GDH Activity Research Articles

Effects of spraying paclobutrazol at different stages on physiological characteristics, yield and quality of peanut

To explore the optimum stage of spraying with paclobutrazol (PBZ) for different peanut cultivars under high yield condition, we investigated the effects of spraying PBZ at different stages on chlorophyll content, root activity, protective enzymes, nitrogen and carbon metabolism enzymes of leaves, pod yield and kernel quality of peanut, with Huayu 20 and Huayu 25 as materials. The results showed that spraying PBZ at different stages increased root activity, chlorophyll content, SOD, POD, CAT, SS, SPS, PEPC activities and decreased the MDA content, NR, GS, GDH and GOGAT activities for pod setting stage of two cultivars. These results suggested that the effects would be more obvious at earlier spray time. For HY25, the most significant effects of spraying PBZ on those parameters of pod filling stage could be obtained when the main stem height was 25 cm. For HY20, spraying PBZ when the main stem height was 25 cm decreased the activities of protective enzymes. Earlier spraying time to HY20 would lead to early senescence, lower chlorophyll content, root activity and carbon metabolism enzyme activity. For HY20, the most significant effects of spraying PBZ on those parameters of pod filling stage could be obtained when the main stem height was 30 cm. Our results indicated that PBZ treatments at the optimum stage could improve pod yield and economic coefficient of both cultivars and enhance the fat content and the relative content of oleic acid and the O/L. Under high yield condition, the optimum stage of spraying PBZ was 25 cm height of the main stem for HY25 and 30 cm for HY20.

Blood and Tissue Enzymatic Activities of GDH and LDH, Index of Glutathione, and Oxidative Stress among Breast Cancer Patients Attending Referral Hospitals of Addis Ababa, Ethiopia: Hospital-Based Comparative Cross-Sectional Study.

The exact cause of breast cancer is unknown; it is a multifactorial disease. It is the most diagnosed and the second killer cancer among women. Breast cancer can be originated from tissues of breast or secondary from other organs via metastasis. Generally, cancer cells show aberrant metabolism and oxidative stress when compared to noncancerous tissues of breast cancer patients. The current study aims at evaluating glutamate and glucose metabolism through GDH and LDH enzyme activities, oxidant, and antioxidative status among breast cancer patients attending referral hospitals of Addis Ababa, Ethiopia. Result. Catalytic activities of glutamate dehydrogenase, lactate dehydrogenase, and oxidative stress index were significantly increased in both serum (4.2 mU/ml, 78.6 mU/ml, and 3.3 : 1, resp.) and cancerous tissues (1.4 mU/ml, 111.7 mU/ml, and 2.15 : 1, resp.) of breast cancer patients as compared to those in serum of control group (3.15 mU/ml, 30.4 mU/ml, and 2.05 : 1, resp.) and noncancerous tissues of breast cancer patients (0.92 mU/ml, 70.5 mU/ml, and 1.1 : 1, resp.) (P ≤ 0.05). Correspondingly, ratios of reduced to oxidized glutathione were significantly decreased in both serum (20 : 1) and cancerous tissues (23.5 : 1) of breast cancer patients when compared to those in serum of control group (104.5 : 1) and noncancerous tissues of breast cancer patients (70.9 : 1) (P ≤ 0.05). Conclusion. Catalytic activities of GDH and LDH, ratios of GSH to GSSG, and concentration of TOS among breast cancer patients were significantly higher than were those among control group and noncancerous tissues of breast cancer patients, while TAC of breast cancer patients is significantly lower than that of control group and normal tissues of breast cancer patients.

