT-407 cells, and 7 of which belong to the functional categories such as Amino Acid transport/Metabolism, nucleotide transport/Metabolism, posttranslational modification/protein turnover/chaperones, and translation. Among the genes encoding the host-induced proteins, disruption of purH, trpD, tsaA, and groEL2 resulted in reduced cytotoxicity. The purH, trpD, and tsaA mutants showed impaired growth in the INT-407 lysate; however, the growth rate of the groEL2 mutant was not significantly changed, indicating that the possible roles of the host-induced proteins in the virulence of V. vulnificus are rather versatile.

IEEE Trans Inf Technol Biomed. 2008 Jul; 12(4): 541-8
Gupta R, Mittal A, Singh K

G-protein coupled receptors (GPCRs) play a vital role in different biological processes, such as regulation of growth, death, and Metabolism of cells. GPCRs are the focus of significant amount of current pharmaceutical research since they interact with more than 50% of prescription drugs. The dipeptide-based support vector machine (SVM) approach is the most accurate technique to identify and classify the GPCRs. However, this approach has two major disadvantages. First, the dimension of dipeptide-based feature vector is equal to 400. The large dimension makes the classification task computationally and memory wise inefficient. Second, it does not consider the biological properties of protein sequence for identification and classification of GPCRs. In this paper, we present a novel-feature-based SVM classification technique. The novel features are derived by applying wavelet-based time series analysis approach on protein sequences. The proposed feature space summarizes the variance information of seven important biological properties of Amino Acids in a protein sequence. In addition, the dimension of the feature vector for proposed technique is equal to 35. Experiments were performed on GPCRs protein sequences available at GPCRs Database. Our approach achieves an accuracy of 99.9%, 98.06%, 97.78%, and 94.08% for GPCR superfamily, families, subfamilies, and subsubfamilies (amine group), respectively, when evaluated using fivefold cross-validation. Further, an accuracy of 99.8%, 97.26%, and 97.84% was obtained when evaluated on unseen or recall datasets of GPCR superfamily, families, and subfamilies, respectively. Comparison with dipeptide-based SVM technique shows the effectiveness of our approach.

Cas Lek Cesk. 2008; 147(5): 261-5
Tomková M, Marohnic CC, Baxová A, Martásek P

Antley-Bixler syndrome (ABS) is a rare congenital disorder characterized by numerous craniofacial, skeletal and, in some cases, urogenital abnormalities resulting from disordered steroidogenesis. Known genetic causes in sporadic cases of ABS include dominant mutations in the fibroblast growth factor 2 receptor gene (FGFR2). Recent research shows surprisingly that symptoms of Antley-Bixler syndrome, combined with disordered steroidogenesis and urogenital anomalies, are caused by mutations in the POR gene that encodes NADPH-cytochrome P450 oxidoreductase (CYPOR). CYPOR is a four domain-containing monomeric flavoprotein that contains two flavins, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), a binding site for NADPH, and the N-terminal sequence of 25 Amino Acids which determines the microsomal localization of the protein. CYPOR is the electron donor to microsomally localized cytochromes P450 that participate in xenobiotic Metabolism and steroidogenesis. Mutations in the POR gene lead to apparent diminished activity of some P450 enzymes. Association of CYPOR with ABS discloses new facts about this disease and recent research shows that patients with ABS-like skeletal anomalies, but with mutations in the POR gene and disordered steroidogenesis, represent a new disorder called POR deficiency.

Environ Health Perspect. 2008 Jul; 116(7): 880-5
Milnes MR, Garcia A, Grossman E, Grün F, Shiotsugu J, Tabb MM, Kawashima Y, Katsu Y, Watanabe H, Iguchi T, Blumberg B

BACKGROUND: Nuclear receptor subfamily 1, group I, member 2 (NR1I2), commonly known as steroid and xenobiotic receptor (SXR) in humans, is a key ligand-dependent transcription factor responsible for the regulation of xenobiotic, steroid, and bile Acid Metabolism. The ligand-binding domain is principally responsible for species-specific activation of NR1I2 in response to xenobiotic exposure. OBJECTIVES: Our objective in this study was to create a common framework for screening NR1I2 orthologs from a variety of model species against environmentally relevant xenobiotics and to evaluate the results in light of using these species as predictors of xenobiotic disposition and for assessment of environmental health risk. METHODS: Sixteen chimeric fusion plasmid vectors expressing the Gal4 DNA-binding domain and species-specific NR1I2 ligand-binding domain were screened for activation against a spectrum of 27 xenobiotic compounds using a standardized cotransfection receptor activation assay. RESULTS: NR1I2 orthologs were activated by various ligands in a dose-dependent manner. Closely related species show broadly similar patterns of activation; however, considerable variation to individual compounds exists, even among species varying in only a few Amino Acid residues. CONCLUSIONS: Interspecies variation in NR1I2 activation by various ligands can be screened through the use of in vitro NR1I2 activation assays and should be taken into account when choosing appropriate animal models for assessing environmental health risk.

