Production of oligosaccharides and cellobionic acid by <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 growing on sugars,cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.     

Aerobiosis and the hemoprotein content of photosynthetic bacteria

Summary Rhodospirillum rubrum and Rhodopseudomonas spheroides, grown under various degrees of illumination, aeration, and iron deprivation, have been assayed for their content of cytochrome c, RHP, catalase, total iron, bacteriochlorophyll, and carotenoids.Concentrations of bacteriochlorophyll and carotenoids were consistent with the findings of Cohen-Bazire et al. (1957).Total iron content, which ranged from 0.017 to 0.04% of the dry weight, reflected the content of the principal hemoproteins but exceeded the amount of iron in these hemoproteins.The catalase content of R. rubrum, on a dry weight basis, was 0.0005% for cells grown anaerobically in the light, and 0.0028% for cells grown in darkness with vigorous aeration; that of Rps. spheroides was 0.006% and 0.25%, respectively. The catalase content in both species rose with increasingly vigorous aeration.Cytochrome c in both species, and RHP in R. rubrum, attained the same levels in cells grown under vigorous aeration as in cells grown anaerobically in the light. In cells grown under limited aeration the levels of these substances were about 50% higher. In Rps. spheroides the RHP content was greatest in cells grown anaerobically, falling under gentle aeration and declining further under more vigorous aeration.Iron deficiency caused a decrease in the catalase content of cells grown anaerobically in the light but not in cells grown aerobically. The content of cytochrome c and of RHP was diminished by iron depletion in aerobic cultures, but not in anaerobic cultures.operated by Union Carbide Corporation for the U.S.Atomic Rnergy Commission.     

The Cultivation of Blastocrithidia triatomae Cerisola et al., 19711

Blastocrithidia triatomae, a species very resistant to cultivation in conventional media, was successfully grown in association with cultured lepidopteran cells. The cultured flagellates were identical to those in the insect host, including the presence of characteristic cysts on the flagellum. Growth rate was better for cultures in the Heliothis zea medium than in IPL-45 medium or modified McConnell's medium.     

The mechanisms of nitrogen assimilation in Pseudomonads

Pseudomonas aeruginosa, Ps. fluorescens and 3 marine psychrophylic pseudomonads were grown in chemostat cultures with nitrate ammonia or glutamate as nitrogen source. In cultures grown on nitrate (either carbon- or nitrogen-limited) and in ammonia nitrogen-limited cultures ammonia was assimilated via the GS/GOGAT pathway. With a excess of ammonia in the culture however ammonia was assimilated via GDH and GS was either present only at low levels or absent. Two distinct GDH activities were detected in all 5 bacteria, one specific to NAD and one to NADP. The presence of these activities was determined by the environment in which cells were grown. These activities showed differences with respect to substrate affinity (Km values) for ammonia, incubation temperature and to a lesser extent pH and may involve separate GDH isoenzymes. GS from the marine bacterium PL₁ had a very high affinity for ammonia (Km of 0.3mm) but a low affinity for glutamate (Km of 19mm).     

Daughter cells as an important factor in determining the physiological state of yeast populations

Cells of Candida utilis grown in a single-stage chemostat at D = 0.05, 0.1, 0.25, and 0.35 hr^?l were separated into a fraction of scar-bearing mother cells and a fraction of scar-free daughter cells. The scar-free cells were transferred into small batch cultures where the length of the maturation phase, changes in length and width of cells, specific growth rate, and specific rate of RNA and protein synthesis were examined for 5 hr. The daughter cells grown at D = 0.05 hr^?1 were very small at the moment of separation from the mother cells (about one-third of the mother cell). Their maturation phase (in a batch culture), at the beginning of which they attain the specific growth rate approaching the μ_max of the strain used, lasts for 3 hr. On the other hand, daughter cells grown at D = 0.35 hr^?1 are almost the same size as the mother cells at the moment of separation. After transfer to a batch culture they begin to bud almost immediately. Similarly, in their other morphological and physiological parameters they differ strikingly from immature daughter cells which are formed at low specific growth rates. The importance of these differences from the point of view of mathematical modeling of growth processes is discussed.     

