Fermentative production of L-alanyl-L-glutamine by a metabolically engineered Escherichia coli strain expressing L-amino acid alpha-ligase

In spite of its clinical and nutritional importance, l-alanyl-l-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing l-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of the dpp and pep genes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologous l-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lal and ald expressed under the control of the uspA promoter) or pPE177 (lal and ald expressed under the control of the rpoH promoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containing d-amino acids were formed.     

Advantages of AlaGln as an additive to cell culture medium: use with anti-CD20 chimeric antibody-producing POTELLIGENT™ CHO cell lines

l-alanyl-l-glutamine (AlaGln) is dipeptide that has better solubility and stability than Glutamine (Gln). In this study, we evaluated the utility of this dipeptide during culture of POTELLIGENT™ Chinese hamster ovary (CHO) cells expressing anti-CD20 chimeric antibody. Although AlaGln in the culture medium lowered the specific growth rate, the MAb titer was maximized when Gln was completely replaced by AlaGln in both the basal and feed media. Moreover, AlaGln augmented production of antibody not only at flask scale but also at spinner scale, although the extent of this effect was dependent on the cell clone. To explore the mechanism responsible for the effect of AlaGln on cell growth, we measured apoptosis in the early phase of cell culture on days 8, 9, and 10. The apoptotic ratio was reduced in medium containing AlaGln. Ammonia was generated in medium containing Gln when it was maintained at 37 °C, which impeded the growth and productivity of the cells. In contrast, AlaGln produced less ammonia under these conditions, which may have been one of the properties associated with its beneficial effects. We conclude that certain dipeptides can serve as superior alternative sources of amino acids in cell culture and antibody production.     

l-Glutamine or l-alanyl-l-glutamine prevents oxidant- or endotoxin-induced death of neonatal enterocytes

This study tested the hypothesis that L-glutamine (Gln) or L-alanyl-L-glutamine (Ala-Gln) prevents oxidant- or endotoxin-induced death of neonatal enterocytes. Enterocytes of neonatal pigs rapidly hydrolyzed Ala-Gln and utilized Gln. To determine whether Gln or Ala-Gln has a cytoprotective effect, IPEC-1 cells were cultured for 24 h in Gln-free Dulbecco’s modified Eagle’s-F12 Ham medium containing 0, 0.5, 2.0 or 5.0 mM Gln or Ala-Gln, and 0, 0.5 mM H₂O₂ or 30 ng/ml lipopolysaccharide (LPS). Without Gln or Ala-Gln, H₂O₂- or LPS-treated cells exhibited almost complete death. Gln or Ala-Gln at 0.5, 2 and 5 mM dose-dependently reduced H₂O₂- or LPS-induced cell death by 14, 54 and 95%, respectively, whereas d-glutamine, alanine, glutamate, ornithine, proline, glucosamine or nucleosides had no effect. To evaluate the effectiveness of Gln or Ala-Gln in vivo, 7-day-old piglets received one-week oral administration of Gln or Ala-Gln (3.42 mmol/kg body weight) twice daily and then a single intraperitoneal injection of LPS (0.1 mg/kg body weight); piglets were euthanized in 24 and 48 h to analyze intestinal apoptotic proteins and morphology. Administration of Gln or Ala-Gln to LPS-challenged piglets increased Gln concentrations in small-intestinal lumen and plasma, reduced intestinal expression of Toll-like receptor-4, active caspase-3 and NFkB, ameliorated intestinal injury, decreased rectal temperature, and enhanced growth performance. These results demonstrate a protective effect of Gln or Ala-Gln against H₂O₂- or LPS-induced enterocyte death. The findings support addition of Gln or Ala-Gln to current Gln-free pediatric amino acid solutions to prevent intestinal oxidative injury and inflammatory disease in neonates.     

