Cell suicide in starving hybridoma culture: survival-signal effect of some amino acids

Two mouse hybridoma cell lines cultured in different basal media with the iron-rich protein-free supplement were subjected to deliberate starvation by inoculation into media diluted with saline to 50% or less. In the diluted media the growth was markedly suppressed and a large fraction of cells died by apoptosis. The cells could be rescued from apoptotic death by individual additions of amino acids, such as glycine, L-alanine, L-serine, L-threonine, L-proline, L-asparagine, L-glutamine, L-histidine, D-serine, -alanine or taurine. Amino acids with hydrophobic or charged side chains were without effect. The apoptosis preventing activity manifested itself even in extremely diluted media, down to 10% of the standard medium. The activity of L-alanine in the protection of cells starving in 20% medium was shown also in semicontinuous culture. In the presence of 2 mM L-alanine the steady-state viable cell density more than doubled, with respect to control, and the apoptotic index dropped from 37% in the control to 16%. It was concluded that the apoptosis-preventing amino acids acted as signal molecules, rather than nutrients, and that the signal had a character of a survival factor. The specificity of present results, obtained with two different hybridomas, supports our view (Frank and Chládková-rámková, 1995) that the membrane transport macromolecules themselves may play the role of the recognition elements in a signal transduction pathway controlling the survival of hybridoma cells.     

Starvation-induced programmed death of hybridoma cells: Prevention by amino acid mixtures

Association of the availability of nutrients with the phenomenon of programmed cell death-apoptosis-was investigated using hybridoma cells cultured in protein-free medium under conditions of starvation, i.e., in RPMl-1640 medium diluted to 50% with saline. Amino acid mixtures, such as MEM essential amino acids or MEM nonessential amino acids were found to prevent starvation death significantly when added to the diluted medium in 1 to 2 mM concentrations, the MEM vitamin mixture was ineffective, and glutamine displayed a moderate growth-supporting effect. The specific monoclonal antibody production rate in cultures supplemented with amino acid mixtures was strikingly low, whereas supplementation with glutamine alone or simultaneously with other amino acids resulted in a specific antibody production rate comparable with the rate observed in undiluted medium. (c) 1995 John Wiley & Sons, Inc.     

Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection

Cell culture and fermentation broth media are used in the manufacture of biotherapeutics and many other biological materials. Characterizing the amino acid composition in cell culture and fermentation broth media is important because deficiencies in these nutrients can reduce desired yields or alter final product quality. Anion-exchange (AE) chromatography using sodium hydroxide (NaOH) and sodium acetate gradients, coupled with integrated pulsed amperometric detection (IPAD), determines amino acids without sample derivatization. AE-IPAD also detects carbohydrates, glycols, and sugar alcohols. The presence of these compounds, often at high concentrations in cell culture and fermentation broth media, can complicate amino acid determinations. To determine whether these samples can be analyzed without sample preparation, we studied the effects of altering and extending the initial NaOH eluent concentration on the retention of 42 different carbohydrates and related compounds, 30 amino acids and related compounds, and 3 additional compounds. We found that carbohydrate retention is impacted in a manner different from that of amino acid retention by a change in [NaOH]. We used this selectivity difference to design amino acid determinations of diluted cell culture and fermentation broth media, including Bacto yeast extract-peptone-dextrose (yeast culture medium) broth, Luria-Bertani (bacterial culture medium) broth, and minimal essential medium and serum-free protein-free hybridoma medium (mammalian cell culture media). These media were selected as representatives for both prokaryotic and eukaryotic culture systems capable of challenging the analytical technique presented in this paper. Glucose up to 10mM (0.2%, w/w) did not interfere with the chromatography, or decrease recovery greater than 20%, for the common amino acids arginine, lysine, alanine, threonine, glycine, valine, serine, proline, isoleucine, leucine, methionine, histidine, phenylalanine, glutamate, aspartate, cystine, and tyrosine.     

Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality

Elevated pCO(2) inhibits cell growth. This growth inhibition is accompanied by a decrease in intracellular pH (pHi), as well as a decrease in glycolysis. Elevated concentrations (mM) of some amino acids have been shown by others to protect cells exposed to two very different environmental stresses: nutrient starvation and hyperosmolality. The fact that many of the amino acids shown to have protective effects against other stresses are transported into the cell through a pHi-sensitive transporter led us to study the possibility of using these amino acids as protective agents under elevated pCO(2). Screening experiments using 5, 15, and 25 mM of each amino acid showed that not all amino acids that protect cells from hyperosmolality protect them from elevated pCO(2). Glycine betaine and glycine were chosen for further characterization in both hybridoma and CHO cells. Asparagine and threonine were also tested in hybridoma and CHO cells, respectively. All amino acids tested under 195 mm Hg pCO(2)/435 mOsm/kg (50% growth inhibition) restored the specific growth rate (mu) in hybridoma cells to that observed under control conditions (40 mm Hg/320 mOsm/kg). Addition of each amino acid resulted in an increase in the consumption rate and intracellular accumulation of that amino acid. In CHO cells, glycine betaine also restored mu to control values, while glycine and threonine partially restored mu. In hybridoma cells, the higher specific antibody productivity obtained at elevated pCO(2) was maintained with the lowest amino acid concentration (5 mM). Productivity decreased toward control values with increasing amino acid concentrations. Elevated pCO(2) decreased the specific tPA productivity in the CHO cell line studied. Only glycine betaine resulted in a 20% increase in productivity at 195 mm Hg/435 mOsm/kg. With the exception of glycine betaine in hybridoma cells, amino acids did not mitigate the associated pHi decrease of at least 0.2 pH units at 195 mm Hg/435 mOsm/kg. pHi in hybridoma cells under elevated pCO(2) in the presence of glycine betaine was about 0.1 pH units below that of control. Amino acids had no effect on the cell size response of hybridoma cells, while they partially offset the increase in CHO cell size at elevated pCO(2). Glycine betaine, asparagine, and glycine increased the specific glucose consumption rate observed at 195 mm Hg/435 mOsm/kg (50% of control) to values greater than 70% of control in hybridoma cells. In CHO cells, only glycine betaine increased q(glc) (by 20%) under elevated pCO(2). All amino acids tested improved the cell yield from glutamine at 195 mm Hg/435 mOsm/kg in both cell lines.     

Decline in ribonucleic acid and protein synthesis with loss of viability during the early hours of imbibition of rye (Secale cereale L.) embryos.

The specificity of amino acid transport in normal (high-glutathione) sheep erythrocytes was investigated by studying the interaction of various neutral and dibasic amino acids in both competition and exchange experiments. Apparent Ki values were obtained for amino acids as inhibitors of L-alanine influx. Amino acids previously found to be transported by high-glutathione cells at fast rates (L-cysteine, L-alpha-amino-n-butyrate) were the most effective inhibitors. D-Alanine and D-alpha-amino-n-butyrate were without effect. Of the remaining amino acids studied, only L-norvaline, L-valine, L-norleucine, L-serine and L-2,4-diamino-n-butyrate significantly inhibited L-alanine uptake. L-Alanine efflux from pre-loaded cells was markedly stimulated by extracellular L-alanine. Those amino acids that inhibited L-alanine influx also stimulated L-alanine efflux. In addition, D-alanine, D-alpha-amino-n-biutyrate, L-threonine, L-asparagine, L-alpha, beta-diaminoproprionate, L-ornithine, L-lysine and S-2-aminoethyl-L-cysteine also significantly stimulated L-alanine efflux. L-Lysine uptake was inhibited by L-alanine but not by D-alanine, and the inhibitory potency of L-alanine was not influenced by the replacement of Na+ in the incubation medium with choline. L-Lysine efflux from pre-loaded cells was stimulated by L-alanine but not by D-alanine. It is concluded that these cells possess a highly selective stero-specific amino acid-transport system. Although the optimum substrates are small neutral amino acids, this system also has a significant affinity for dibasic amino acids.     

