Glutamine and Glutamate Transport in Cyanophora paradoxa

The present investigation showed that isolated cyanelles from Cyanophora paradoxa selectively enriched glutamine from the external medium, whereas glutamate poorly penetrated into these organelles. Glutamine uptake proceeded in two phases, presumably involving a low and a high affinity system. The uptake of glutamine was significantly enhanced by 2-oxoglutarate and light. Inhibitor experiments indicated that glutamine and 2-oxoglutarate were converted to glutamate by a ferredoxin-dependent glutamate synthase (GOGAT) reaction inside the cyanelles, and the glutamate formed at best slowly left these organelles. Such results were obtained independently of each other by measuring either the ¹⁴C-glutamine uptake or the 2-oxoglutarate and glutamine-dependent O₂ evolution. Glutamine is suggested to be the N-compound which is supplied to the eukaryotic host. Glutamine could be exported jointly with 2-oxoglutarate, possibly employing a common carrier. Cyanelles have apparently evolved glutamine (and oxoglutarate) carrier(s) with properties not yet described for any other organism.     

Inhibition of Glutamate Transport in Synechococcus cedrorum by Glutamine

The absence of glutamate transport in glutamine-grown cells of Synechococcus cedrorum R7 was due to feedback inhibition and not repression of transport activity.     

草坪草离蠕孢叶枯病菌生物学特性的研究

对草坪离蠕孢叶枯病病原菌进行分离鉴定,并对其生物学特性进行了研究。结果表明:该病害由禾草离蠕孢(Bipolaris sorokiniana)引起。该病原菌的菌丝生长及产孢的最适温度为30℃,孢子萌发最适温度为25℃,菌丝的致死温度为65℃,而孢子的致死温度则为55℃;该菌对酸碱度的适应能力较强,中性偏酸性的条件对菌丝的生长有利,而pH值为8.0时最易产孢;各碳源对菌丝的生长均有促进作用,但不同碳源对产孢量的影响很大,单糖和双糖利于产孢,多糖对产孢的影响不大。氮源对菌丝生长和产孢量非常重要,无机氮效果较好,硝态氮好于氨态氮,有机氮效果最差。花粉、叶面物质和草坪草汁液可促进孢子萌发。     

Sugar transport in the light leaf spot pathogen Pyrenopeziza brassicae

Abstract In the light leaf spot fungus Pyrenopeziza brassicae , the kinetics of uptake of sucrose, glucose and fructose are all biphasic. At low and high concentrations, glucose and fructose share a high-affinity and a low-affinity hexose carrier respectively, with K _m s of 3.5 and 4.6 μM for uptake of glucose and fructose respectively by the high-affinity system, and K _s s of 690 and 750 uM for uptake of glucose and fructose by the low-affinity system. The data also suggest the existence of separate high-affinity and low-affinity uptake systems for sucrose. P. brassicae possesses both soluble and paniculate invertase activity, with pH optima of 5.0 and 4.0 respectively. Activity of the particulate invertase is considerably in excess of the highest rates of sucrose uptake.     

Differential Effects of Nitrate and Light on the Expression of Glutamine Synthetases and Ferredoxin-Dependent Glutamate Synthase in Maize

The effects of nitrate and light on the expression of genesfor glutamine synthetase (GS) isoproteins and ferredoxin-dependentglutamate synthase (Fd-GOGAT) were studied in different organsof maize seedlings by analyzing the levels of the respectivepolypeptides and mRNAs. In roots, the levels of plastidic GSand of a novel, root-specific GS molecule localized in the extraplastidiccompartment were increased markedly by nitrate, whereas Fd-GOGATand cytosolic GS remained at their initial levels. Ammonia wasnot effective in inducing the plastidic GS and Fd-GOGAT butit did induce the novel GS isoprotein. In leaves, cytosolicand plastidic GSs and Fd-GOGAT were present in both mesophyllcells (MC) and bundle sheath cells (BSC). Upon addition of nitrate,the level of plastidic GS increased preferentially in MC, andupon exposure of etiolated seedlings to light, the levels ofplastidic GS and Fd-GOGAT increased in BSC in a coordinatedmanner. The relationship between the expression of genes forGSs and Fd-GOGAT and the physiological role of the GS/GOGATcycle is discussed in terms of the characteristics of nitrogenmetabolism in roots, MC, and BSC. (Received August 11, 1992; Accepted September 21, 1992)     

Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Tissue Distribution of Glutamate Synthase and Glutamine Synthetase in Rice Leaves : Occurrence of NADH-Dependent Glutamate Synthase Protein and Activity in the Unexpanded, Nongreen Leaf Blades

To further explore the function of NADH-dependent glutamate synthase (GOGAT), the tissue distribution of NADH-GOGAT protein and activity was investigated in rice (Oryza sativa L.) leaves. The distributions of ferredoxin (Fd)-dependent GOGAT, plastidic glutamine synthetase, and cytosolic glutamine synthetase proteins were also determined in the same tissues. High levels of NADH-GOGAT protein (33.1 μg protein/g fresh weight) and activity were detected in the 10th leaf blade before emergence. The unexpanded, nongreen portion of the 9th leaf blade contained more than 50% of the NADH-GOGAT protein and activity per gram fresh weight when compared with the 10th leaf. The expanding, green portion of the 9th leaf blade outside of the sheath contained a slightly lower abundance of NADH-GOGAT protein than the nongreen portion of the 9th blade on a fresh weight basis. The fully expanded leaf blades at positions lower than the 9th leaf had decreased NADH-GOGAT levels as a function of increasing age, and the oldest, 5th blade contained only 4% of the NADH-GOGAT protein compared with the youngest 10th leaf blade. Fd-GOGAT protein, on the other hand, was the major form of GOGAT in the green tissues, and the highest amount of Fd-GOGAT protein (111 μg protein/g fresh weight) was detected in the 7th leaf blade. In the nongreen 10th leaf blade, the content of Fd-GOGAT protein was approximately 7% of that found in the 7th leaf blade. In addition, the content of NADH-GOGAT protein in the 10th leaf blade was about 4 times higher than that of Fd-GOGAT protein. The content of plastidic glutamine synthetase polypeptide was also the highest in the 7th leaf blade (429 μg/g fresh weight) and lowest in nongreen blades and sheaths. On the other hand, the relative abundance of the cytosolic glutamine synthetase polypeptide was the highest in the oldest leaf blade, decreasing to 10 to 20% of that value in young, nongreen leaves. These results suggest that NADH-GOGAT is important for the synthesis of glutamate from the glutamine that is transported from senescing source tissues through the phloem in the nongreen sink tissues in rice leaves.     

A Detached Cotyledon Test for the Isolation of Mutants of Pyrenopeziza brassicae Defective in Pathogenicity Determinants

A detached cotyledon pathogenicity test was devised for the isolation of UV-induced mutants of the hemibiotrophie ascomycete pathogen of Brassica spp., Pyrenopeziza brassicae defective in pathogenicity determinants. At least 95 % of cotyledons of suitable susceptible cultivars of oilseed rape (Brassica napus L. ssp. oleifera cvs Shogun or Bolko) showed symptoms of disease (white spore pustules [conidiomata] on the surface of the cotyledon) within three weeks of inoculation of cotyledons with 10⁴ conidia of P. brassicae. The test allowed rapid screening of UV-induced mutants with a low frequency of false negatives. From 1,700 survivors of UV mutagenesis tested, 20 non-pathogenic mutants were obtained.     

Sulphur supply and infection with Pyrenopeziza brassicae influence L-cysteine desulphydrase activity in Brassica napus L

Different field surveys have shown that sulphur (S) fertilization can increase the resistance of agricultural crops against fungal pathogens. The mechanisms of this sulphur-induced resistance (SIR) are, however, not yet known. Volatile S compounds are thought to play an important role because H(2)S is toxic to fungi. A field experiment was conducted to analyse the influence of S fertilization and the activity of H(2)S-releasing enzymes on fungal infections. Two levels of N and S fertilizers and two varieties of oilseed rape were investigated with respect to their potential to release H(2)S by the enzymatic activity of L-cysteine desulphydrase (LCD) and O-acetyl-L-serine(thiol)lyase (OAS-TL). LCD releases H(2)S during cysteine degradation, while OAS-TL consumes H(2)S during cysteine synthesis and free H(2)S is only released in a side reaction. All plots of the field trial showed an infection with Pyrenopeziza brassicae and leaf disc samples were taken from visibly infected leaf areas and apparently uninfected areas to investigate the reaction to the infection in relation to the treatments. Different S fractions and the activities of LCD and OAS-TL were measured to evaluate the potential to release H(2)S in relation to S nutrition and fungal infection. S fertilization significantly increased the contents of total S, sulphate, organic S, cysteine, and glutathione in the plants, but decreased LCD activity. Infection with P. brassicae increased cysteine and glutathione contents, as well as the activity of LCD. Therefore crops were able to react to a fungal infection with a greater potential to release H(2)S, which is reflected by an increasing LCD activity with fungal infection.     

