Uptake of glutamine and glutamate by the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120

Abstract Whole cells of the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120 exhibited K _m values for l -glutamine and l -glutamate of 33 μM and 0.5 mM, respectively. V _max of uptake was ca. 30 nmol mg⁻¹ (chlorophyll) min⁻¹ for both amino acids. The similar pattern of sensitivity to other amino acids exhibited by both transport activities suggests that a common transport system is involved in glutamine and glutamate uptake by this cyanobacterium.     

l-[3H]Nitroarginine and l-[3H]Arginine Uptake into Rat Cerebellar Synaptosomes: Kinetics and Pharmacology

Abstract: Characteristics of the transport of the nitric oxide synthase substrate l -arginine and its inhibitor, N ^G-nitro- l -arginine ( l -NOARG), into rat cerebellar synaptosomes were studied. Uptake of both l -arginine and l -NOARG was linear with increasing amount of protein (up to 40 µg) and time of incubation (up to 5 min) at 37°C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of l -NOARG (650 pmol/mg of protein) was three to four times higher than that of l -arginine (170 pmol/mg of protein). l -NOARG uptake showed biphasic kinetics ( K _{m 1} = 0.72 m M , V _{max 1} = 0.98 nmol/min/mg of protein; K _{m 2} = 2.57 m M , V _{max 2} = 16.25 nmol/min/mg of protein). l -Arginine uptake was monophasic with a K _m of 106 µ M and a V _max of 0.33 nmol/min/mg of protein. l -NOARG uptake was selectively inhibited by l -NOARG, N ^G-nitro- l -arginine methyl ester, and branched-chain and aromatic amino acids. l -Alanine and l -serine also inhibited l -NOARG uptake but with less potency. Uptake of l -arginine was selectively inhibited by N ^G-monomethyl- l -arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, l -NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas l -arginine is transported by the basic amino acid carrier system y⁺ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of l -NOARG and l -arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.     

Characterization of Neuronal Amino Acid Transporters: Uptake of Nitric Oxide Synthase Inhibitors and Implication for Their Biological Effects

Abstract: In the present study we investigated uptake of the nitric oxide (NO) synthase inhibitors N ^G-methyl- l -arginine and N ^G-nitro- l -arginine by the mouse neuroblastoma × rat glioma hybrid cell line NG108-15. Uptake of N ^G-methyl- l -arginine was characterized by biphasic kinetics ( K _m1 = 8 µmol/L, V _max1 = 0.09 nmol × mg⁻¹× min⁻¹; K _m2 = 229 µmol/L, V _max2 = 2.9 nmol × mg⁻¹× min⁻¹) and was inhibited by basic but not by neutral amino acids. Uptake of N ^G-nitro- l -arginine followed Michaelis-Menten kinetics ( K _m = 265 µmol/L, V _max = 12.8 ± 0.86 nmol × mg⁻¹× min⁻¹) and was selectively inhibited by aromatic and branched chain amino acids. Further characterization of the transport systems revealed that uptake of N ^G-methyl- l -arginine is mediated by system y⁺, whereas systems L and T account for the transport of N ^G-nitro- l -arginine. In agreement with these data on uptake of the inhibitors, l -lysine and l -ornithine antagonized the inhibitory effects of N ^G-methyl- l -arginine on bradykinin-induced intracellular cyclic GMP accumulation, whereas l -tryptophan, l -phenylalanine, and l -leucine interfered with the effects of N ^G-nitro- l -arginine. These data suggest that rates of uptake are limiting for the biological effects of NO synthase inhibitors.     

Inhibition, by 2-Oxo Acids That Accumulate in Maple-Syrup-Urine Disease, of Lactate, Pyruvate, and 3-Hydroxybutyrate Transport Across the Blood-Brain Barrier

Abstract: Data are presented in support of the transport of (-)- d -3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were V_max 2.0 μmol.g⁻¹.min⁻¹, K _m 29 m M , and K _D 0.024 ml.g⁻¹.min⁻¹. The value for V_max was the same as that for l -lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-0x0-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The K _m values for the 2-oxo acids were severalfold lower than the respective K _m values. 2-oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.     