Apoptosis and nutrition: Involvement of amino acid transport system in repression of hybridoma cell death

Mouse hybridoma cells cultured on the verge of starvation-induced apoptosis, i.e. in a medium diluted with saline, proved to serve as a sensitive screening system for apoptosis-suppressing activity of nutrient medium components. Conventional amino acid mixtures were found to suppress the starvation-induced apoptosis, whereas a vitamin mixture was ineffective. (Frank F (1995) Biotechnol. Bioeng. 45: 86–90). Recent experiments showed that suppression of apoptosis, and concurrent resumption of growth, could be achieved by addition of single substances at millimolar concentrations. The set of active substances included certain coded L-amino acids (glycine, alanine, serine, threonine, proline, asparagine, glutamine, histidine), non-coded amino acids (-alanine, taurine, 4-aminobutyric acid), and a non-metabolizable analogue (2-aminoisobutyric acid). This finding shows that some amino acids do not act solely as nutrients, but also as specific signal molecules. The specificity of the effect points to the involvement of adaptively regulated amino acid transport systems A and N in maintaining the balance between triggering and suppression of starvation-induced apoptosis.     

Amino acids metabolism by VO 208 hybridoma cells: some aspects of the culture process and medium composition influence

In the present study an approach has been developed in order to examine the consequence of essential and non essential amino acid supplementation on VO208 hybridoma cells behaviour. The effect of amino acid enrichment has been studied taking into account the culture process, i.e., batch or continuous culture mode and the medium composition, i.e., a home made serum-free medium or a serum containing one. A group of 4 amino acids, i.e., Ser, Pro, Gly and Arg presented atypical evolution pattern of their extracellular concentration depending on the type of the medium and on the culture mode. Some amino acids were probably involved in the limitation of the cellular proliferation. Met was one of the amino acids that appears to may have been at limiting concentration in all cases. In continuous culture mode, an enrichment of amino acids resulted in a rapid improvement of the viable cell density in both media, with or without the presence of serum. For most amino acids, supplementation during continuous culture induced an increase of the amino acid uptake rate. A comparative analysis of amino acids utilisation, depending on the culture conditions studied in the present study, has been performed in order to propose an overall picture of amino acids metabolism by VO 208 Hybridoma cell line. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification and characterization of rat kidney L-alanine: 4,5-dioxovalerate transaminase and inhibition by hemin

Rat kidney L-alanine:4,5-dioxovalerate transaminase (EC 2.6.1.43), which may be involved in the formation of aminolevulinic acid in mammalian cells, was purified 82-fold to apparent homogeneity with a 19% yield. Molecular weight of the enzyme, as estimated by gel filtration, was found to be 225 000. In polyacrylamide gel electrophoresis under denaturing conditions, the enzyme moved as a single band corresponding to an Mr of 37 000, suggesting that the enzyme is composed of six identical subunits. The Km values of L-alanine and 4,5-dioxovalerate are 2.9 and 0.25 mM, respectively. The enzyme had an optimum activity at pH 6.6 and was most active at 65 degrees C. Among some amino acids tested, L-alanine proved to be the most efficient amino donor, and the enzyme was also stereospecific for the L-isomer. The effect of intermediate metabolites of heme biosynthesis, for example, delta-aminolevulinic acid, protoporphyrin, hemin and bilirubin has been studied on purified L-alanine:4,5-dioxovalerate transaminase. Amongst these metabolites, hemin and protoporphyrin were found to be effective inhibitors.     

Peptone,a low-cost growth-promoting nutrient for intensive animal cell culture

The effect of addition of peptone to serum-free and serum supplemented media for the growth of hybridoma cells in various systems was studied. Supplementation of defined medium with either proteose peptone or meat peptone resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system. Other peptones were either inactive or less effective. In continuous culture, using medium supplemented with new born calf serum, the addition of peptone resulted in 125% and 150% increases in cell and antibody concentrations respectively. Similar increase in cell number (128%) was also obtained in spin-filter perfusion culture when medium was supplemented with peptone. By comparison, the substitution of a defined 1xMEM amino acids mixture resulted in only a 50% increase. At higher perfusion rates the cell number maintained in steady state using peptone supplement could be increased to 1.3×10⁷ cells ml^–1 while the serum concentration was reduced from 5% to 1% at a perfusion rate of 2.5 volumes per day.     