Comp Funct Genomics. 2005; 6(7-8): 345-56
Maicas S, Moreno I, Nieto A, Gómez M, Sentandreu R, Valentín E

A total of 6047 open reading frames in the Candida albicans genome were screened for Zn(II)(2)C(6)-type zinc cluster proteins (or binuclear cluster proteins) involved in DNA recognition. These fungal proteins are transcription regulators of genes involved in a wide range of cellular processes, including Metabolism of different compounds such as sugars or Amino Acids, as well as multi-drug resistance, control of meiosis, cell wall architecture, etc. The selection criteria used in the sequence analysis were the presence of the CysX(2)CysX(6)CysX(5-16)CysX(2)CysX(6-8)Cys motif and a putative nuclear localization signal. Using this approach, 70 putative Zn(II)(2)C(6) transcription factors have been found in the genome of C. albicans.

Mol Syst Biol. 2008; 4: 205
Martin FP, Wang Y, Sprenger N, Yap IK, Rezzi S, Ramadan Z, Peré-Trepat E, Rochat F, Cherbut C, van Bladeren P, Fay LB, Kochhar S, Lindon JC, Holmes E, Nicholson JK

Gut microbiome-host metabolic interactions affect human health and can be modified by probiotic and prebiotic supplementation. Here, we have assessed the effects of consumption of a combination of probiotics (Lactobacillus paracasei or L. rhamnosus) and two galactosyl-oligosaccharide prebiotics on the symbiotic microbiome-mammalian supersystem using integrative metabolic profiling and modeling of multiple compartments in germ-free mice inoculated with a model of human baby microbiota. We have shown specific impacts of two prebiotics on the microbial populations of HBM mice when co-administered with two probiotics. We observed an increase in the populations of Bifidobacterium longum and B. breve, and a reduction in Clostridium perfringens, which were more marked when combining prebiotics with L. rhamnosus. In turn, these microbial effects were associated with modulation of a range of host metabolic pathways observed via changes in lipid profiles, gluconeogenesis, and Amino-Acid and methylamine Metabolism associated to fermentation of carbohydrates by different bacterial strains. These results provide evidence for the potential use of prebiotics for beneficially modifying the gut microbial balance as well as host energy and lipid homeostasis.

J Clin Endocrinol Metab. 2008 Jul 15;
Riis AL, Jørgensen JO, Ivarsen P, Frystyk J, Weeke J, Møller N

Context: Hyperthyroidism increases energy expenditure, glucose turnover, lipolysis and protein breakdown. Objective: To test whether increased protein breakdown occurs independently of other catabolic effects in mild experimental hyperthyroidism. Design: Single blind, randomized, placebo controlled, crossover. Protein dynamics of the whole body and of the forearm muscles were measured by Amino Acid tracer dilution technique ((15)N-Phenylalanine and (2)H4-Tyrosine). All subjects underwent a 3 hour study in the basal state followed by a 3 hour euglycemic clamp study. Setting: University clinical research unit. Participants: Eight healthy women (24-46 years). Intervention: 6 days thyroid hormone (thyroxine 50 microg and tri-iodothyronine 0.67 microg/kg/day) or placebo administration. Results: Thyroid hormone administration led to mild T3-hyperthyroidism with more than a doubling of T3 levels and suppression of TSH. Energy expenditure and body composition was unchanged. Glucose infusion rates, forearm glucose uptake and levels of lipid intermediates were also alike. Basal whole body phenylalanine flux and tyrosine flux (reflecting whole body protein breakdown) were increased (p < 0.05) as were whole body protein synthesis rate (p = 0.05). Basal forearm rate of appearance and disappearance for phenylalanine (reflecting muscle protein breakdown and synthesis) were similar. Conclusions: Mild short term experimental hyperthyroidism increases whole body protein turnover and breakdown, before any measurable changes in energy expenditure, glucose and fat Metabolism, suggesting that Amino Acid and protein Metabolism is an early and primary target for thyroid hormone action in humans.