N-System Amino Acid Transport at the Blood-CSF Barrier

Abstract: Despite l -glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in l -[¹⁴C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na⁺-dependent mechanism. The Na⁺-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na⁺-dependent uptake was inhibited neither by the A-system substrate α-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and l -glutamate γ-hydroxamate, three N-system substrates. Replacement of Na⁺ with Li⁺ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The V _max and K _max for glutamine transport by this system were 8.1 ± 0.3 nmol/mg/min and 3.3 ± 0.4 m M , respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.     

Characterization of ammonium and methylamine transport systems in phototrophic sulfur bacteria

Abstract Transport of ammonium and methylamine into the cells of green sulfur bacterium Chlorobium limicola and purple sulfur bacterium Thiocapsa roseopersicina is carried out by a common transport system. This system has (for C. limicola and T. roseopersicina , respectively) pH optimum 7.0 and 7.5; V _max 0.6 and 4.2 nmol min⁻¹ (mg protein)⁻¹; K_m 5.9 × 10⁻⁵ M and 1.3 × 10⁻⁵ M, and is capable of forming 120- and 600-fold methylamine gradients. The methylamine transport can be energized by the artificially imposed transmembrane K⁺ diffusive potential and is inhibited by tetraphenylphosphonium or valinomycin and K⁺. The data presented indicate that methylamine transport in both studied species is exclusively driven by the membrane potential gradient (ΔΨ).     

Neuropeptide Y stimulates retinal neural cell proliferation--involvement of nitric oxide

Neuropeptide Y (NPY) is a 36 amino acid peptide widely present in the CNS, including the retina. Previous studies have demonstrated that NPY promotes cell proliferation of rat post-natal hippocampal and olfactory epithelium precursor cells. The aim of this work was to investigate the role of NPY on cell proliferation of rat retinal neural cells. For this purpose, primary retinal cell cultures expressing NPY, and NPY Y₁, Y₂, Y₄ and Y₅ receptors [Álvaro et al. , (2007) Neurochem. Int., 50, 757] were used. NPY (10–1000 nM) stimulated cell proliferation through the activation of NPY Y₁, Y₂ and Y₅ receptors. NPY also increased the number of proliferating neuronal progenitor cells (BrdU⁺/nestin⁺ cells). The intracellular mechanisms coupled to NPY receptors activation that mediate the increase in cell proliferation were also investigated. The stimulatory effect of NPY on cell proliferation was reduced by l -nitroarginine-methyl-esther ( l -NAME; 500 μM), a nitric oxide synthase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ; 20 μM), a soluble guanylyl cyclase inhibitor or U0126 (1 μM), an inhibitor of the extracellular signal-regulated kinase 1/2 (ERK 1/2). In conclusion, NPY stimulates retinal neural cell proliferation, and this effect is mediated through nitric oxide–cyclic GMP and ERK 1/2 pathways.     

Cation-selectivity of the l-glutamate transporters of Escherichia coli, Bacillus stearothermophilus and Bacillus caldotenax: dependence on the environment in which the proteins are expressed

l -Glutamate transport by the H⁺-glutamate and Na⁺-glutamate symport proteins of Escherichia coli K-12 (GltP_Ec and GltS_Ec, respectively) and the Na⁺-H⁺-glutamate symport proteins of Bacillus stearothermophilus (GltT_Bs) and Bacillus caldotenax (GltT_Bc) was studied in membrane vesicles derived from cells in which the proteins were either homologously or heterologously expressed. Substrate and inhibitor specificity studies indicate that GltP_Ec, GltT_Bs and GltT_Bc fall into the same group of transporters, whereas GltS_Ec is distinctly different from the others. Also, the cation specificity of GltS_Ec is different; GltS_Ec transported l -glutamate with (at least) two Na⁺, whereas GltP_Ec, GltT_Bs and GltT_Bc catalysed an electrogenic symport of l -glutamate with ≥two H⁺, i.e. when the proteins were expressed in E. coli Surprisingly studies in membrane vesicles of B. stearothermophilus and B. caldotenax indicated a Na⁺-H⁺- l -glutamate symport for both GltT_Bs and GltT_Bc. The Na⁺ dependency of the GltT transporters in the Bacillus strains increased with temperature. These observations suggest that the conformation of the transport proteins in the E. coli and the Bacillus membranes differs, which influences the coupling ion selectivity.     

