Glutamine In Medium Research Articles

Utilization of Nitrate for Early Androgenic Embryogenesis of Brassica napus L.

The utilization of nitrate in microspore culture of Brassica napus was examined. The microspores developed into embryos in a medium containing glutamine as a sole source of nitrogen (medium A) as well as in a medium containing nitrate and glutamine (medium C). On the other hand, no embryo could develop from the microspores in a medium with nitrate as a sole source of nitrogen (medium B). Transfer experiments from one medium to another during various periods of culture indicated that the microspores cultured in medium A stopped growing as soon as they were transferred to medium B. On the other hand, the microspores cultured in medium B did not show any change even after transfer to medium A on the 4th day of culture. Although the amount of nitrate in medium B increased within the first 14 days, the increase was less pronounced in medium C. The activity of nitrate reductase in vivo was not detected in the developing androgenic embryos before the 8th day of culture, whereas 15 days-old embryos showed a higher activity of nitrate reductase than leaf. From these results it was concluded that nitrate was not used for early androgenic embryogenesis in B. napus before 15 days of culture because of the lack of nitrate reductase activity. Negative effect of the presence of nitrate on androgenic embryogenesis was not observed, either. On the contrary, the presence of nitrate promoted the growth of heart-shaped or more developed embryos after rapid and complete consumption of glutamine in the culture medium C. Nitrate in the medium for microspore culture of B. napus was considered to be an ideal source of nitrogen at the later stages of androgenic embryogenesis.

Caspase-mediated Cleavage of DNA Topoisomerase I at Unconventional Sites during Apoptosis

Previous studies have demonstrated that topoisomerase I is cleaved late during apoptosis, but have not identified the proteases responsible or examined the functional consequences of this cleavage. Here, we have shown that treatment of purified topoisomerase I with caspase-3 resulted in cleavage at DDVD146 downward arrowY and EEED170 downward arrowG, whereas treatment with caspase-6 resulted in cleavage at PEDD123 downward arrowG and EEED170 downward arrowG. After treatment of Jurkat T lymphocytic leukemia cells with anti-Fas antibody or A549 lung cancer cells with topotecan, etoposide, or paclitaxel, the topoisomerase I fragment comigrated with the product that resulted from caspase-3 cleavage at DDVD146 downward arrowY. In contrast, two discrete topoisomerase I fragments that appeared to result from cleavage at DDVD146 downward arrowY and EEED170 downward arrowG were observed after treatment of MDA-MB-468 breast cancer cells with paclitaxel. Topoisomerase I cleavage did not occur in apoptotic MCF-7 cells, which lack caspase-3. Cell fractionation and band depletion studies with the topoisomerase I poison topotecan revealed that the topoisomerase I fragment remains in proximity to the chromatin and retains the ability to bind to and cleave DNA. These observations indicate that topoisomerase I is a substrate of caspase-3 and possibly caspase-6, but is cleaved at sequences that differ from those ordinarily preferred by these enzymes, thereby providing a potential explanation why topoisomerase I cleavage lags behind that of classical caspase substrates such as poly(ADP-ribose) polymerase and lamin B1.

Glutamine requirement of proliferating T lymphocytes

Glutamine is required for lymphocyte proliferation but the site of glutamine action is not yet known. In this study, the effect of glutamine on key events that occur during lymphocyte activation [interleukin-2 (IL-2) production, IL-2 use, IL-2 receptor expression, transferrin receptor expression] was investigated. Rat or mouse spleen lymphocytes were cultured in the presence of the T-cell mitogen concanavalin A (Con A) and various concentrations of glutamine. There was a trend (not significant) for the ratio of CD4 +:CD8 + spleen lymphocytes to increase (from 1.9 to 2.6) as the concentration of glutamine in culture medium increased from 0 to 2 mmol/L. As the concentration of glutamine increased, there was an increase in the proportion of cells expressing the IL-2 receptor (from 30 to 45%) and the transferrin receptor (from 34% to 55%). As the concentration of glutamine increased there was a 2.7-fold increase in the concentration of IL-2 in the culture medium. The IL-2 concentration was decreased when an IL-2 receptor-blocking antibody was included in the culture medium; the IL-2 concentrations measured were taken to indicate the initial Con A-stimulated production of IL-2. In these conditions, the IL-2 concentration in the medium increased 39-fold as the glutamine concentration increased. The use of IL-2 by an IL-2-dependent cell line was dependent on the glutamine concentration in the culture medium. Thus, all four components of lymphocyte activation investigated (IL-2 production, IL-2 use, IL-2 receptor expression, transferrin receptor expression) were dependent on the concentration of glutamine present in the culture medium. Thus, glutamine might provide an early signal in the lymphocyte activation process.