The enhancement of specific antibody production rate in glucose- and glutamine-controlled fed-batch culture

The concentration effects of certain amino acids (Asp, Ile, Leu, Lys, Met, Val, Phe and Gln which were highly consumed during cultivation), and glucose on cell growth and antibody productivity were investigated using dish culture. From these experiments, it was found that only glutamine enrichment enhanced the specific antibody production rate. The other amino acids described above did not affect either the specific growth rate or specific antibody production rate. Thus we investigated the quantitative effects of glutamine concentration in the range of 0.4∼33.3 mmol·1⁻¹ on kinetic parameters in fed-batch culture which kept both glucose and glutamine concentration constant. As a result the specific growth rate decreased with increase in glutamine concentration in the range larger than 20 mmol·1⁻¹. The specific antibody production rate had a maximum value at about 25 mmol·1⁻¹ glutamine concentration.     

Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-²H₃]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.     

Dietary alanyl-glutamine improves growth performance of weaned piglets through maintaining intestinal morphology and digestion–absorption function

Alanyl-glutamine (Ala-Gln), a highly soluble and stable glutamine dipeptide, is known to improve gut integrity and function. The aim of this study was to evaluate whether dietary Ala-Gln supplementation could improve growth performance, intestinal development and digestive-absorption function in weaned piglets. A total of 100 purebred Yorkshire piglets weaned at 21 days of age were assigned randomly to four dietary treatment groups and fed a basal diet (control group) or a basal diet containing 0.15%, 0.30% and 0.45% Ala-Gln, respectively. Compared with the control group, piglets fed the Ala-Gln diets had higher average daily gain and lower feed : gain and diarrhea rate (P < 0.05). Moreover, dietary Ala-Gln supplementation increased villous height and villous height : crypt depth ratio in duodenum and jejunum (P < 0.05), as well as the activities of maltase and lysozyme in jejunum mucosa (P < 0.05). In addition, a decrease in serum diamine oxidase activity and crypt depth in duodenum and jejunum was observed in piglets fed the Ala-Gln diets (P < 0.05). Serum cytosolic phospholipase A2 (cPLA2) concentration and gene expression of cPLA2, Na⁺-dependent glucose transporter 1, glucose transporter 2 and peptide transporter 1 in jejunum were increased by feeding Ala-Gln diets relative to control diet (P < 0.05). These results indicated that feeding Ala-Gln diet has beneficial effects on the growth performance of weaned piglets, which associated with maintaining intestinal morphology and digestive-absorption function.     

Regulation of the High-Affinity Nitrate Transport System in Wheat Roots by Exogenous Abscisic Acid and Glutamine

Nitrate is a major nitrogen （N） source for most crops. Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological and molecular levels. Understanding how nitrate uptake is regulated will help us engineer crops with improved nitrate uptake efficiency. The present study investigated the regulation of the high-affinity nitrate transport system （HATS） by exogenous abscisic acid （ABA） and glutamine （Gin） in wheat （Triticum aestivum L.） roots. Wheat seedlings grown in nutrient solution containing 2 mmol/L nitrate as the only nitrogen source for 2weeks were deprived of N for 4d and were then transferred to nutrient solution containing 50 μmol/L ABA, and 1 mmol/L Gin in the presence or absence of 2 mmol/L nitrate for 0, 0.5, 1, 2, 4, and 8 h. Treated wheat plants were then divided into two groups. One group of plants was used to investigate the mRNA levels of the HATS components NRT2 and NAR2 genes in roots through semi-quantitative RT-PCR approach, and the other set of plants were used to measure high-affinity nitrate influx rates in a nutrient solution containing 0.2 mmol/L ^15N-labeled nitrate. The results showed that exogenous ABA induced the expression of the TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNAR2.1, and TaNAR2.2 genes in roots when nitrate was not present in the nutrient solution, but did not further enhance the induction of these genes by nitrate. Glutamine, which has been shown to inhibit the expression of NRT2 genes when nitrate is present in the growth media, did not inhibit this induction. When Gin was supplied to a nitrate-free nutrient solution, the expression of these five genes in roots was induced. These results imply that the inhibition by Gin of NRT2 expression occurs only when nitrate is present in the growth media. Although exogenous ABA and Gin induced HATS genes in the roots of wheat, they did not induce nitrate influx.     