Red-cell amino acid transport. Evidence for the presence of system ASC in mature human red blood cells.

The properties of Na+-dependent L-alanine transport in human erythrocytes were investigated using K+ as the Na+ substitute. Initial rates of Na+-dependent L-alanine uptake (0.2 mM extracellular amino acid) for erythrocytes from 22 donors ranged from 40 to 180 mumol/litre of cells per h at 37 degrees C. Amino acid uptake over the concentration range 0.1-8 mM was consistent with a single saturable component of Na+-dependent L-alanine transport. Apparent Km and Vmax. values at 37 and 5 degrees C measured in erythrocytes from the same donor were 0.27 and 0.085 mM respectively, and 270 and 8.5 mumol/litre of cells per h respectively. The transporter responsible for this uptake was identified as system ASC on the basis of cross-inhibition studies with a series of 42 amino acids and amino acid analogues. Apparent Ki values for glycine, L-alpha-amino-n-butyrate, L-serine and L-leucine as inhibitors of Na+-dependent L-alanine uptake at 37 degrees C were 4.2, 0.12, 0.16 and 0.70 mM respectively. Reticulocytes from a patient with inherited pyruvate kinase deficiency were found to have a 10-fold elevated activity of Na+-dependent L-alanine uptake compared with erythrocytes from normal donors. Separation of erythrocytes according to cell density (cell age) established that even the oldest mature erythrocytes retained significant Na+-dependent L-alanine transport activity. Amino acid transport was, however, a more sensitive indicator of cell age than acetylcholinesterase activity. Erythrocytes were found to accumulate L-alanine against its concentration gradient (distribution ratio approx. 1.5 after 4 h incubation), an effect that was abolished in Na+-free media. Na+-dependent L-alanine uptake was shown to be associated with L-alanine-dependent Na+ influx, the measured coupling ratio being 1:1.     

Influence of amino acids,mammalian serum,and osmotic pressure on the proliferation of Drosophila cell lines

In the D22 medium of ECHALIER and OHANESSIAN for the culture of Drosophila cell lines lactalbumin hydrolysate could be replaced by a synthetic amino acids mixture. In spite of the presence of yeast extract and fetal calf serum the omission of any one of arginine, asparagine, cysteine, histidine, methionine, proline, serine, or threonine prevented cell proliferation. Of these eight amino acids cysteine had to be added in concentrations higher than 0.1 mM. Without much effect on cell proliferation foetal calf serum could be reduced from 10% to 2% or be replaced by 1% horse serum or 1% porcine serum. Cells could grow in media of osmolarities from 225 mOsm up to 400 mOsm depending on the osmotic agent used. Chloride concentrations up to 80 mM were compatible with proliferation as was a wide range of sodium/potassium ratios.     

Nutrient enrichment and in-situ waste removal through electrical means for hybridoma cultures

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.     

Growth limitation in hybridoma cell cultures: The role of inhibitory or toxic metabolites

Hybridoma cells usually grow to fairly low cell densities in batch cultures (1–3×10⁶ cells/ml). The reason for this is either that essential nutritional components of the medium are consumed, or that the cells produce some kind of inhibitory or toxic metabolite. This investigation presents evidence for the latter. Spent medium from cultures of hybridoma cells did not support growth of cells at lower cell densities (1–3×10⁵ cells/ml). The ability to support cell growth could not be restored by adding additional serum, energy sources (glucose, pyruvate) or L-glutamine. Furthermore, the consumption of amino acids could not account for this growth inhibition. On the contrary, the spent medium contained a substance that inhibited cell growth. This substance or metabolite was found in a fraction eluted from a gel filtration column when spent medium was applied to the column. This substance was found in the spent medium from all hybridoma and myeloma cell lines that were tested. The molecular weight of the substance was about 5 kD. Spent medium from two hybridoma cell lines also contained a substance that was eluted in the same fraction as albumin (67 kD). It is likely that this (or these) substance(s) is responsible for the growth limitation in hybridoma cell cultures.Abbreviations PBS phosphate buffered saline     

Effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney-21 cells.

The effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney (BHK-21) cells were investigated. The cells were incubated with labelled choline in the presence of an amino acid or ethanolamine. The uptake of labelled choline was noncompetitively inhibited by amino acids. Glycine, L-alanine, L-serine, L-leucine, L-aspartate, and L-arginine were effective inhibitors and a maximum of 22% inhibition of choline uptake was obtained with 5 mM glycine. Analyses of the labelings in the choline-containing metabolites revealed that the conversion of choline to CDP-choline and subsequently phosphatidylcholine was not affected by the presence of amino acids. The uptake of choline was also inhibited by ethanolamine in a concentration-dependent manner. Kinetic studies on the uptake of choline indicated that the inhibition by ethanolamine was competitive in nature. Although ethanolamine is a potent inhibitor of choline kinase, analyses of the labelings in the choline-containing metabolites indicated that the conversion of choline to phosphocholine was not affected in the cells incubated with ethanolamine. Ethanolamine did not change the pool sizes of phosphocholine and CDP-choline. Based on the specific radioactivity of CDP-choline and the labeling of phosphatidylcholine, the rates of phosphatidylcholine biosynthesis were not significantly different between the control and the ethanolamine-treated cells. In view of the concentrations of amino acids (millimolar) and ethanolamine (micromolar) in most cell culture media, it appeared that only amino acids were important metabolites for the regulation of choline uptake in BHK-21 cells. We conclude that both amino acids and ethanolamine have no direct effect on the biosynthesis of phosphatidylcholine.     

Enhancing the sensitivity of multidimensional NMR experiments by using triply-compensated π pulses

An improved expression protocol is proposed for amino acid type-specific [¹³C], [¹⁵N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449–456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents.     

Bovine colostrum or milk as a serum substitute for the cultivation of a mouse hybridoma

A mouse-mouse hybridoma was grown in serum-free medium supplemented with bovine milk or colostrum. Bovine colostrum supported growth of the hybridoma whereas bovine milk alone did not support cellular proliferation. For growth in medium supplemented with colostrum, the maximum cell concentration achieved was 1.4 x 10(6) cells/mL in 2.2% colostrum, which is 44% of that obtained in 9% serum. When cells were grown in media containing milk and low amounts of serum (<1%) the maximum cell concentration in 2.2% milk with 0.4% serum was 2 x 10(6) cells/ml, whereas it was only 0.2 x 10(6) cells/ml and 1.3 x 10(6) cells/ml in 2.2% milk alone and 0.4% serum alone, respectively. Similar behavior was observed for growth in media containing colostrum and low amounts of serum. The monoclonal antibody production in media containing combinations of serum and milk or colostrum was comparable to that obtained in media with higher serum concentrations. Experiments performed with conditioned media suggest that the rapid decrease in viability, after the maximum cell concentration has been reached, is partially due to the presence of some inhibitory components generated during the cell culture rather than due to depletion of some serum components.     

Dibasic amino acid interactions with Na+-independent transport system asc in horse erythrocytes. Kinetic evidence of functional and structural homology with Na+-dependent system ASC