Increase in the production of allelopathic substances by Prymnesium parvum cells grown under N- or P-deficient conditions

The haptophyte Prymnesium parvum is known to produce a set of highly potent exotoxins, commonly called prymnesins. These toxins have been shown to have several biological activities, including ichthyotoxic, neurotoxic, cytotoxic, hepatotoxic and hemolytic activity towards a range of marine organisms. In addition, recent studies have shown that the toxicity of P. parvum is enhanced when the cells are grown under N- or P-deficient conditions. In this study, the influence of prymnesium toxins on the growth of other phytoplankton species was investigated by addition of cell-free filtrate of P. parvum cultures grown under nutrient-deficient (N or P) or non-deficient conditions. Addition of cell-free filtrate from P. parvum cultures grown under N or P limitation inhibited the growth of Thalassiosira weissflogii, Prorocentrum minimum and Rhodomonas cf. baltica. In contrast, a strain of Prymnesium patelliferum known to produce prymnesium toxins was not negatively affected under any conditions. Furthermore, addition of filtrates from nutrient-sufficient P. parvum cultures did not negatively influence the growth of any of the tested species. These findings suggest that prymnesium toxins may play an allelopathic role, and that the production of allelopathic substances is regulated by the availability of nutrients.     

Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and β -galactosidase expression by Saccharomyces cerevisiae

The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne β -galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (Δμ) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited Δμ -dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. Δμ decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or Δμ, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased Δμ values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of Δμ may have conferred was counteracted by an increased R value. Increased β-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity. Received 08 July 1996/ Accepted in revised form 14 January 1997     

Influence of fructose and glucose on the production of exopolysaccharides and the activities of enzymes involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772

 The effect of fructose and glucose on the growth, production of exopolysaccharides and the activities of enzymes involved in the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus grown in continuous culture was investigated. When grown on fructose, the strain produced 25 mg l^-1 exopolysaccharide composed of glucose and galactose in the ratio 1:2.4. When the carbohydrate source was switched to a mixture of fructose and glucose, the exopolysaccharide production increased to 80 mg l^-1, while the sugar composition of the exopolysaccharide changed to glucose, galactose and rhamnose in a ratio of 1:7.0:0.8. A switch to glucose as the sole carbohydrate source had no further effect. Analysis of the enzymes involved in the synthesis of sugar nucleotides indicates that in cell-free extracts of glucose-grown cells the activity of UDP-glucose pyrophosphorylase was higher than that in cell-free extracts of fructose-grown cells. The activities of dTDP-glucose pyrophosphorylase and the rhamnose synthetic enzyme system were very low in glucose-grown cultures but could not be detected in fructose-grown cultures. Cells grown on a mixture of fructose and glucose showed similar enzyme activities as cells grown on glucose. Analysis of the intracellular level of sugar nucleotides in glucose-grown cultures of L. delbrueckii subsp. bulgaricus showed the presence of UDP-glucose and UDP-galactose in a ratio of 3.3:1, respectively, a similar ratio and slightly lower concentrations were found in fructose-grown cultures. The lower production of exopolysaccharides in cultures grown on fructose may be caused by the more complex pathway involved in the synthesis of sugar nucleotides. The absence of activities of enzymes leading to the synthesis of rhamnose nucleotides in fructose-grown cultures appeared to result in the absence of rhamnose monomer in the exopolysaccharides produced on fructose. Received: 1 February 1996/Received revision: 31 May 1996/Accepted: 2 June 1996     

Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841

Bacteroids of R. leguminosarum MNF3841 isolated from pea nodules using Percoll gradients had activities of TCA cycle enzymes up to 6-fold higher than those measured in free-living cells grown on fumarate or sucrose. Activities of sugar catabolic enzymes on the other hand were 2–14-fold lower in isolated bacteroids than in sucrose-grown free-living cells. In continuous culture, cells of strain MNF3841 grown on sucrose under P _i limitation had 2–3-fold higher activities of invertase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes and 6-phosphogluconate dehydrogenase, than cells grown on fumarate. With one exception O₂ limited cultures had similar activities of the carbon catabolic enzymes to P _i-limited cultures grown in the same substrate. Glucose-6-phosphate dehydrogenase in O₂-limited cells grown of fumarate was 50% lower than in P _i-limited cells. Co-utilization of fumarate and sucrose occurred with chemostat cultures supplied with both under a variety of conditions.Abbreviations E-D Entner-Doudoroff - EMP Embden-Meyerhof-Parnas - PEPCK phosphoenolpyruvate carboxy kinase - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid]     

THE EFFECT OF LIGHT QUALITY ON THE CARBON METABOLISM AND EXTRACELLULAR RELEASE OF CHLAMYDOMONAS REINHARDTII DANGEARD1

The influence of red, blue, green, and white light on growth and photosynthetic rates, carbon metabolism, and rates of release of extracellular compounds in the freshwater alga Chlamydomonas reinhardtii Dangeard was examined. Relative growth constants were 0.28, 0.32, 0.40, and 0.41 in green, white, blue, and red light, respectively. Photosynthetic rates were higher in white, blue, or red than in green light of the same intensity. More than 66% of the ¹⁴CO₂ assimilated by cells grown under blue or green light was incorporated into the ethanol-insoluble fraction, compared with about 50% in cells grown under white or red light. The percentage of sugars in this fraction was significantly higher in cells grown under green or red light than in cells cultured in white or blue light, while the percentage of proteins was highest in blue light. Light quality also influenced the composition of the ethanol-soluble fraction. The percentage of organic acids was highest in cells grown in green and white light, while amino acids were highest in blue and green cultures. The percentage of ethanol-soluble sugars was greatest in cultures grown in blue and red light. The percentage release of dissolved organic carbon into the medium was highest in white light and lowest in blue or red light. The nature of the extracellular products varied according to the quality of light under which the cells were cultured, but had no consistent relation to the nature or concentration or components in the ethanol-soluble fraction.     

Fatty-acid composition and biosynthesis in cell suspension cultures of Glycine max (L.) Merr., Catharanthus roseus G. Don and Nicotiana tabacum L.

The fatty-acid composition of C. roseus and N. tabacum cell suspension cultures was unaffected by subculture on Wood and Braun, Murashige and Skoog, or Gamborg B5C media. However, placing the cultures — which were normally grown at 25° C — at 15° C reduced growth but resulted in enhanced formation of oleic and linolenic acids in C. roseus cultures and increased levels of linoleic and linolenic acids in cultures of G. max and N. tabacum, respectively. The incorporation of [¹⁴C]acetate into [¹⁴C]linoleic acid was more rapid in N. tabacum cells than in G. max cells, but was very poor in C. roseus where the [¹⁴C] label was distributed mainly between palmitic and oleic acids.     

GROWTH CHARACTERISTICS OF PHYTOPLANKTON DETERMINED BY CELL CYCLE PROTEINS: PCNA IMMUNOSTAINING OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

By immunohistochemistry and immunofluorescence methods, we observed that the analog of proliferating cell nuclear antigen (PCNA) in Dunaliella tertiolecta Butcher (Chlorophyceae) was exclusively located in the nucleus. Among positively stained cells, PCNA abundance varied, being highest in S-phase cells, lower in others, and undetectable in early G1- or late M-phase cells. In exponentially growing and partially synchronized cultures, the percentage of PCNA-stained cells (% PCNA-stained cells) oscillated in the photocycle (12:12 h LD). It increased during the light period and reached a peak (75%) before the onset of the dark period when the culture was mainly (71%) in the S phase of the cell cycle. The DNA synthesis inhibitor, hydroxyurea, depressed PCNA abundance, whereas no effect was detected for the mitosis inhibitor colchicine. We conclude that PCNA in D. tertiolecta is associated with the S phase of the cell cycle where it is accumulated and functioning. PCNA was used to characterize the growth pattern of cultures grown in different media, temperatures, and growth stages. The time lag between the PCNA-stained phase and the M phase was very short in a continuous culture grown in reduced f/2 medium at 22°C and was considerably longer in the cultures grown in f/2 at 15°C. When an exponentially growing culture grew older, % PCNA-stained cells decreased. In a late stationary culture where there was no net growth, a small number of cells were still cycling through the PCNA-stained phase and cell division. In the continuous culture grown at 22°C, the duration of the PCNA-stained phase (T_s) was 13 h. Calculations with this T_s and % PCNA-stained cells yielded a growth rate of 0.77 d^?1, which was close to that obtained by cell counts (0.69 d^?1). Taken together, the results suggest that PCNA is a useful indicator of growth status and a promising cell cycle marker for estimation of species-specific growth rate.     