Uptake and metabolism of taurine in the green alga Chlorella fusca

Taurine entered the alga Chlorella fusca Shihira et Krauss strain 21l-8b via a pH and energy-dependent system ("permease"). Transport followed triphasic kinetics from 10⁻⁶ to 10⁻² M with K_m values for taurine of 5.4 × 10⁻⁵, 4.1 × l0⁻⁴ and l.5 × 10⁻³ M. This uptake system was specific for sulfonic acids and showed no affinity for α- and β -amino acids or Na⁺; thus the permease of C. fusca is different from all known taurine transport systems with respect to structural specificity and lack of Na⁺ -dependence. Uptake was not observed in sulfate-grown algae but developed as a response to sulfate limitation within 2 h. Sulfate addition caused a rapid decline in taurine transport capacity. Labeled taurine was rapidly metabolized in C. fusca to sulfate and ethanolamine, suggesting oxidative hydrolysis as the mechanism of C-S bond cleavage. Further incorporation of these catabolic products in C - and S -metabolism was demonstrated. Taurine catabolism was also detected in other green algae and some cyanobacteria.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Response of nitrogen metabolism to boron toxicity in tomato plants

Boron (B) toxicity has become important in areas close to the Mediterranean Sea where intensive agriculture has been developed. The objective of this research was to study the effects of B toxicity (0.5 m m and 2.0 m m B) on nitrogen (N) assimilation of two tomato cultivars that are often used in these areas. Leaf biomass, relative leaf growth rate (RGR_L), concentration of B, nitrate (NO₃⁻), ammonium (NH₄⁺), organic N, amino acids and soluble proteins, as well as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthase (GS), glutamate synthetase (GOGAT) and glutamate dehydrogenase (GDH) activities were analysed in leaves. Boron toxicity significantly decreased leaf biomass, RGR_L, organic N, soluble proteins, and NR and NiR activities. The lowest NO₃⁻ and NH₄⁺ concentration in leaves was recorded when plants were supplied with 2.0 m m B in the root medium. Total B, amino acids, activities of GS, GOGAT and GDH increased under B toxicity. Data from the present study prove that B toxicity causes inhibition of NO₃⁻ reduction and increases NH₄⁺ assimilation in tomato plants.     

Anion exchange in the giant erythrocytes of African lungfish

Carbon dioxide transport in African lungfish Protopterus aethiopicus blood conformed to the typical vertebrate scheme, implying a crucial and rate-limiting role of erythrocyte Cl^–/HCO₃^– exchange. The rate coefficient for unidirectional Cl^– efflux via the anion exchanger ( k , s⁻¹) increased with temperature in African lungfish, but values were well below those reported in other species. The erythrocytes of African lungfish were, however, very large (mean cellular volume = 6940 µm³), and the ratio of cell water volume to membrane surface area was high ( V _w A _m⁻¹ = 1·89). Hence, the apparent Cl^– permeability ( P _Cl =  kV _w A _m⁻¹, µm s⁻¹) was close to that in other vertebrates. The plot of ln P _Cl against the inverse absolute temperature was left-shifted in the tropical African lungfish compared to the temperate rainbow trout Oncorhynchus mykiss , which supports the idea that P _Cl is similar among animals when compared at their preferred temperatures. Also, Q ₁₀ for anion exchange calculated from P _Cl values in African lungfish was 2·0, supporting the idea that the temperature sensitivity of erythrocyte anion exchange matches the temperature sensitivity of CO₂ production and transport in ectothermic vertebrates.     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.     