Alteration of mammalian cell metabolism by dynamic nutrient feeding.

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10(7) cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity.

On-line simultaneous monitoring of ammonia and glutamine in a hollow-fiber reactor using flow injection analysis

A FIA method has been developed and fully characterized for the simultaneous detection of ammonia and glutamine in culture media. Ammonia detection is based upon a chemical method and does not require an electrode. This species diffuses across a hydrophobic porous membrane into an indicator solution, the absorbance of which is continuously monitored using a LED and a phototransistor. Glutamine was determined by a difference method in which ammonia was measured before and after passage through a module containing immobilized glutaminase. Very good linearity was observed in the range 0–4 mM for both species. Total analysis time was 22 min. This FIA method was used to accurately monitor ammonia and glutamine on line for over 600 h for a hybridoma culture performed in a hollow-fiber reactor. FIA measurements were in good agreement with off-line measurements. No instrument failures occurred, thanks to systematic cleaning and maintenance procedures. This FIA method is a very attractive tool for the monitoring, and possibly control, of continuous cultures over extended periods.

Glutamine is not an essential amino acid for Sf-9 insect cells

Spodoptera frugiperda (Sf-9) insect cells are fully capable of growth and proliferation in a glutamine, glutamate and aspartate-free medium, provided that ammonium ions are supplied. S. frugiperda (Sf-21) and Mamestra brassicae cells (IZD-MB-0503) also grow in glutamine-free media but not Trichoplusia ni cells (BTI-TN 5B1-4). The yield of β-galactosidase in Sf-9 cells infected with a recombinant baculovirus under glutamine-free conditions was even higher than the yield obtained in glutamine containing cultures.

Glutamine requirement of proliferating T lymphocytes.

Glutamine is required for lymphocyte proliferation but the site of glutamine action is not yet known. In this study, the effect of glutamine on key events that occur during lymphocyte activation [interleukin-2 (IL-2) production, IL-2 use, IL-2 receptor expression, transferrin receptor expression] was investigated. Rat or mouse spleen lymphocytes were cultured in the presence of the T-cell mitogen concanavalin A (Con A) and various concentrations of glutamine. There was a trend (not significant) for the ratio of CD4+:CD8+ spleen lymphocytes to increase (from 1.9 to 2.6) as the concentration of glutamine in culture medium increased from 0 to 2 mmol/L. As the concentration of glutamine increased, there was an increase in the proportion of cells expressing the IL-2 receptor (from 30 to 45%) and the transferrin receptor (from 34% to 55%). As the concentration of glutamine increased there was a 2.7-fold increase in the concentration of IL-2 in the culture medium. The IL-2 concentration was decreased when an IL-2 receptor-blocking antibody was included in the culture medium; the IL-2 concentrations measured were taken to indicate the initial Con A-stimulated production of IL-2. In these conditions, the IL-2 concentration in the medium increased 39-fold as the glutamine concentration increased. The use of IL-2 by an IL-2-dependent cell line was dependent on the glutamine concentration in the culture medium. Thus, all four components of lymphocyte activation investigated (IL-2 production, IL-2 use, IL-2 receptor expression, transferrin receptor expression) were dependent on the concentration of glutamine present in the culture medium. Thus, glutamine might provide an early signal in the lymphocyte activation process.

Correlation between steady‐state cell concentration and cell death of hybridoma cultures in chemostat

In the present study, the steady-state cell density (X) of chemostat cultures of murine hybridoma was varied by the concentration of glucose and glutamine in culture medium and the dissolved oxygen partial pressure. Except at low glutamine and low oxygen levels, the specific death rate (k(d)) of the cultures was found to decrease with increasing dilution rate (D). However, the plot of k(d) vs. X/D yielded linear relation, which suggests that cell death was due to a non-growth-linked inhibitory product of the cells. The k(d) value measured at low glutamine and low oxygen levels remained practically unchanged over a wide range of D between 0.020 and 0.029 h(-1). The k(d) for low oxygen cultures was always lower than the values obtained in low glucose and low glutamine cultures. A low-molecular-weight component of possibly less than 3000 MW was detected to be cell-death-inducing in the supernatant of exponentially growing cultures. It was neither lactate nor ammonium. The autoinhibitor was not cell-line specific. (c) 1995 John Wiley & Sons, Inc.