Growth measurements of Chromatium cultures

Summary The optical density of Chromatium cultures grown anaerobically in the light with sulfide as electron donor is mainly determined by the sulfur content of the cells. Since the sulfur content varies, growth cannot be followed in this way. However, optical density measurements are very useful to characterize the moment of sulfide depletion and maximal sulfur storage. In addition to protein or cell nitrogen, increase in structural cell material in growing cultures of Chromatium can easily be followed by bacteriochlorophyll determinations, provided that the illumination intensity is higher than the saturation intensity.     

Influence of carbon source on production of a heat stable protease from Thermomonospora fusca YX

Summary Thermomonospora fusca YX produced a very active heat stable protease when incubated in media containing cellulose as the substrate. Cultures grown on Solka-floc generated the highest amount of protease whereas the protease was produced at significantly lower levels when T. fusca YX was grown on cellobiose or glucose. Negligible growth or protease production was observed when protein was used as a carbon source. The production of the protease did not appear to be constitutive. While rapid growth was observed on either cellobiose or glucose, protease levels were at least two to fourfold lower than for the T. fusca YX cultures grown on Solka-floc wich generated 33% less cell mass. Protease production was four times lower in cultures which employed casein hydrolysate (tryptone) or xylan as carbon sources than for cellulose.     

VARIABILITY IN NITRATE UPTAKE KINETICS IN THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Short-term (1–9 min) nitrate uptake kinetics were measured in Thalassiosira pseudonana (Hust.) Hasle & Heimdal grown in nitrate-limited, ammonium-limited, and nitrate-sufficient continuous cultures. For all cultures, maximal nitrate uptake rates did not develop until approximately 3 min after nitrate addition; thereafter, nitrate uptake rates remained constant or declined slightly. The K_s and V_max for the nitrate-limited cultures were higher at any growth rate than those for the ammonium-limited or nitrate-sufficient cultures. Thus, much higher nitrate concentrations would be required to saturate nitrate uptake in nitrate-limited Thalassiosira pseudonana than is usually considered necessary. The lack of data for other species grown under a range of environmental conditions makes it difficult to generalize about the effect of preconditioning on nitrate uptake kinetics.     

Light- and sucrose-dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape (Brassica napus L.)

Light-dependent gene expression was analysed in photomixotrophic cell suspension cultures of rape (Brassica napus L.) growing in media containing either 2.0% or 0.6% sucrose. During growth in darkness phytochrome type I and NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) accumulated in both cell culture lines to a similar extent. Illumination with continuous white, blue or red light, but not with far-red light, resulted in disappearance of both chromoproteins within 24 h in both cell cultures. Further analysis showed that the phytochrome system of rape cell cultures reacts in a similar way to that of re-etiolated dicotyledonous plants, showing rapid P_fr destruction and rapid P_fr dark reversion. In contrast, the light-dependent expression of genes encoding the major chlorophyll a- and b-binding protein (CAB) and the re-accumulation of chlorophyll were found to be strongly dependent on sucrose concentration in culture media. Whereas cells grown in darkness in medium containing 2.0% sucrose showed, after exposure to continuous white light, a very weak re-induction of CAB mRNA, CAB protein and chlorophyll accumulation, the cells in medium containing 0.6% sucrose reacted very strongly. It was also possible to demonstrate that phytochrome (by high irradiance response, HIR, and by low fluence response, LF) and the blue/UV-A receptor are involved in the light-dependent gene expression of CAB. Similar to complete cells, protoplasts derived from the two different cell cultures showed an almost identical sucrose concentration-dependent and light-quality-dependent regulation of CAB mRNA accumulation. As the dark-grown photomixotrophic cells and protoplasts reflect some typical photoregulatory characteristics known from dark-grown plants it is supposed that this system will be an excellent tool for studying biochemical and molecular biological aspects of light-dependent signal transduction in cells of higher plants.     

