Metabolic conversion of exogenous14C-aspartate and14C-gIutamate in the dark byFucus serratus L

The rates of uptake of exogenous L[U-¹⁴C] aspartate and glutamate into tissues of vegetative growing tips ofFucus serratus and their metabolism were studied in the dark. In these non-photosynthetic conditions, aspartate was fixed and metabolically converted more rapidly than glutamate. Radioactivity from¹⁴C-aspartate was principally transferred into glutamate. On the other hand, metabolism of absorbed¹⁴C-glutamate was very slow and its rate did not increase during incubation time, but produced more diversified soluble radioactive compounds. Thus inF. serratus, glutamate principally seems to be in the dark more a temporary¹⁴CO₂ storage product coming from β-carboxylation than a rapidly turned over intermediate.     

Glutamate Uptake and Metabolism in C-6 Glioma Cells: Alterations by Potassium Ion and Dibutyryl cAMP

Abstract: Uptake and metabolism of glutamate was studied in the C-6 glioma cell line grown in the absence or presence of dibutyryl cyclic AMP (dbcAMP). Glutamate and aspartate uptake were competitive in cells grown under both conditions. Increased [K⁺] in the medium caused a significant decrease in the uptake of both amino acids. A small part of this decrease (<25%) was due to an enhanced efflux of tissue amino acid. The effects of increased [K⁺] were observed whether or not the [Na⁺] in the medium was concomitantly decreased. In cells grown in the presence of 1 mM dbcAMP for 48 h, glutamate uptake and metabolism were altered. Tissue levels of glutamate, aspartate, glutamine, GABA, and alanine were generally less in treated than in naive cells. When incubated with 50 μM [U-¹⁴C]glutamate, there was significantly less incorporation of radioactivity into treated cells with time, resulting in greatly lowered specific radioactivities of glutamate, aspartate, and GABA. However, the rate of labeling of glutamine was greatly increased; this was consistent with the previously observed doubling in glutamine synthetase activity in dbcAMP-treated C-6 cells. Tissue glutamate decarboxylase activity was halved in treated cells, accounting for the large decrease in GABA labeling. The metabolic data suggested a decreased uptake of exogenous glutamate; in studies on initial rates of uptake, the V_max of high-affinity glutamate uptake was decreased by 40%. This decrease was of the same order of magnitude as that observed in the metabolic experiments. Thus, in this glial model, both rapid, acute changes and slower, long-term changes in neuroactive amino acid metabolism were observed. Each of these conditions mimics a stimulus of neuronal origin, and the resulting changes could modulate extrasynaptic activity of neuroactive amino acids.     

Protein patterns in the oat coleoptile as influenced by auxin and by protein turnover

Synthesis of growth-limiting proteins (GLP) is required for continued auxin-induced elongation of oat (Avena sativa L.) coleoptiles. In order to determine whether GLP synthesis is dependent or independent of auxin, a double-labeling ratio technique, coupled with disc-gel electrophoresis, has been used to assess the effect of auxin on the pattern of protein synthesis. Sections were peeled to enhance amino-acid uptake; proteins were labeled with [¹⁴C]- or [³H] leucine in the presence or absence of indole-3-acetic acid for 40 min to 6 h, and were separated into soluble, membrane-associated, and wall-associated fractions. Regardless of the conditions used, or the protein fraction examined, no changes in response to auxin were detected in the pattern of protein synthesis. In order to escape detection by this technique an auxin-induced protein would have to comprise less than 0.75% of the total newly synthesized protein. Thus the synthesis of GLP appears to be independent of auxin. The same technique has been used to follow protein turnover. During the chase, proteins are initially degraded at an average rate of 8% h^?1, and some protein bands showed as much as 14% h^?1 degradation. No protein was detected which had a turnover rate as rapid as the GLP.     