Carbon-nitrogen relations during batch growth ofNannochloropsis oculata (Eustigmatophyceae) under alternating light and dark

Nannochloropsis oculata (strain CCAP 849/1) was sampled at least every 12 h over a 26-d period of batch culture growth in a 12 h/12 h light/dark illumination cycle. Exponential cell-specific growth rate was 0.5 d^–1. Cell division occurred during the dark phase, while ammonium uptake, pigment synthesis and cell volume increase occurred mainly during the light. Stationary phase cells were on average larger that the largest exponentially growing cells. The lag phase prior to cell division was short with the C/N ratio returning to 6.25 (from 28) within 2 d of refeeding with ammonium. Significant Chl.a synthesis commenced after this period; net synthesis of Chl.a ceased on exhaustion of the N-source with a 40% fall in levels by the end of the stationary phase. Levels of carotenoids per cell also declined during N-deprivation although per ml of culture levels remained constant. Ammonium-refeeding of N-deprived cells resulted in a very rapid rise in glutamine (Gln) and very high ratios of glutamine/glutamate (Gln/Glu peaking at 35 within 1 h); peak Gln/Glu was lower in cells refed in the dark or after a shorter period of N-deprivation. The major intracellular amino acids during exponential phase were Glu, Gln, alanine and arginine, but on exhaustion of the N-source, levels of Gln fell rapidly (Gln/Glu falling to below 0.1 from 0.5–0.9 in the light and 0.3 in darkness during exponential growth). During N-deprivation tyrosine accumulated within the cells. Comparisons are drawn with the growth ofIsochrysis galbana, another alga used in aquaculture, under identical conditions.Author for correspondence     

Auxin-facilitated utilization of glutamic acid by tobacco crown-gall teratoma cells

Summary Teratoma tissues obtained by inoculating Nicotiana tabacum cv. Turkish with a moderately virulent strain of the crown-gall bacterium require the synthetic auxin, -naphthaleneacetic acid (NAA) when glutamic acid is used as a sole nitrogen source in the culture medium. In contrast, growth on culture media containing ammonium ion, nitrate ion or glutamine as an N source does not require NAA. Moreover, added NAA does not stimulate teratoma tissue grown on these N sources. Glutamic acid did not inhibit growth of teratoma tissue on media containing NO ₃ ^- . Growth on mixtures of glutamic acid and NO ₃ ^- was additive in the presence of NAA indicating that NAA promotes the utilization of glutamic acid in the culture medium. Increased concentration of potassium ion in the culture medium was required for growth on glutamic acid in the absence of added auxin. K⁺ did not stimulate growth on glutamine. When teratoma tissues were grown on media containing glutamic acid and varying concentrations of both K⁺ and NAA increasing concentrations of NAA reduced the stimulating effect of K⁺ and, conversely, increasing concentrations of K⁺ reduced the stimulating effect of NAA. It is concluded that K⁺ and auxin act either directly or indirectly at a common site to promote glutamic acid utilization.     

Adaptation of mammalian cells to non-ammoniagenic media

Although glutamine is used as a major substrate for the growth of mammalian cells in culture, it suffers from some disadvantages. Glutamine is deaminated through storage or by cellular metabolism, leading to the formation of ammonia which can result in growth inhibition. Non-ammoniagenic alternatives to glutamine have been investigated in an attempt to develop strategies for obtaining improved cell yields for ammonia sensitive cell lines.Glutamate is a suitable substitute for glutamine in some culture systems. A period of adaptation to glutamate is required during which the activity of glutamine synthetase and the rate of transport of glutamate both increase. The cell yield increases when the ammonia accumulation is decreased following culture supplementation with glutamate rather than glutamine. However some cell lines fail to adapt to growth in glutamate and this may be due to a low efficiency transport system.The glutamine-based dipeptides, ala-gln and gly-gln can substitute for glutamine in cultures of antibody-secreting hybridomas. The accumulation of ammonia in these cultures is less and cell yields in dipeptide-based media may be improved compared to glutamine-based controls. In murine hybridomas, a higher concentration of gly-gln is required to obtain comparable cell growth to ala-gln or gln-based cultures. This is attributed to a requirement for dipeptide hydrolysis catalyzed by an enzyme with higher affinity for ala-gln than gly-gln.     