Role of glutamine synthetase, glutamine and NH4+ in the regulation of glutamine uptake in the cyanobacterium Anabaena cycadeae.

The role of glutamine synthetase (GS), glutamine and/or NH4+ in the regulation of glutamine uptake was studied in the cyanobacterium Anabaena cycadeae and its glutamine auxotrophic mutant lacking GS activity. The uptake pattern was found to be biphasic in both the strains consisting of an initial rapid phase lasting up to 60s followed by a slower second phase. The glutamine uptake was found to be maximum in glutamine medium whereas the cells incubated in N2 and NO3- media showed similar uptake activities in both wild-type and mutant strain. The glutamine uptake system was found to be NH4+-repressible. NH4+ and glutamine did not inhibit the glutamine uptake in the glutamine auxotrophic mutant lacking GS activity, suggesting that NH4+ and glutamine are not the repressor signals for glutamine uptake. The glutamine auxotrophic mutant also had higher level of glutamine uptake as compared to wild-type strain, indicating that GS activity is not necessarily involved in the uptake process of glutamine. Azaserine, an inhibitor of glutamate synthase (GOGAT), effectively inhibited the glutamine uptake in both the strains. Thus, it is suggested that the increased intracellular glutamine level regulates its own uptake in the cyanobacterium Anabaena cycadeae.

Stimulatory and inhibitory effects of amino acids on the development of hamster eight-cell embryos in vitro.

Amino acids were added to a simple medium (TALP) in an effort to enhance the development of hamster eight-cell embryos in vitro. The addition of 1.0 mM glutamine to TALP medium markedly increased the percentage of embryos reaching the blastocyst stage. The addition of groups of 4 or 20 amino acids to TALP + glutamine medium produced differential effects, some groups inhibiting development while others stimulated it. The beneficial effect of glutamine was on the eight-cell to morula transition as well as on blastocyst formation, while the effects of the other 20 amino acids studied were only on blastocyst development. This study represents further progress made in identifying the culture requirements for growth of hamster preimplantation embryos in vitro.

An L-methionine—D,L-sulfoximine-resistant mutant of the cyanobacterium Nostoc muscorum showing inhibitor-resistant γ-glutamyl-transferase, defective glutamine synthetase and producing extracellular ammonia during N 2 fixation

The role of γ-glutamyl-transferase in regulation of N 2-fixation and ammonia assimilation in Nostoc muscorum was examined by isolating mutants in which the enzyme was resistant to L-methionine—D,L-sulfoximine. Mutant and wild-type were compared with respect to nitrogenase activity, extracellular production of nitrogenase-catalysed ammonia, γ-glutamyl-transferase activity and growth in N 2 and glutamine media. While the production of inhibitor-resistant enzyme with defective γ-glutamyl-transferase activity fully explains the inhibitor-resistant growth phenotype, the present results also suggest close metabolic linkage between γ-glutamyl transferase, nitrogenase and assimilation and extracellular production of N 2-derived ammonia.

Isolation of molybdenum cofactor defective cell lines of Nicotiana tabacum

Thirty-nine chlorate resistant cell lines were isolated after plating ethylmethane sulphonate treated allodihaploid cells of Nicotiana tabacum cv. Xanthi on agar medium containing 20 mM chlorate. Thirty-two of these cell lines grew as well on nitrate medium as on amino acid medium and three other cell lines grew well on amino acid medium but poorly on nitrate medium. Four other cell lines, 042, P12, P31 and P47 which could grow on amino acid medium, but not on nitrate medium, were examined further. They lacked in vitro nitrate reductase activity but were able to accumulate nitrate. All lines possessed nitrite reductase activity. Lines 042, P12, and P31 had a cytochrome c reductase species which was the same size as the wild type nitrate reductase associated cytochrome c reductase species, whilst the cytochrome c reductase species in line P47 was slightly smaller. All four lines lacked xanthine dehydrogenase activity and neither nitrate reductase nor xanthine dehydrogenase activity was restored by subculture of the four lines into either nitrate medium or glutamine medium supplemented with 1 mM sodium molybdate. These four lines are different from other molybdenum cofactor defective cell lines so far described in N. tabacum and possess similar properties to certain other cnx mutants described in Aspergillus nidulans.