Enhancement in dopamine uptake and release induced by monosodium l-glutamate from caudate nucleus under in vitro conditions

l-Glutamate has an excitatory and cytotoxic effect on the central nervous system. It was shown previously that norepinephrine and dopamine uptake and release were affected by in vivo administration of glutamate to adult rats. The kinetic parameters, K_m and V_max of [¹⁴C]DA uptake and release were measured on synaptosomal and slices from caudate nucleus under in vitro conditions at different glutamate concentrations. Results showed an important increase in [¹⁴C]DA uptake on synaptosomal (> 100%) and slices by lower glutamate concentrations, the affinity for transport system was increased (100%) and its release of high potassium evoked was also increased at 0.5 μM of glutamate. The results suggest the possibility that glutamate may modify DA uptake and release interacting with the DA transporter complex at the synaptic level.     

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward. During training and incorporation for 20-30 min, both [³H]leucine and [1-¹⁴C]leucine underwent a significant increase in catabolism, resulting in a decreased radioactivity in the free amino acids. [2-²H]Methionine underwent a rapid loss of isotope, so that 90% of the radioactivity was in the form of tritiated water at the end of training, and this phenomenon masked any possible effect of training. The brain uptake of [³⁵S]methionine increased during the training, resulting in an increased radioactivity in the proteins. Uptake of [³H]lysine increased slightly during training only after 1 h incorporation and not after 20 or 30 min, as judged from a time course of radioactivity in the free amino acids. Incorporation into nuclear proteins increased selectively during 20 min, and into nuclear and cytosol proteins after 60 min incorporations. It is concluded that changes in the observed rate of incorporation of a precursor into brain subcellular proteins under the influence of behaviour might be the result of changes in precursor catabolism or uptake, or both, and that each amino acid behaves in a different way. Even the same amino acid gives different results depending on the isotope and its position in the amino acid.     

Effects of temperature,irradiance, and daylength on the marine diatom leptocylindrus danicus cleve. I. Photosynthesis and cellular composition

Rates of ¹⁴C uptake and cellular composition of C, N, and Chl a in the marine diatom Leptocylindrus danicus Cleve were measured in axenic batch culture under 49 combinations of temperature (5, 10, 15, 20 °C), daylength (15: 9, 12: 12, 9: 15 LD), and irradiance (at least four irradiances per daylength). ¹⁴C uptake exhibited a temperature-dependent daylength effect. Similar P-I curves characterized cells grown under 15: 9 and 12: 12 LD; P_max values were 17.2, 11.2, 4.3, and 1.8 pg C. pg Chl a^?1. h^?1 at 20, 15, 10, and 5°C, respectively. Under 9:15 LD at 20 and 15°C, the lightsaturated photosynthetic rate was ≈50% that in cells grown under longer daylengths. ¹⁴C uptake was independent of daylength at 10 and 5°C. The initial slope, a, of cells grown under long daylengths increased by five-fold between 5 and 20 °C. α values of cells grown under 9: 15 LD at 15 and 20 °C were depressed relative to longer daylengths. Chl a was inversely related to irradiance, and increased with temperature from 10 to 20 °C, whereas cell carbon and nitrogen showed a similar temperature dependence but was not influenced by irradiance or daylength. The C : N ratio and cell volume were independent of temperature, irradiance, and daylength. Both the C : Chl a and N : Chl a ratios increased with irradiance by greater amounts at lower temperatures.     