Physiological changes during the adaptation of hybridoma cells to low serum and serum-free media

Two murine hybridoma cell lines (167.4G5.3 and S3H5/gamma2bA2) were adapted to grow in low-serum and serum-free media by a weaning procedure. The changes in cell growth, metabolic, and antibody production rates with adaptation were examined using biochemical and flow cytometric analyses. After adaptation to a particular serum level, the short-term serum response of the cells was experimentally determined. Specific growth rates, glucose and glutamine uptake and lactate and ammonia production rates, and specific antibody production rates were evaluated from the data. For both cell lines, an improvement in cell growth was observed after adaptation, and both higher growth rates and higher cell concentrations were obtained. The specific glucose and glutamine uptake rates and the lactate and ammonia production rates changed insignificantly with adaptation. Conversely, changes in the specific antibody production rate of the two cell lines differed. Cell line 167.4G5.3 showed a loss in antibody productivity at low serum levels, while the S3H5/gamma2bA2 kept its original productivity in low-serum-containing media. The intracellular antibody content for S3H5/gamma2bA2 cells remained unaltered by adaptation, but a low antibody containing cell population appeared in the 167.4G5.3 culture. The loss of specific antibody productivity in this cell line was due to the appearance of this population.     

Insulin-like growth factor-I stimulates amino acid transport in a glutamine-deprived human neuroblastoma cell line

It is still unknown how insulin-like growth factor-I (IGF-I) regulates cancer cell growth in the condition of the limited availability of key nutrients, such as glutamine. We investigated the effects of IGF-I on cell growth and amino acid transport in a glutamine-deprived human neuroblastoma cell line, SK-N-SH. Cell growth was measured, and ³H-labeled amino acid transport was assayed after treatment with or without IGF-I (50 ng/ml) in 2 mM (control) and 100 μM glutamine concentrations. Cell growth rates were dependent on glutamine concentrations. IGF-I stimulated cell growth in both 2 mM and 100 μM glutamine. IGF-I stimulated glutamine transport in 100 μM glutamine with the mechanism of increasing carrier V_max, but had no effect in 2 mM glutamine. IGF-I also stimulated leucine, glutamate and 2-(methylamino)isobutyric acid transport in 100 μM glutamine. There were significant increases in [³H]thymidine and [³H]leucine incorporation in IGF-I-treated cells in both 2 mM and 100 μM glutamine. These data suggest that IGF-I stimulates cell growth by increasing amino acid transport in the condition of low glutamine levels in a human neuroblastoma cell line. This mechanism may allow to maintain cell growth even in nutrient-deprived tumor tissues.     

GROWTH FACTOR INTERACTIONS IN THE TISSUE CULTURE OF TUMOROUS AND NONTUMOROUS NICOTIANA GLAUCA-LANGSDORFFII

Schaeffer , Gideon W., Harold H. Smith and Marion P. Perkus . (Brookhaven Natl. Lab., Upton, N. Y.) Growth factor interactions in the tissue culture of tumorous and nontumorous Nicotiana glauca-langsdorffii. Amer. Jour. Bot. 50(8): 766–771. Illus. 1963.—Tissues representing tumorous and nontumorous Nicotiana glauca-langsdorffii were cultured on high (5 ×) and low (1 ×) concentrations of a modified White's basal medium containing 2.9 × 10^–6m indoleacetic acid. The growth responses of tissues of both the tumorous and nontumorous genotypes to supplements of kinetin, glutamine, inositol and nucleic acid constituents added singly and in all combinations were noted on high-salt media. The nucleic acid components inhibited growth and were omitted from low-salt media. The best growth response was observed with glutamine and inositol for tissues from the tumorous hybrid and with glutamine, inositol and kinetin in the nontumorous type. Kinetin was a distinct and consistent requirement for rapid growth of nontumorous tissues, but no appreciable kinetin effect could be observed with tissues from the tumorous genotype.     