Effects of Light and Inhibitors on Glutamate Metabolism in Leaf Discs of Vicia faba L: Sources of ATP for Glutamine Synthesis and Photoregulation of Tricarboxylic Acid Cycle Metabolism

Metabolism of [¹⁴C]glutamate was studied in leaf discs of Vicia faba L. in light and in darkness. In white light glutamine was the main labeled product. In the dark label was principally in compounds closely associated with tricarboxylic acid cycle metabolism, predominantly aspartate. Entry of label from glutamate into tricarboxylic acid metabolism appeared to be at least partially by decarboxylation of glutamate to γ-amino butyric acid, followed by conversion to succinate. 3-(3,4-dichlorophenyl)-1, 1-Dimethylurea inhibited light-enhanced synthesis of glutamine and caused reversion toward the dark pattern of metabolism. Methionine sulfoximine severely inhibited glutamine synthesis and caused accumulation of labeled malate.     

Genetic Analysis of Glutamate Transport and Glutamate Decarboxylase in Escherichia coli

The location of the Escherichia coli K-12 genes determining or regulating glutamate transport, and the location of the gene determining glutamate decarboxylase synthesis, were established by conjugation. The ability to grow on glutamate as the sole source of carbon and energy was used to select for glutamate transport recombinants. Two genes determining the ability to grow on glutamate as the sole source of carbon and energy were mapped. One (gltC) is located near mtl (mannitol), and the other (gltH) appears to be located between the gal (galactose) and trp (tryptophan) loci. The glutamate decarboxylase gene (gad) is strongly linked to gltC. The gltC(+) recombinants grow on glutamate much faster and accumulate this amino acid to a greater extent than do the gltH(+) recombinants. The gltH(+) gene functioned only in one female strain (P678), whereas the gltC gene functioned in all the female strains tested (P678, C600, W1).     

A Two-Translocator Model for the Transport of 2-Oxoglutarate and Glutamate in Chloroplasts during Ammonia Assimilation in the Light

This study examines the transport of 2-oxoglutarate (2-OG) and other dicarboxylates during ammonia assimilation in illuminated spinach chloroplasts. The transport of all dicarboxylates examined was strongly inhibited by NH₄Cl preincubation in the light. Treatment with NH₄Cl caused a rapid depletion of the endogenous glutamate pool and a corresponding increase in endogenous glutamine content. The inhibition of transport activity by NH₄Cl was apparently linked to its metabolism in the light because inhibition of glutamine synthetase activity by the addition of l-methionine sulfoximine or carbonylcyanide-m-chlorophenylhydrazone abolished this affect. Measurements of endogenous metabolite pools showed that malate was most rapidly exchanged during the uptake of all exogenous dicarboxylates examined. Depending on the exogenous substrates used, the apparent half-times of efflux measured for endogenous malate, aspartate and glutamate were 10, 10 to 30, and 15 to 240 seconds, respectively. The transport of 2-OG was also inhibited by malate. But chloroplasts preincubated with malate in the presence or absence of NH₄Cl were found to have high transport activity similar to untreated chloroplasts. A two-translocator model is proposed to explain the stimulation of 2-OG transport as well as the stimulation of (NH₃, 2-OG)-dependent O₂ evolution by malate (KC Woo, CB Osmond 1982 Plant Physiol 69: 591-596) in isolated chloroplasts. In this model the transport of 2-OG on the 2-OG translocator and glutamate on the dicarboxylate translocator is coupled to malate counter-exchange in a cascade-like manner. This results in a net 2-OG/glutamate exchange with no net malate transport. Thus, during NH₃ assimilation the transport of 2-OG into and the export of glutamate out of the chloroplast occurs via the 2-OG and the dicarboxylate translocators, respectively.     