Photoheterotrophy and light-dependent uptake of organic and organic nitrogenous compounds by Planktothrix rubescens under low irradiance

1. Planktothrix rubescens is the dominant photoautotrophic organism in Lake Zürich, a prealpine, deep, mesotrophic freshwater lake with an oxic hypolimnion. Over long periods of the year, P. rubescens accumulates at the metalimnion and growth occurs in situ at irradiance near the photosynthesis compensation point. Experiments were conducted to evaluate the contribution of photoheterotrophy, heterotrophy and light‐dependent uptake of nitrogenous organic compounds to the carbon and nitrogen budget of this cyanobacterium under conditions of restricted availability of light quanta. 2. We used both purified natural populations of P. rubescens from the depth of 9 m and an axenic culture grown under low irradiance at 11 μmol m^?2 s^?1 on a light : dark cycle (10 : 14 h) to determine the uptake rates of various amino acids, urea, glucose, fructose, acetate and inorganic carbon. The components were added to artificial lake water in low amounts that simulated the naturally occurring potential concentrations. 3. The uptake rates of acetate and amino acids (glycine, serine, glutamate and aspartate) were strongly enhanced at low irradiance as compared with the dark. However, no difference was observed in the uptake of arginine, which was taken up at high rates under both treatments. The uptake rates of glucose, fructose and urea were very low under all conditions. Similar results were obtained for both axenic P. rubescens and for purified natural populations of P. rubescens that were separated from bacterioplankton and other phytoplankton. 4. Metalimnetic P. rubescens that was stratified at low irradiance for weeks exhibited much higher uptake rates than filaments that were entrained in the deepening surface mixed layer and experienced higher irradiance. The added organic compounds contributed up to 62% to the total carbon uptake of metalimnetic P. rubescens. On the basis of a molar C : N ratio of 4.9, the nitrogen uptake as organic compounds satisfied up to 84% of the nitrogen demand. 5. The experiments indicate that photoheterotrophy and light‐dependent uptake of nitrogenous organic compounds may contribute significantly to the carbon and nitrogen budget of filaments at low irradiance typical for growth of P. rubescens in the metalimnion and at the bottom of the surface mixed layer.     

Effect of low irradiance on the photosynthetic performance and spiking of Phalaenopsis

Lowering irradiance can delay the flower stalk, i.e., spike development, in order to schedule flowering time of Phalaenopsis; however, the effect on photosynthetic performance and spiking inhibition remains poorly understood. We compared light and shade treatments of Phalaenopsis aphrodite subsp. formosana in order to determine how limiting light affects day-night changes in the photosynthetic capacity of leaves and the carbon pool of leaves and stems resulting in delayed spiking. The low irradiance treatment [20 μmol(photon) m^?2 s^?1] for six weeks did not affect potential functions of photosynthetic apparatus estimated by chlorophyll a fluorescence analysis, but it significantly reduced the net CO₂ uptake and O₂ evolution rates, carbohydrate and organic acid concentrations, and amplitudes of CAM activity in new and fully expanded leaves of Phalaenopsis and delayed the spiking compared with the control kept at 150 μmol(photon) m^?2 s^?1. The shortened stem contained a remarkably high sucrose concentration, accounting for more than 80% of total soluble sugars for both treatments throughout the day. Moreover, the sucrose concentration was unaffected by the lowering of irradiance. The relationship between the sucrose content and spiking seemed to be loose; the major factor(s) for spiking in Phalaenopsis remained to be ascertained as the flower stalk bud is attached to the shortened stem.     

Hypoxia suppresses astrocyte glutamate transport independently of amyloid formation

Sustained hypoxia alters the expression of numerous proteins and predisposes individuals to Alzheimer’s disease (AD). We have previously shown that hypoxia in vitro alters Ca²⁺ homeostasis in astrocytes and promotes increased production of amyloid β peptides (Aβ) of AD. Indeed, alteration of Ca²⁺ homeostasis requires amyloid formation. Here, we show that electrogenic glutamate uptake by astrocytes is suppressed by hypoxia (1% O₂, 24 h) in a manner that is independent of amyloid β peptide formation. Thus, hypoxic suppression of glutamate uptake and expression levels of glutamate transporter proteins EAAT1 and EAAT2 were not mimicked by exogenous application of amyloid β peptide, or by prevention of endogenous amyloid peptide formation (using inhibitors of either β or γ secretase). Thus, dysfunction in glutamate homeostasis in hypoxic conditions is independent of Aβ production, but will likely contribute to neuronal damage and death associated with AD following hypoxic events.     