Stability of Minimum Essential Medium functionality despite l-glutamine decomposition

l-Glutamine (l-Gln) instability in liquid media is a well-known fact. Also, negative effect of ammonia, one of the l-Gln degradation products, on viability of many cell cultures and on replication of different viruses has been described. However, negative effects of ammonia have been reported in doses excessively exceeding those that could be generated in regularly used liquid culture media due to spontaneous l-Gln breakdown (below 2 mM). Traditional virus vaccine production processes have been established and registered involving l-Gln containing media use. Eventual culture media replacement in the regular production process belongs to the major regulative changes that require substantial financial expenses. The aim of this study was to evaluate the effect of storage of Minimum Essential Media with Hanks salts on their relevant biological functions during virus vaccine production process in relation to l-Gln decrease. Our results show a cell type dependent effect of spontaneous l-Gln degradation during medium storage. They also suggest that for cell cultures used in measles, mumps, and rubella virus production the media retain their functionality in respect to cell viability or virus growth over a certain time window despite l-Gln degradation.     

Effect of the nitrogen source on glutamine and alanine biosynthesis in Neurospora crassa. An in vivo 15N nuclear magnetic resonance study

The influences of different nitrogen sources on the relative rates of biosynthesis of glutamine and alanine have been studied by 15N nuclear magnetic resonance spectroscopy of intact Neurospora crassa mycelia suspensions. The rate of glutamine synthesis was fastest after growth in media deficient in free ammonium ion, whereas it was slowest following growth in media containing both glutamic acid and glutamine. The reverse trend was observed for the biosynthesis of alanine. A competition between the two biosynthetic pathways for the same substrate, glutamic acid, was found to limit the rate of alanine synthesis when glutamine synthesis was rapid. The observed in vivo rates of these reactions are compared to the reported specific activities of the enzymes catalyzing the reactions, and implications of these results for nitrogen regulation of these pathways under various physiological conditions are discussed.     

Metabolic changes associated with adaptation of plant cells to water stress

Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from ¹⁵N labeling kinetics after substituting [¹⁵N]ammonium and [¹⁵N]nitrate for the ¹⁴N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and γ-aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and γ-aminobutyrate are synthesized at rates greatly in excess of those simply required to maintain the free pools with growth, indicating that these amino acids are rapidly turned over. Thus, both synthesis and utilization rates for alanine and γ-aminobutyrate are increased in adapted cells. Adaptation to stress leads to increased rates of synthesis of valine and leucine apparently at the expense of isoleucine. Remarkably low ¹⁵N flux via the aspartate family amino acids was observed in these experiments. The rate of synthesis of threonine appeared too low to account for threonine utilization in protein synthesis, pool maintenance, and isoleucine biosynthesis. It is possible that isoleucine may be deriving carbon skeletons from sources other than threonine. Tentative models of the nitrogen flux of these two contrasting cell lines are discussed in relation to carbon metabolism, osmoregulation, and nitrogenous solute compartmentation.     

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1 g L⁻¹ increased the maximal cell density by 70 %, while a mixture of YE (1 g L⁻¹) and YP.A (4 g L⁻¹) increased IgG production by 180 %. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20 %. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.     

The adaptation of BHK cells to a non-ammoniagenic glutamate-based culture medium.