Amino acid transport in horse erythrocytes is regulated by three co-dominant allelomorphic genes coding for high-affinity transport activity (system asc1), low-affinity transport activity (system asc2) and transport-deficiency, respectively. The asc systems are selective for neutral amino acids of intermediate size, but unlike conventional system ASC, do not require Na+ for activity. In the present series of experiments we have used a combined kinetic and genetic approach to establish that dibasic amino acids are also asc substrates, systems asc1 and asc2 representing the only mediated routes of cationic amino acid transport in horse erythrocytes. Both transporters were found to exhibit a strong preference for dibasic amino acids compared with neutral amino acids of similar size. Apparent Km values (mM) for influx via system asc1 were L-lysine (9), L-ornithine (27), L-arginine (27), L-alanine (0.35). Corresponding Vmax estimates (mmol/l cells per h, 37 degrees C) were L-lysine (1.65), L-ornithine (2.15), L-arginine (0.54), L-alanine (1.69). Apparent Km values for L-lysine and L-ornithine influx via system asc2 were approximately 90 and greater than 100 mM, respectively, with Vmax values greater than 2 and greater than 1 mmol/l cells per h, respectively. Apparent Km and Vmax values for L-alanine uptake by system asc2 were 14 mM and 6.90 mmol/l cells per h. In contrast, L-arginine was transported by system asc2 with the same apparent Km as L-alanine (14 mM), but with a 77-fold lower Vmax. This dibasic amino acid was shown to cause cis- and trans-inhibition of system asc2 in a manner analogous to its interaction with system ASC, where the side-chain guanidinium group is considered to occupy the Na+-binding site on the transporter. Concentrations of extracellular L-arginine causing 50% inhibition of zero-trans L-alanine influx and half-maximum inhibition of L-alanine zero-trans efflux were 14 mM (extracellular L-alanine concentration 15 mM) and 3 mM (intracellular L-alanine concentration 15.5 mM), respectively. We interpret these observations as evidence of structural homology between the horse erythrocyte asc transporters and system ASC. Physiologically, intracellular L-arginine may function as an endogenous inhibitor of system asc2 activity.     

Glutamine and the regulation of DNA replication and cell multiplication in fibroblasts

Several studies indicate that glutamine is a critical requirement for cell growth in vitro. Growing and quiescent (serum-starved) 3T3-fibroblasts were exposed to media (Dulbecco's modified Eagle's minimal essential medium) in which the concentration of the 13 essential amino acids had been lowered to 1/100 or 1/1,000 of that in DMEM - either all together or one by one. The effects on DNA synthesis were measured by autoradiographic determinations of the percentage of labeled cells after 24 hours exposure to ³H-thymidine. A reduction of all 13 essential amino acids to 1/100 or 1/1,000 of the normal concentration in the medium resulted only in a minor growth inhibitory effect during the first cell cycle. A similar growth inhibitory effect was caused by the depletion of one of the 13 essential amino acids (except glutamine) from the medium. However, a depletion of glutamine from the medium resulted in a marked inhibition of growth. Conversely, a relative excess of glutamine, when the other 12 amino acids were lowered to 1/1,000 of the normal concentration, counteracted the growth inhibitory effect of serum starvation. It was even possible to stimulate quiescent cells to undergo DNA synthesis by exposing them to a serum-depleted (0.5% serum) medium with a relative excess of glutamine.     

Insulin regulation of amino acid transport in mesenchymal cells from avian and mammalian tissues.

Insulin regulation of amino acid transport across the cell membrane was studied in a variety of mesenchymal cell directly isolated from avian and mammalian tissues or collected from confluent cultures. Transport activity of the principal systems of mediation in the presence and absence of insulin was evaluated by measuring the uptake of representative amino acids under conditions approaching initial entry rates. Insulin enhanced the transport rate of substrate amino acids from the A system(alpha-aminoisobutyric acid, L-proline, glycine, L-alanine and L-serine) in fibroblasts and osteoblasts from chick-embryo tissues, in mesenchymal cells (fibroblasts and smooth muscle cells) from immature rat uterus, in thymic lymphocytes from young rats and in chick-embryo fibroblasts from confluent secondary cultures. In these tissues, the uptake of amino acid substrates of transport systems L and Ly+ (L-leucine, L-phenylalanine, L-lysine) was not affected by the presence of the hormone. No insulin control of amino acid transport was detected in chick-embryo chondroblasts and rat peritoneal macrophages. These observations identify the occurrence of hormonal regulatory patterns of amino acid transport for different mesenchymal cells types and indicate that these properties emerge early during cell differentiation.     

Effect of Bcl-2 Expression on Hybridoma Cell Growth in Serum-Supplemented, Protein-Free and Diluted Media

Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps. This revised version was published online in June 2006 with corrections to the Cover Date.