Effects of cyclic nucleotides upon the germination of Mucor racemosus sporangiospores

This study describes the effects of exogenous cyclic nucleotides upon the germination of sporangiospores of Mucor racemosus in a defined medium containing a variety of carbon and nitrogen sources. Adenosine 3′5′-cyclic monophosphate (cAMP) enhanced germination, with maximal stimulation at concentrations greater than 5 m m. N 6-Monobutyryl cAMP, 8-bromo-cAMP, and adenosine also stimulated germination but 5′-adenosine monophosphate (AMP) did not. N 6, O 2′-Dibutyryl cAMP blocked spore germination in cellobiose, maltose, galactose, 2-ketoglutarate, leucine, and lysine, a result that was also obtained when sporangiospores were incubated in a glucose medium without an exogenous nitrogen source. In glucose, dibutyryl cAMP retarded but did not arrest germination. Dibutyryl cAMP effected rapid and synchronous germination in alanine, aspartate, glutamate, and glutamine media. We conclude that cAMP nonspecifically stimulated the rate of sporangiospore germination while dibutyryl cAMP has a specific stimulatory effect on the initiation of germination of Mucor sporangiospores.

Growth and Composition of Kernels Developing on Excised Oat Panicles in Liquid Culture1

Excised oat (Avena sativa L., cv. ‘Froker’) panicles were cultured in defined media for 12 days, beginning 8 days after heading, to determine effects of supplied culture nutrients on growth, development, and composition of the grain. The sterile media contained 55.6 mM sucrose, mineral salts, vitamins, and various sources and levels of N. Dry weight increases of the groats were near normal when the N source was mixed amino acids, glutamine, or NH4NO3. The N concentration of the groats was increased to 3.85, 4.30, and 5.08% with mixed amino acids, glutamine, and NH4 NO3, respectively, after 12 days in culture, as compared to a N concentration of about 2.96% for groats on intact plants. Electrophoresis and amino acid analysis of total groat protein revealed no marked differences between cultured and intact plants. The percentage of N in the bran fraction was increased by mixed amino acids medium, and that of the.endosperm by glutamine medium. There was no increase in nitrate in groats of the cultured panicles. Levels of soluble α‐amino N were increased slightly by the glutamine medium. It was concluded that the panicle culture technique is valid for study of the effects of supplied nutrients on grain composition. Groat N is responsive to the level of media N, and glutamine or NH4NO3 adequate as a N source.

Mutation in the Blue-green Alga Nostoc muscorum II. L-methionine-DL-sulfoximine-resistant Mutants

Summary The blue-green alga Nostoc muscorum and its Em-R het- nif2- mutant are completely sensitive to 15 ppm concentration of L-methionine-DL-sulfoximille (MSO). Growth-toxicity created by MSO is independent of the inorganic nitrogen sources, i.e., NO3-, NO2- and NH4+. The pa.rent N. muscorum does not form heterocysts in inorganic nitrogen media lacking MSO, but does form them in all these media (except glutamine) containing MSO. MSO-R strain does not form heterocysts in NO3-, NO2-, NH4+ or glutamine medium containing or lacking MSO.

An azide-resistant mutant of the blue-green algaNostoc muscorum producing heterocyst and nitrogenase in the presence of fixed inorganic nitrogen source

The N2, NO 3 − , NO 2 − , NH 4 + and glutamine growing cultures of parentNostoc muscorum are found more or less equally sensitive to azide inhibition of growth. A mutant strain resistant to sodium azide was isolated from the parent strain in NO 3 − medium and the two strains were compared with regard to their heterocyst formation and nitrogenase activity in NO 3 − , NO 2 − , NH 4 + and glutamine media. While the parent strain stops production of both heterocyst and nitrogenase in all the fixed nitrogen media, the azide resistant strain forms both in the fixed inorganic nitrogen media but only heterocyst and no nitrogenase in the glutamine medium. Clearly a single genetic determinant of regulatory nature appears to mediate azide-resistance as well as relief of heterocyst and nitrogenase formation from inhibition by the fixed inorganic nitrogen source. The results of glutamine effect on the heterocyst and nitrogenase formation of the two strains indicate the operation of two levels of glutamine-sensitive regulation, one which operates through the common genetic determinant of heterocyst and nitrogenase regulation and the other exclusive to nitrogenase regulation. The in vivo functional nitrogenase does not appear to be the reason for azide-resistance and neither ammonia nor glutamine or its close metabolic product seems to function in the control of heterocyst spacing.