Although glutamine is a major carbon source for mammalian cells in culture, its chemical decomposition or cellular metabolism leads to an undesirable excess of ammonia. This limits the shelf-life of glutamine-supplemented media and may reduce the cell yield under certain conditions. We have attempted to develop a less ammoniagenic medium for the growth of BHK-21 cells by a mole-to-mole substitution of glutamine by glutamate. This results in a medium that is thermally stable but unable to support an equivalent growth yield. However, supplementation of the glutamate-based medium with asparagine (3 mM) and a minimal level of glutamine (0.5 mM) restored the original growth capacity of the cultures. Substitution of the low level of glutamine with the glutamine dipeptides, ala-gln (1 mM), or gly-gln (3 mM) resulted in an equivalent cell yield and in a thermally stable medium. The ammonia accumulation in cultures with glutamate-based medium was reduced significantly (>60%). Factors mediating growth and adaptation in medium substituted with glutamate were also investigated. The maximum growth capacity of the BHK-21 cells in glutamate-based medium (without glutamine) was achieved after a period of adaptation of 5 culture passages from growth in glutamine-based cultures. Adaptation was not influenced by increases in glutamate uptake which was constitutively high in BHK cells. Adaptation was associated with changes in the activities of enzymes involved in glutamate or glutamine metabolism. The activities of glutamine synthetase (GS) and alanine aminotransferase (ALT) increased significantly and the activity of phosphate-activated glutaminase (PAG) decreased significantly. The activity of glutamate dehydrogenase (GDH) showed no significant change after adaptation to glutamate. These changes resulted in an altered metabolic profile which included a reduced ammonia production but an increased alanine production. Alanine production is suspected of being an alternative route for removal of excess nitrogen.     

Cell death of prostate cancer cells by specific amino acid restriction depends on alterations of glucose metabolism

Selective amino acid restriction targets mitochondria resulting in DU145 and PC3 prostate cancer cell death. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met) differentially modulates glucose metabolism, glycogen synthase kinase 3β (GSK3β), p53, and pyruvate dehydrogenase (PDH) in these two cell lines. In DU145 cells, Gln and Met restriction increase glucose consumption, but Tyr/Phe restriction does not. Addition of glucose to culture media diminishes cell death induced by Tyr/Phe‐restriction. Addition of pyruvate reduces cell death due to Tyr/Phe and Gln restriction. Tyr/Phe, Gln and Met restriction increase phosphorylation of GSK3β‐Ser⁹, phosphorylation of p53‐Ser¹⁵ and reduce the mitochondrial localization of PDH. Addition of glucose or pyruvate to cultures significantly reverses the alterations in GSK3β, p53 and PDH induced by amino acid restriction. In p53‐null PC3 cells, Tyr/Phe, Gln and Met restriction decreases glucose consumption, reduces phosphorylation of Akt‐Ser⁴⁷³, and increases phosphorylation of GSK3β‐Ser⁹. Addition of pyruvate or glucose reduces death of Met‐restricted cells. Addition of glucose increases phosphorylation of Akt‐Ser⁴⁷³ in amino acid‐restricted cells reduces phosphorylation of GSK3β‐Ser⁹ in Tyr/Phe and Gln restricted cells and increases phosphorylation of GSK3β‐Ser⁹ in Met restricted cells. Addition of pyruvate reduces phosphorylation of GSK3β‐Ser⁹ in all amino acid‐restricted cells. In summary, cell death induced by specific amino acid restriction is dependent on or closely related to the modulation of glucose metabolism. GSK3β (DU145 and PC3) and p53 (DU145) are crucial switches connecting metabolism and these signaling molecules to cell survival during amino acid restriction. J. Cell. Physiol. 224: 491–500, 2010. © 2010 Wiley‐Liss, Inc.     

Nutritional Study of Three Strains of Naegleria gruberi*

Naegleria gruberi strains cloned from amebas isolated from a Vero cell culture (“TS”), a sewer drainage ditch (“PD”), and an established laboratory line (“S”) were morphologically identical except for differences in size and flagellate transforming ability. Cultivation on a Trypticase-yeast extract-glucose medium (“TYG”) fortified with autoclaved E. coli resulted in increased cell size of 2 strains. Differences also were noted in growth rates and optimal growth temperatures. The autoclaved E. coli in TYG medium was replaceable with serum only for strains TS and PD. A basal salts medium + autoclaved E. coli supported growth of all 3 strains, but the basal salts medium + serum would not support growth of any of the strains.