Glutamine Transport in Mouse Cerebral Astrocytes

Abstract: We measured initial influx and exchange of [¹⁴C]glutamine in primary astrocyte cultures in the presence and absence of Na⁺. Kinetic analysis of transport in Na⁺-free solution indicated two saturable Na⁺-independent components, one of which was identifiable functionally as system L₁ transport. In the presence of Na⁺, multiple hyperbolic components were not resolvable from the kinetic data. Nevertheless, other evidence supported participation by at least three Na⁺-dependent neutral amino acid transporters (systems A, ASC, and N). System A transport of glutamine was usually absent or minimal, based on lack of inhibition by α-(methylamino)isobutyric acid. However, vigorous system A-mediated transport emerged after derepression by substrate deprivation. Participation by system ASC was indicated by trans-acceleration of Na⁺-dependent uptake, preferential inhibition of an Li⁺-intolerant component of uptake by cysteine, and inhibition by cysteine of a component resistant to inhibition by histidine and α-(methylamino)isobutyric acid. Because nonsaturable transport of glutamine appeared negligible, and system L transport of glutamine was suppressed in the presence of Na⁺, low-affinity system ASC transport may be the major route of export of glutamine from astrocytes. At 700 µ M glutamine, the primary uptake route was system N transport, identified on the basis of selective inhibition by histidine and asparagine, pH sensitivity, and tolerance of Li⁺ in place of Na⁺.     

Phosphate-Activated Glutaminase and Mitochondrial Glutamine Transport in the Brain

A review of the properties of purified and tissue bound phosphate activated glutaminase (PAG) in brain and kidney (pig and rat) is presented, based on kinetic, electron microscopic and immunocytochemical studies. PAG is a mitochondrial enzyme and two pools can be separated, a soluble and membrane associated one. Intact mitochondria appear to express PAG accessible only to the outer phase of the inner mitochondrial membrane. This PAG has properties similar to that of the membrane fraction and polymeric form of purified enzyme. PAG in the soluble fraction has properties similar to that of the monomeric form of purified enzyme and is assumed to be dormant due to the high matrix concentration of the inhibitor glutamate. A hypothetical model for the localization of PAG in the mitochondria is presented. The activity of PAG in vivo is assumed to be regulated by cytosolic glutamate and other compounds, that affect the activation by phosphate. Glutamine is transported into brain and kidney mitochondria by a protein catalyzed energy requiring process, which may be mediated by more than one protein. There is no correlation between glutamine hydrolysis and transport.     

Comparative studies of light leafspot (Pyrenopeziza brassicae) epidemics on the growth and yield of winter oilseed rape

Comparisons of epidemics of light leafspot of differing duration and time of initiation were made in two experiments using a single cultivar of Brassica napus. Fungicide was applied before introduction of disease to prevent infection or some time after inoculation to stop further disease development. In the first experiment, substantial reductions in green leaf area and total plant dry-matter were found at flowering when disease was introduced in the autumn or in January. Plant dry weight at maturity was also greatly reduced in these treatments. The detrimental effect of an epidemic initiated in the autumn was avoided to a large extent if fungicide application began in February. Epidemics initiated in March had only small effects on final dry-matter yield. Seed yield was negatively correlated with the length of the epidemic. In a second experiment, early epidemics initiated in the autumn were halted after different time intervals. Commencing fungicide application even as early as December failed to prevent some loss of dry weight at flowering. At maturity, however, dry weight and seed yield were reduced significantly when fungicide application was delayed until February. Failure to control the disease resulted in a 46% loss of seed yield.     

Identification of the New Pathogen (Stemphylium lycopersici) Causing Leaf Spot on Pepino (Solanum muricatum)

Pepino (Solanum muricatum var. pepino) plants were found affected by an extensive leaf spot caused by plant pathogenic fungi during a survey in the Cameron highlands, Pahang state, Malaysia. Symptomatic leaf samples were collected from infected pepino plants and cultivated on PDA medium, and the pathogen was isolated and purified; then, consequently, all isolates were identified as Stemphylium lycopersici on the basis of their cultural and morphological characteristics and combined sequences of the internal transcribed spacer (ITS) and glyceraldehyde‐3‐phosphate dehydrogenase (gpd) regions. A pathogenicity assay on detached leaves further confirmed that S. lycopersici causes leaf spot disease. To the best of our knowledge, this is the first report of S. lycopersici causing leaf spot on pepino in Malaysia and worldwide.     