New Phytol. 2008 Jun 27;
de Azevedo Souza C, Barbazuk B, Ralph SG, Bohlmann J, Hamberger B, Douglas CJ

* The plant enzyme 4-coumarate:coenzyme A ligase (4CL) is part of a family of adenylate-forming enzymes present in all organisms. Analysis of genome sequences shows the presence of '4CL-like' enzymes in plants and other organisms, but their evolutionary relationships and functions remain largely unknown. * 4CL and 4CL-like genes were identified by blast searches in Arabidopsis, Populus, rice, Physcomitrella, Chlamydomonas and microbial genomes. Evolutionary relationships were inferred by phylogenetic analysis of aligned Amino Acid sequences. Expression patterns of a conserved set of Arabidopsis and poplar 4CL-like acyl-CoA synthetase (ACS) genes were assayed. * The conserved ACS genes form a land plant-specific class. Angiosperm ACS genes grouped into five clades, each of which contained representatives in three fully sequenced genomes. Expression analysis revealed conserved developmental and stress-induced expression patterns of Arabidopsis and poplar genes in some clades. * Evolution of plant ACS enzymes occurred early in land plants. Differential gene expansion of angiosperm ACS clades has occurred in some lineages. Evolutionary and gene expression data, combined with in vitro and limited in vivo protein function data, suggest that angiosperm ACS enzymes play conserved roles in octadecanoid and fatty Acid Metabolism, and play roles in organ development, for example in anthers.

Mol Microbiol. 2008 Jun 27;
Eylert E, Schär J, Mertins S, Stoll R, Bacher A, Goebel W, Eisenreich W

The intracellular Metabolism of Listeria monocytogenes was studied by (13)C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino Acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-(13)C(6)]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-(13)C(6)]glucose was provided during the infection period, (13)C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C(3)-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-(13)C(6)]glucose into bacterial Amino Acids under the same experimental settings. The (13)C pattern suggests that (i) significant fractions (50-100%) of bacterial Amino Acids were provided by the host cell, (ii) a C(3)-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.

Biotechnol Bioeng. 1996 Mar 20; 49(6): 667-74
Farfán MJ, Martín-Rendón E, Calderón IL

In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the Amino Acid production by Saccharomyces cerevisiae. The genes used were wild-type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3-R2 and HOM3-R6), that code for feedback-insensitive aspartate kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate kinase and that, probably, a second and less important regulation takes place at the level of the homoserine kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3-R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine Metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully. (c) 1996 John Wiley & Sons, Inc.

J Natl Cancer Inst. 1967 Aug; 39(2): 187-91
Holoubek V, Hnilica LS

Attempts were made to isolate a factor from tumor tissue which, after injection, enhances the 3H-thymidine incorporation into the DNA of mice. This factor was in the cell nuclei and was extractable together with Acid-soluble nuclear proteins (histones). Additional purification showed that this factor was either closely bound with the very lysine-rich histone (F1), or it was this histone itself. Acid-soluble nuclear proteins prepared from liver and thymus did not influence the incorporation of 3H-thymidine into DNA after injection. The very lysine-rich histone (F1) in these extracts was in an inactive form. It could be activated by separation from other Acid-soluble nuclear proteins. Purified, the very lysine-rich histone (F1) enhanced the 3H-thymidine incorporation into DNA, regardless of its being prepared from tumor or nontumor tissues. By enhancing the incorporation of 3H-thymidine into DNA after injection, the very lysine-rich histone (F1) significantly differed from arginine-rich (F2aF3) and lysine-rich (F2b) histones which suppressed the 3H-thymidine incorporation.

Biotechnol Bioeng. 1996 Jan 20; 49(2): 111-29
Marx A, de Graaf AA, Wiechert W, Eggeling L, Sahm H

To determine the in vivo fluxes of the central Metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of (1)H-detected (13)C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-(13)C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 Amino Acids isolated from the cell. Since precursor metabolites of the central Metabolism are incorporated in an exactly defined manner in the carbon skeleton of Amino Acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central Metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic Acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic Acid cycle to the carbon 3 units of glycolysis of 30.6%. (c) 1996 John Wiley & Sons, Inc.

Biotechnol Bioeng. 1995 Aug 5; 47(3): 319-26
Chang YH, Grodzinsky AJ, Wang DI

In-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 x 10(6) to 9.1 x 10(6) 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.(c) 1995 John Wiley & Sons, Inc.

Biotechnol Bioeng. 1995 Jun 20; 46(6): 579-87
Zhou W, Rehm J, Hu WS

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as Amino Acids were maintained at low levels during the cell growth phase. During the cultivation, cell Metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and Amino Acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.