Cadmium increases the activity levels of glutamate dehydrogenase and cysteine synthase in Chlamydomonas reinhardtii

The presence of 300 μM Cd²⁺ in culture medium was found to be toxic to Chlamydomonas reinhardtii, reducing growth and productivity by about 48%. Approximately 30% of the total cadmium in the medium was accumulated by the alga resulting in 0.88% of the algal dried biomass. Elemental analysis indicated a cadmium-dependent decrease in the C (about 3.2%) and N content (about 7.1%) within C. reinhardtii, while the S content increased by approximately 7.5%. In parallel, Cd²⁺ produced a significant activation of the aminating glutamate dehydrogenase (EC 1.4.1.2) activity and also NAD⁺- and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42, respectively) activities upon 24 h of the exposure to 150 μM of the metal. These data are consistent with the key role of the glutamate dehydrogenase/isocitrate dehydrogenase system to supply the glutamate required for the Cd²⁺-induction of phytochelatin synthesis in the alga. Moreover, the presence of cadmium in the culture medium enhances the sulfate uptake rate and the components of the cysteine synthase complex within the cells such as the serine acetyltransferase (EC 2.3.1.30) and O-acetyl-L-serine (thiol)lyase (EC 4.2.99.8) activities.     

Metabolism of phenylalanine and tyrosine in tobacco cell lines resistant and sensitive to p-fluorophenylalanine

Chromatography of soluble polyphenols of p-fluorophenylalanine-sensitive and -resistant tobacco cells revealed that the 10-fold increased level found in the resistant line was largely due to the accumulation of two unidentified polyphenols. The uptake of Phe-[U-¹⁴C] and Tyr-[U-¹⁴C] by the resistant line was ca 10 % that by the sensitive line. About 90 % of the phenylalanine-[¹⁴C] which was taken up by both cell lines could be accounted for as free phenylalanine in protein, soluble polyphenols or CO₂. The fate of Tyr-[¹⁴C] was similar to that of phenylalanine except that the incorporation was into insoluble polymeric forms of polyphenols rather than into soluble polyphenols. The resistant line incorporated 9-times more phenylalanine-[¹⁴C] into soluble polyphenols than did the sensitive line. The different ¹⁴C-aromatic amino acid accumulation and incorporation patterns noted with the two cell lines indicates that there are different active pools. Differential uptake rates by the two cell lines might affect the distribution of the absorbed amino acid among the different pools.     

Asparagine biosynthesis by Oryza sativa seedlings

l-Aspartate-[U-¹⁴C] was quickly metabolized in rice seedlings into amino acids, organic acids and sugars. On feeding simultaneously with ammonium for 2 hr, about 1% of the total soluble radioactivity was recovered as asparagine. Major amino acids labelled were aspartate, glutamate, glutamine and alanine in both shoots and roots. On the other hand, on feeding l-aspartate-[U-¹⁴C] to rice seedlings precultured in an ammonium medium, asparagine accounted for 35% of the total soluble radioactivity in the roots. Different labelling patterns in amino acids from those of non-precultured tissues were observed, and the main amino acids labelled in this case were asparagine and γ-aminobutyrate in the roots; glutamate, asparagine and glutamine in the shoots. It was observed in the roots that this increase of asparagine labelling was associated with a decrease of label in glutamate.     