Studies on the control of cell division in a green alga,Ankistrodesmus gracilis (Chlorococcales)

The effects of various kinds of nitrogen compounds on the proliferation patterns ofAnkistrodesmus gracilis were examined. Among the tested compounds, the addition of peptone, arginine or glutamine to the N-free medium was the most effective on cell growth. The effectiveness was also shown in media containing one of several kinds of amino acid. In both peptone and arginine media the alga proliferated with multiple fission type of cell division, whereas the cultures grown in glutamine or serine medium contained 2-celled colonies at high frequencies. The latter was an efficient culture condition for cell reproduction with binary fission. The evidence for the behavior of nuclear and cytoplasmic division in these growth patterns were obtained from observations of thin sectioned materials.

In vitro synthesis of peritrophic membranes of the blowfly, Calliphora erythrocephala

Tyrode solution containing added glutamine and Leloup's medium 1 has been used as a basic medium for the in vitro culture of the so-called proventriculus of adult Calliphora erythrocephala to elucidate some of the factors controlling the synthesis of peritrophic membranes (PM) in vitro. The formation rate was chosen as a quantitative criterion for the evaluation of the modifications of the incubation media. After systematic variation of osmolarity, pH, and temperature optimal formation rates were obtained in media with an osmolarity of 320 to 360 mOsmol, a pH of 6·8, and an incubation temperature of 27°C. Under these conditions the average rate of formation was in the modified Tyrode solution 3·0±1·1 mm PM/hr, and in Leloup's medium 3·6±0·8 mm PM/hr. In the modified Tyrode solution the formation of PM was complete after 5 to 7 hr, whereas in Leloup's medium it continued up to 24 hr. The addition of β-ecdysone caused an increase of the formation rate of PM to 4·5 to 5·5 mm PM/hr. The results obtained led to the hypothesis that an osmotically regulated enzyme system could be the limiting factor of the formation rate of peritrophic membranes, i.e. a system which could regulate the internal osmolarity of the formative cells by the interconversion of a bulk polymer and its monomer which are needed for the synthesis of PM.

Utilization of amino acids as nitrogen sources, and their effects on nitrate reductase in the marine diatom Cyclotella cryptica.

Several amino acids supplied at 0.1 mM allow good growth of Cyclotella cryptica. The cells can grow as well on arginine and glutamine as on nitrate. Glutamate, proline, ornithine, and asparagine also allow good growth, while glycine, alanine, and aspartate support relatively low initial growth rates. Isoleucine appears not be utilized at all. Ammonium and urea are excellent nitrogen sources for growth.There is good correlation between chlorophyll and protein content in cells grown on different nitrogen sources. Cells grown in nitrate, arginine, and glutamine media contain the highest amount of protein and the best pigmentation. Although both glutamate and proline allowed good growth, cells grown in these media have low levels of protein and chlorophyll, indicating nitrogen limitation. No growth inhibition was observed for any of the amino acids tested at 0.1 mM in the presence of nitrate. It is estimated that amino acids at naturally occurring concentrations may contribute significantly to the nitrogen economy of this diatom when more common nitrogen supplies such as nitrate and ammonia are scarce.Nitrate reductase in C. cryptica appears to be found only in the presence of nitrate, and is repressed by other nitrogen compounds. The extent of repression by amino acids, urea, and ammonium is related to their ability to promote growth when supplied as sole sources of nitrogen.

RNA metabolism in human cells during amino acid deprivation: II. Synthesis of ribosomal and transfer RNA

The effects of amino acid deprivation on the rate of synthesis of cytoplasmic rRNA and tRNA, prepared by phenol extraction, were studied. Incubation of the cells in a glutamine or leucine free medium resulted in a 60–90 % decrease in the rate of formation of rRNA. The rate of synthesis of tRNA was inhibited by 35–50 %. Restitution of the complete medium also had characteristically different effects on the two species of RNA. After addition of the missing amino acid, the rate of synthesis of rRNA increased, following a lag period of about 1 h. On the other hand, the rate of synthesis of tRNA exhibited a marked decrease during the first hours of nutritional restitution and a longer lag period, prior to the expected acceleration. Therefore, the effect of nutritional shifts on the synthesis of tRNA and rRNA was non-coordinate. The results are discussed with reference to different regulatory mechanisms for protein synthesis in eu- and prokaryote cells.