Diagnosis of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in the UK

Light leaf spot lesions were generally first observed as light green areas on leaves of UK winter oilseed rape crops in January or February and later became brittle and bleached. Elongated lesions, which were brown with indistinct edges, developed on stems in the spring and summer, when lesions were also observed on flower buds, pedicels and pods. Development of diagnostic white pustules (spore masses of Pyrenopeziza brassicae, which erupt through surfaces of infected tissues) for confirmation of light leaf spot infection on symptomless plants or plants with indistinct or ambiguous symptoms in the autumn, winter or spring was enhanced by incubating plants in polyethylene bags. In experiments with artificially inoculated plants, glasshouse-grown plants exposed in infected crops and plants sampled from crops, white pustules developed at all incubation temperatures from 2^oC to 20^oC on infected leaves of different cultivars. The period of incubation required before the appearance of pustules decreased as the time that had already elapsed since the initial infection increased. The longest periods of incubation were required at the lowest temperatures (2^oC or 5^oC) but leaves senesced and abscised from plants most quickly at the highest temperatures (15^oC or 20^oC), suggesting that the optimal incubation temperature was between 10^oC and 15^oC.     

Alteration of Striatal Glutamate Release After Glutamine Synthetase Inhibition

The effect of the glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), on glutamate levels in, and glutamate release from, rat striatal tissue was examined. Tissue levels of glutamate were unchanged 24 h after an intraventricular injection of MSO, but tissue glutamine levels were decreased 50%. Calcium-dependent, potassium-stimulated glutamate release was diminished in tissue prisms from animals pretreated with MSO compared to controls. The decreased release of glutamate correlated over time with the inhibition of GS following an intraventricular injection of MSO. The maximum diminution of calcium-dependent, potassium-stimulated glutamate release (50%) and the maximum inhibition of GS activity (51%) were observed 24 h after MSO. The addition of 0.5 mM glutamine to the perfusion medium completely reversed the effects of MSO pretreatment on calcium-dependent, potassium-stimulated glutamate release. Since GS is localized in glial cells and the measured glutamate release is presumed to occur from neurons, the data support the contention that astroglial glutamine synthesis is an important contributor to normal neuronal neurotransmitter release.     

海南菠萝一种叶斑病病原菌的分离与鉴定及多基因序列分析

为明确菠萝叶斑病病原菌可可毛色二孢菌在海南省各市县的亲缘关系及遗传差异,从海南省的海口、澄迈、儋州、三亚、保亭等16个市县进行病样采集和病样分离,根据科赫氏法则鉴定,获得42株病原菌.观察形态特征,并基于多基因联合序列分析其遗传多样性.结果 表明,通过形态学鉴定和ITS与TUB2基因序列联合进化树分析发现,其中来自海口、澄迈、儋州、三亚、保亭等16个市县的16株代表病原菌均鉴定为可可毛色二孢菌(Lasiodiplodia theobromae),其分生孢子平均大小为(22.06～31.07) μm× (11.77～16.48) μm;通过ITS、TUB2、EF-1α、GAPDH、CHS-1和ACT基因拼接序列聚类分析,结果分为3个类群,海南岛的中部(儋州,昌江,白沙,五指山,万宁,琼海等地)聚为一个类群,中北部(屯昌,临高)聚为一个类群,西南部(东方,乐东,三亚等地)聚为一个类群.该结果说明来源于不同产地不同菠萝品种上的可可毛色二孢菌遗传多样性丰富,在海南省16个市县菠萝叶斑病菌中可可毛色二孢菌L.theobromae为优势种.     

The Role of Glutamine Synthetase and Glutamate Dehydrogenase in Cerebral Ammonia Homeostasis

In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [¹³N]ammonia have shown that about 25?% of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of α-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [¹³N]ammonia in rat brain is incorporated into glutamate and the α-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/α-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/α-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: l-Aspartate?+?GTP?+?H₂O?→?Fumarate?+?GDP?+?P_i?+?NH₃) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research.