Amino Acid Cycling in Colonies of the Planktonic Marine Cyanobacterium Trichodesmium thiebautii

We examined diel trends in internal pools and net efflux of free amino acids in colonies of the nonheterocystous, diazotrophic cyanobacterium Trichodesmium thiebautii, freshly collected from waters of the Caribbean and the Bahamas. The kinetics of glutamate uptake by whole colonies were also examined. While intracolonial pools of most free amino acids were relatively constant through the day, pools of glutamate and glutamine varied over the diel cycle, with maxima during the early afternoon. This paralleled the daily cycle of nitrogenase activity. We also observed a large net release of these two amino acids from intact colonies. Glutamate release was typically 100 pmol of N colony^-1 h^-1. This is about one-fourth the concurrent rate of N₂ fixation during the day. However, while nitrogenase activity only occurs during the day, net release of glutamate and glutamine persisted into the night and may therefore account for a greater loss of recently fixed N on a daily basis. This release may be an important route of new N input into tropical, oligotrophic waters. Whole colonies also displayed saturation kinetics with respect to glutamate uptake. The K_s for whole colonies varied from 1.6 to 3.2 μM, or about 100-fold greater than typical ambient concentrations. Thus, uptake systems appear to be adapted to the higher concentrations of glutamate found within the intracellular spaces of the colonies. This suggests that glutamate may be a vehicle for N exchange among trichomes in the colony.     

Human adipose-derived stem cells enhance the glutamate uptake function of GLT1 in SOD1-bearing astrocytes

Impaired glutamate uptake function of astrocytes associated with accumulation of extracellular glutamate is a well-documented feature of amyotrophic lateral sclerosis (ALS). Enhancing the uptake function of astrocytic glutamate transport 1 (GLT1) may be a potential treatment for this disease. Human adipose-derived stem cells (hADSCs) are capable of secreting a large number of cytokines which exhibit diverse pharmacological effects. Therefore, we investigate the influence of the soluble factors released by hADSCs on the GLT1 in primary astrocytes cultured from SOD1^G93A mice, a widely studied mutant human SOD1 transgenic model of ALS. Our data indicate that soluble factors from hADSCs significantly upregulate the expression of GLT1 in SOD1^G93A-bearing astrocytes, which result in enhanced glutamate uptake function. The upregulation of GLT1 is accompanied by the inhibition of caspase-3 activation in mutant astrocytes. In addition, we find that hADSCs cocultured with SOD1^G93A-bearing astrocytes produce more VEGF, HGF and IGF-1, which are reported to have neuroprotective effects. Our results suggest that hADSCs may be a potential candidate in cellular therapy for ALS.     

METABOLIC PROPERTIES OF A PURIFIED PREPARATION OF LARGE FRAGMENTS OF THE CEREBELLAR GLOMERULI: GLUCOSE METABOLISM AND AMINO ACID UPTAKE

Glomerulus particle preparations contain large fragments of the cerebellar glomeruli and are composed almost exclusively of well-defined neuronal processes (Balázs et al., 1975). The metabolic competence of the glomerulus particles was demonstrated by their ability to convert [¹⁴C]glucose to ¹⁴CO₂ and lactate at a linear rate for over 1 h. The preparations also transported deoxyglucose via an high affinity uptake system (K_T= 0.2-0.5 mM). The kinetics of uptake of various labelled amino acids were also studied. Apparently high affinity uptake systems (K_T values about 10^-5 M) were found for thc putative transmitters GABA, glycine, glutamate, and aspartate, but not for leucine, serine, and tyrosine. The maximal velocity of high affinity uptake was the greatest for GABA (about 15 nmol/mg protein per 10 min), while glycine was taken up at about 50%, and aspartate and glutamate at only 13% of the rate obtained with GABA. High affinity uptake of glycine required Na⁺ (half maximal uptake at 70 mM-NaCl). Inhibition of glucose transport and glycolysis, electron transport, or oxidative phosphorylation also depressed high affinity uptake of glycine. 2,4-Diaminobutyric acid was a potent competitive inhibitor of GABA uptake (K₁ approx 22 μM), while β-alanine and glycine had a relatively minor inhibitory effect on the uptake of GABA.     