Mitochondrial disorders in multiple sclerosis

To determine the mitochondrial dysfunction in multiple sclerosis (MS). Fourteen adult patients with MS and 23 healthy people were examined. Cytochemical analysis of lymphocytes in peripheral blood was carried out. The activity of the mitochondrial enzymes involved in metabolism of carbohydrates (lactate dehydrogenase, LDH), amino acids (glutamate dehydrogenase, GDH), fatty acids (alpha-glycerophosphate, α-GPDH) and II complex of the mitochondrial respiratory chain (succinate dehydrogenase, LDH), and the level of lactate in the blood were measured. The activity of α-GPDH was reduced in 62.5% patients and increased in 37.5%. GDH activity in all patients was lowered. LDH activity was reduced in 71.4% patients and compensatory increased in 28.6%. In 66.7% patients, LDH activity was reduced and in 33.3% compensatory increased. Increased blood lactate was observed in 33.3% patients before meal and 44.4% after carbohydrate loading. After carbohydrate loading, in 33.3% patients lactate levels in the blood increased and in 11.1% increased above normal values. Therefore, the enzyme activity have decreased in most patients that suggests the decompensation of mitochondrial function. The results indicate the advisability of administering the energotrophic drugs carnicetin and coenzyme Q10 (idebenone) in patients with MS.

Effects of Salicylic Acid on Gaba Metabolism in Pepper Plants under Stress Conditions

Salicylic acid (SA) is a potential plant hormone that enhances the capacity of plants to cope with various stress factors. Gamma-aminobutyric acid (GABA) is an important non-protein amino acid, which is increased by stress conditions. However, the interaction between SA and GABA is not well known. In this response the aim of this study was to evaluate the effects of SA on GABA metabolism in pepper plants (Capsicum annuum L.) under individual and combined salt (NaCl) and polyethylene glycol (PEG) induced osmotic stresses. Total chlorophyll content, the activities of glutamate dehydrogenase (GDH, EC 1.4.1.4) and glutamate decarboxylase (GAD, EC 4.1.1.15), GABA content were analyzed in the leaves of pepper plants. Total chlorophyll content was decreased by combined salt and PEG-induced osmotic stresses. Individual SA, NaCl and PEG applications increased GDH activity. Under stress conditions, SA did not enhance GDH activity; however, GAD activity was increased within these conditions. Our results showed that salicylic acid application did not enhance GABA accumulation during individual and combined stress conditions. The dose and application process of SA could lead to this result. However further studies should be done to identify the interaction between SA and GABA under various stress conditions.

Effects of acclimation to high environmental temperatures on intermediary metabolism and osmoregulation in the sub-Antarctic notothenioid Eleginops maclovinus

Eleginops maclovinus is a sub-Antarctic fish endemic to the South American coast and is a monotypic species of the Eleginopidae family. A scarce amount of scientific information exists regarding the effects of temperature on E. maclovinus physiology. In this study, E. maclovinus specimens were acclimated for 2 weeks at 10 °C (control), 14, and 18 °C. Posterior assessments were performed for the intermediary metabolism of carbohydrates, lipids, and amino acids (in the plasma, liver, gills, and kidney) and on the osmoregulatory capacity [plasma osmolality and gill, kidney, and foregut Na+, K+-ATPase (NKA) activities]. In the plasma, only lactate and total amino acid levels were affected by the temperature. NKA activity presented a linear relationship with the increased temperatures. The liver exhibited no change in the carbohydrates metabolism, but the glycogen/glucose metabolite levels did differ. Amino acid metabolism showed a direct relationship between temperature and GDH and Asp-AT activities, and an inverse relationship between temperature and Ala-AT activity. Lipid metabolism was similar to Ala-AT. In the gills, carbohydrate metabolism (G6PDH and HK activities) presented a direct relationship with the increased temperatures. In the kidney, carbohydrate metabolism (only G6PDH activity) was greater at higher temperatures, and amino acid metabolism (GDH activity) showed a direct relationship with temperature. Lipid metabolism (G3PDH activity) was greatest at 14 °C, presenting significant differences compared to 10 °C. The obtained results suggest that E. maclovinus can acclimate to the projected thermal increase of climate change, with a reorganization of the intermediary metabolism components being tissue-dependent and osmoregulatory effects being affected by NKA activity and greater rates of nitrogenous waste extrusion.

Enhanced diastereoselective synthesis of t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate by using aldo-keto reductase and glucose dehydrogenase co-producing engineered Escherichia coli.