Redox modulation of homomeric rho1 GABA receptors

The activity of many receptors and ion channels in the nervous system can be regulated by redox-dependent mechanisms. Native and recombinant GABA_A receptors are modulated by endogenous and pharmacological redox agents. However, the sensitivity of GABA_C receptors to redox modulation has not been demonstrated. We studied the actions of different reducing and oxidizing agents on human homomeric GABAρ₁ receptors expressed in Xenopus laevis oocytes. The reducing agents dithiothreitol (2 mM) and N -acetyl- l -cysteine (1 mM) potentiated GABA-evoked Cl⁻ currents recorded by two-electrode voltage-clamp, while the oxidants 5-5'-dithiobis-2-nitrobenzoic acid (500 μM) and oxidized dithiothreitol (2 mM) caused inhibition. The endogenous antioxidant glutathione (5 mM) also enhanced GABAρ₁ receptor-mediated currents while its oxidized form GSSG (3 mM) had inhibitory effects. All the effects were rapid and easily reversible. Redox modulation of GABAρ₁ receptors was strongly dependent on the GABA concentration; dose–response curves for GABA were shifted to the left in the presence of reducing agents, whereas oxidizing agents produced the opposite effect, without changes in the maximal response to GABA and in the Hill coefficient. Our results demonstrate that, similarly to GABA_A receptors and other members of the cys-loop receptor superfamily, GABA_C receptors are subjected to redox modulation.     

Kinetic characterization of sulphur-containing excitatory amino acid uptake in primary cultures of neurons and astrocytes

The uptake of the neuroactive sulphur amino acids -cysteine sulphinate, -cysteate, -homocysteine sulphinate and -homocysteate was investigated in astrocytes cultured from the prefrontal cortex; in neurons, cultured from cerebral cortex; and, in granule cells, cultured from cerebellum. It was shown that each amino acid acted as a substrate for a plasma membrane transporter in both neurons and astrocytes. Astrocytes and neurons exhibited a high-affinity uptake for -cysteine sulphinate and -cysteate with K_m values ranging from 14–100 μM, and a low-affinity uptake for -homocysteine sulphinate and -homocysteate, with K_m values ranging from 225–1210 μM. The uptake of all transmitter candidates studied was partially sodium-dependent. This sodium-dependency was most evident at low (< 100 μM) concentrations of each substrate. The apparent uptake measured in the absence of sodium was included as a component in corrections made for non-saturable influx. With the exception of -cysteine sulphinate, uptake of each sulphur amino acid was greatest in astrocytes, with V_max values ranging between 15–32 nmol min⁻¹ mg⁻¹ cell protein. Moreover, the uptake of each sulphur amino acid in cerebellar granule cells (V_max values ranging between 10–25 nmol min⁻¹ mg⁻¹ cell protein) was consistently greater than that in cerebral cortex neurons (V_max values ranging between 1.5–6 nmol min⁻¹ mg⁻¹ cell protein).     

Effect of gibberellic acid on K+ (Rb+) uptake and transport in sunflower roots

Four-week-old sunflower plants ( Helianthus annuus L. cv. Halcón), grown in different nutrient solutions, were used to study the effects of gibberellic acid (GA₃) on K⁺ (Rb⁺) uptake by roots or transport to the shoot. Gibberellic acid application to the nutrient solution did not affect the exudation process of excised roots. When GA₃ was sprayed on leaves 2 to 6 days before excising the roots, the rate of exudation and the K⁺ flux increased. When the exudation study was done keeping the roots in a nutrient solution in which Rb⁺ replaced K⁺, the GA₃ effects were evident also on Rb⁺ uptake and transport. In intact plants, GA₃ increased the Rb⁺ transported to the shoot but did not affect Rb⁺ accumulation in the root. It is suggested that these GA₃ effects can be explained if it is assumed that GA₃ acts on the transport of ions to the xylem vessels.     