Control of glutamate oxidation in brain and liver mitochondrial systems

1. Glutamate oxidation in brain and liver mitochondrial systems proceeds mainly through transamination with oxaloacetate followed by oxidation of the α-oxoglutarate formed. Both in the presence and absence of dinitrophenol in liver mitochondria this pathway accounted for almost 80% of the uptake of glutamate. In brain preparations the transamination pathway accounted for about 90% of the glutamate uptake. 2. The oxidation of [1-¹⁴C]- and [5-¹⁴C]-glutamate in brain preparations is compatible with utilization through the tricarboxylic acid cycle, either after the formation of α-oxoglutarate or after decarboxylation to form γ-aminobutyrate. There is no indication of γ-decarboxylation of glutamate. 3. The high respiratory control ratio obtained with glutamate as substrate in brain mitochondrial preparations is due to the low respiration rate in the absence of ADP: this results from the low rate of formation of oxaloacetate under these conditions. When oxaloacetate is made available by the addition of malate or of NAD⁺, the respiration rate is increased to the level obtained with other substrates. 4. When the transamination pathway of glutamate oxidation was blocked with malonate, the uptake of glutamate was inhibited in the presence of ADP or ADP plus dinitrophenol by about 70 and 80% respectively in brain mitochondrial systems, whereas the inhibition was only about 50% in dinitrophenol-stimulated liver preparations. In unstimulated liver mitochondria in the presence of malonate there was a sixfold increase in the oxidation of glutamate by the glutamate-dehydrogenase pathway. Thus the operating activity of glutamate dehydrogenase is much less than the `free' (non-latent) activity. 5. The following explanation is put forward for the control of glutamate metabolism in liver and brain mitochondrial preparations. The oxidation of glutamate by either pathway yields α-oxoglutarate, which is further metabolized. Since aspartate aminotransferase is present in great excess compared with the respiration rate, the oxaloacetate formed is continuously removed by the transamination reaction. Thus α-oxoglutarate is formed independently of glutamate dehydrogenation, and the question is how the dehydrogenation of glutamate is influenced by the continuous formation of α-oxoglutarate. The results indicate that a competition takes place between the α-oxoglutarate-dehydrogenase complex and glutamate dehydrogenase, probably for NAD⁺, resulting in preferential oxidation of α-oxoglutarate.     

Effect of garlic oil on incorporation of amino acids into proteins of Culex pipiens quinquefasciatus Say larvae

Incorporation of [¹⁴C]leucine into proteins of 3rd instar Culex pipiens quinquefasciatus Say larvae increases linearly with time between 1 and 4 h. Garlic oil as well as the active larvacidal principle from it, viz. diallyl disulphide, inhibits significantly synthesis of the larval proteins. The maximum reduction in incorporation is observed during the first hour of treatment. The incorporation of [¹⁴C]phenylalanine is also inhibited by garlic oil and the effect is irreversible. Garlic oil does not seem to have any effect on proteins already labelled and it does not suppress substantially oxygen uptake by the larvae.     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. Effects of castration and androgen replacement

Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [¹⁴C]glycine and [¹⁴C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99–100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3α-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. `non-specific'. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.     

Primary metabolites of Phaseolus vulgaris nodules

More ethanol soluble material (carbohydrate and amino nitrogen) was found in both host cell and bacteroid components of Phaseolus vulgaris nodules from plants grown at 28 W/m² than from plants grown at 7 W/m². The range of compounds identified was similar at the two irradiances. On feeding ¹⁴CO₂ to the plant tops at either irradiance the labelling patterns of carbohydrates and organic acids in the nodule host cells and bacteroids suggested that any or all of the following substances could be donated by the host to the bacteroids for general metabolism: sucrose, fructose, glucose, an unidentified carbohydrate, malic acid and an organic acid co-chromatographing with 6-phosphogluconate. Distribution and labelling patterns of nodule amino compounds were consistent with the hypothesis that ammonia is the primary product of nitrogen fixation within bacteroids, and that this ammonia is transported to host cells for assimilation, initially into glutamine and glutamate.