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min^–1 (mg protein)^–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K _m of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V _max nor the d,l-isocitrate K _m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. Received: 10 October 1997 / Accepted: 16 January 1998     

Human oral epithelial cell culture II. Keratin expression in fetal and adult gingival cells

Summary Epithelial cells from human fetal and adult gingiva were cultured in keratinocyte growth medium (KGM), a serum-free medium. The expression of keratin proteins in these cells was evaluated using immunohistochemistry and SDS-PAGE-immunoblot analysis and compared with expression in the tissue. Keratins 5, 6, 14, 16, and 19 were identified in cells cultured from both fetal and adult tissues. K19 was localized in basal cells of fetal oral tissue but was not seen in adult gingiva (except for scattered Merkel cells). K1 and K10 were expressed in tissue, but not in cultured cells. The keratin profiles of cultured epithelial cells from several adult donors were similar and were identical in cultures from primary through Passage 5. K13, a differentiation-specific keratin, was expressed in all suprabasal cells of fetal oral epithelium, but shows only spotty expression in adult gingival tissue. K13 was expressed in cultures of fetal cells, but very weakly or not at all in cultures of adult cells. K13 expression was greater in cultures grown with physiologic calcium concentrations (1.2 mM) than in those grown at 0.15 mM or less. Our findings are consistent with basal-like characters of these cells in 0.15 mM calcium growth conditions. Differentiation of fetal oral cells in culture to the suprabasal basal cell stage in 1.2 mM Ca²⁺ is shown by the expressionof K13. This work was supported by Biomedical Research grant RR05346, National Institutes of Health grant DE04660, University of Washington Graduate Fund and Hack Foundation Fund, Department of Periodontology, University of Washington.     

Isolation and ultrastructure of freshwater strains ofPlanctomyces

Four strains of a freshwaterPlanctomyces species—different in a number of respects from those hitherto described—have been isolated and their morphology and ultrastructure examined by transmission electron microscopy. The ovoid or spherical prokaryotic cells have a cell envelope consisting of outer and inner membranes, but apparently lacking a peptidoglycan wall layer. The cell envelopes of these osmotically sensitive organisms are stabilized in the presence of 5 mM MgSO₄ or CaCl₂; in the absence of divalent cations, autolysis is a common occurrence. Reproduction of these motile, stalked bacteria occurs by an asymmetric budding process in nonaxenic enrichment cultures and in pure cultures grown in very dilute (0.005% or less) peptone medium; but in higher concentrations of nutrients, division is more frequently symmetric and the multifibrillar stalks or appendages are seldom detectable. The cell diameters and the proportion of motile, flagellated cells as a stage of the life cycle are variable features, dependent on cultural conditions.     

Growth Conditions Differentially Regulate the Expression of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate (AMPA) Receptor Subunits in Cultured Neurons

We have studied the expression of a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K^± (10, 25, or 40 mM) with or without 100 μM N-methyl-D-aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K^± (K10) and increased dramatically in cultures grown in 25 mM K^± (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 ± NMDA). In cultures grown in 40 mM K^± (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and -3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA-stimulated ^4SCa^2± influx and 40-[³H]phorbol 12, 13-dibutyrate binding in intact cells. Both functional parameters increased along with the K^± concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits. These results indicate that functional diversity of AMPA receptors can be generated by the degree of chronic depolarization and/or exposure to NMDA in neurons developing in primary culture.