Choline Uptake in Cultured Human Y79 Retinoblastoma Cells: Effect of Polyunsaturated Fatty Acid Compositional Modifications

Abstract: Choline uptake in Y79 human retinoblastoma cells occurs through two kinetically distinguishable processes. The high-affinity system shows little sodium or energy dependence, and it does not appear to be linked to acetyl CoA: choline O -acetyltransferase. When the cells are grown in a culture medium containing 10% fetal bovine serum, the high-affinity system has a K' _m= 2.16 ± 0.13 μ m and V' _max= 27.0 ± 2.9 pmol min⁻¹ mg⁻¹, whereas the low-affinity system has K' _m= 20.4 ± 1.3 μ m and V' _max= 402 ± 49 pmol min⁻¹ mg⁻¹. Under these conditions, the polyunsaturated fatty acid content of the cell membranes is relatively low. When the polyunsaturated fatty acid content of the microsomal membrane fraction was increased by supplementing the culture medium with linolenic or docosahexaenoic acids (n-3 polyunsaturated fatty acids) or arachidonic acid (n-6 polyunsaturated fatty acid), the K' ^m of the high-affinity choline transport system was reduced by 40–60%. The V' _max also was reduced by 20–40%. Supplementation with oleic acid, the most prevalent monounsaturated fatty acid, did not affect either kinetic parameter. The results suggest that one functional effect of the high unsaturated fatty acid content of neural cell membranes is to facilitate the capacity of the high-affinity choline uptake system to transport low concentrations of choline. This effect appears to be specific for polyunsaturated fatty acids but not for a single type, for it is produced by members of both the n-3 and n-6 classes of polyunsaturated fatty acids.     

Anaerobic sulfur oxidation in the absence of nitrate dominates microbial chemoautotrophy beneath the pelagic chemocline of the eastern Gotland Basin, Baltic Sea

Oxic–anoxic interfaces harbor significant numbers and activity of chemolithoautotrophic microorganisms, known to oxidize reduced sulfur or nitrogen species. However, measurements of in situ distribution of bulk carbon dioxide (CO₂) assimilation rates and active autotrophic microorganisms have challenged the common concept that aerobic and denitrifying sulfur oxidizers are the predominant autotrophs in pelagic oxic–anoxic interfaces. Here, we provide a comparative investigation of nutrient, sulfur, and manganese chemistry, microbial biomass distribution, as well as CO₂ fixation at the pelagic redoxcline of the eastern Gotland Basin, Baltic Sea. Opposing gradients of oxygen, nitrate, and sulfide approached the detection limits at the chemocline at 204 m water depth. No overlap of oxygen or nitrate with sulfide was observed, whereas particulate manganese was detected down to 220 m. More than 70% of the bulk dark CO₂ assimilation, totaling 9.3 mmol C m⁻² day⁻¹, was found in the absence of oxygen, nitrite, and nitrate and could not be stimulated by their addition. Maximum fixation rates of up to 1.1 μmol C L⁻¹ day⁻¹ were surprisingly susceptible to altered redox potential or sulfide concentration. These results suggest that novel redox-sensitive pathways of microbial sulfide oxidation could account for a significant fraction of chemolithoautotrophic growth beneath pelagic chemoclines. A mechanism of coupled activity of sulfur-oxidizing and sulfur-reducing microorganisms is proposed.     

SO2 and assimilatory sulfate reduction in beech leaves

The effect of SO₂ on the extractable activity of ATP sulfurylase (EC 2.7.7.4.). adenosine 5'-phosphosulfate sulfotransferase, ribulosebisphosphate carboxylase, chlorophyll, protein, sulfate, and amino acids was examined in leaves of potted grafts of beech ( Fagus sylvatica L.) treated in outdoor fumigation chambers. Addition of 0.025 and 0.075 μl SO₂ 1⁻¹ to unfiltered ambient air caused a decrease in the extractable activity of adenosine 5'-phosphosulfate sulfotransferase to about 20 to 30% of the controls. Neither the extractable activity of ATP sulfurylase and ribulosebisphosphate carboxylase nor the content in chlorophyll, total amino acids and protein were significantly affected by SO₂, but there was an increase in the sulfate content. Leaves treated with 0.075 μl SO₂ 1⁻¹ contained more alanine and cysteine and less serine than the controls. After transfer of the SO2-treated beech trees to control chambers there was an increase in adenosine 5'-phosphosulfate sulfotransferase activity, but no significant decrease in SO₂₋⁴-sulfur.