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo-keto reductase mutant KlAKR-Y295W/W296L (KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity (dep >99.5%) toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) co-producer E. coli BL 21(DE3) pETDuet-esgdh (MCS1)/pET-28b (+)-klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet-esgdh (MCS1)/pET-28b (+)-klakrm peaked at 249.9U/g DCW (dry cellular weight) and 29100U/g DCW, respectively. It completely converted (5R)-1 at substrate loading size of up to 60.0g/L (5R)-1 in the absence of exogenous NADH, which was one-fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3R,5R)-2 with productivity of 243.2kg/m3 d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co-expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235-1242, 2017.

Glutamate dehydrogenase is essential to sustain neuronal oxidative energy metabolism during stimulation

The enzyme glutamate dehydrogenase (GDH; Glud1) catalyzes the (reversible) oxidative deamination of glutamate to α-ketoglutarate accompanied by a reduction of NAD+ to NADH. GDH connects amino acid, carbohydrate, neurotransmitter and oxidative energy metabolism. Glutamine is a neurotransmitter precursor used by neurons to sustain the pool of glutamate, but glutamine is also vividly oxidized for support of energy metabolism. This study investigates the role of GDH in neuronal metabolism by employing the Cns-Glud1−/− mouse, lacking GDH in the brain (GDH KO) and metabolic mapping using 13C-labelled glutamine and glucose. We observed a severely reduced oxidative glutamine metabolism during glucose deprivation in synaptosomes and cultured neurons not expressing GDH. In contrast, in the presence of glucose, glutamine metabolism was not affected by the lack of GDH expression. Respiration fuelled by glutamate was significantly lower in brain mitochondria from GDH KO mice and synaptosomes were not able to increase their respiration upon an elevated energy demand. The role of GDH for metabolism of glutamine and the respiratory capacity underscore the importance of GDH for neurons particularly during an elevated energy demand, and it may reflect the large allosteric activation of GDH by ADP.

Characterization and mechanism of phosphoinositide 3-kinases (PI3Ks) members in insulin-induced changes of protein metabolism in yellow catfish Pelteobagrus fulvidraco

In the present study, seven phosphoinositide 3-kinase (PI3K) members (PI3KCa, PI3KCb, PI3KCd, PI3KCg, PI3KC2a, PI3KC2b and PI3KC3, respectively) were isolated and characterized from yellow catfish Pelteobagrus fulvidraco, and their roles in insulin-induced changes of protein metabolism were determined. These seven PI3Ks can be divided into three classes, class I (including PI3KCa, PI3KCb, PI3KCd and PI3KCg), class II (including PI3KC2a and PI3KC2b) and class III (only including PI3KC3). Compared with mammals, all of these members share similar domain structure. Their mRNAs were widely expressed across ten tested tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, intestine, heart, kidney and ovary), but at variable levels. In the in vivo study, insulin treatment significantly increased hepatic protein content at 3h, accompanied with reduced plasma total amino acid contents and liver ALT activity, and with increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in liver. At 6h and 12h, insulin injection showed no significant effect on liver protein content and plasma total amino acid, but reduced liver ALT activity and increased liver total RNA and the mRNA levels of AKT2, mTORC1 and S6K1 in liver at 6h. In the in vitro study, insulin incubation also tended to increase protein content of hepatocytes, accompanied with reduced cell medium total amino acid contents and hepatocytes ALT activity, and increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in hepatocytes. However, insulin treatment showed no significant effect on GDH activity and mRNA expression of PI3KCa, PI3KCd, PI3KCg, PI3KC2b, PI3KC3 and eEF2 both in the in vivo and in vitro studies. Effects of insulin on the mRNA levels of eIF-4E and 4E-BP1 were different between the in vivo and in vitro studies, and also time-dependent. Compared to single insulin group, insulin+wortmannin group increased ALT activity at 6h but reduced T-RNA content at 6 and 12h. AKT2 and S6K1 mRNA levels at 6 and 12h, mRNA levels of mTORC1, 4E-BP1 and eEF2 at 3 and 6h, and EIF-4E mRNA levels at 3 and 12h, PI3KCb and PI3KC2a mRNA levels were significantly lower in insulin+wortmannin group than those in single insulin group. Thus, our study demonstrated that among seven PI3K members, PI3KCb and PI3KC2a were more sensitive to the insulin signaling pathway, and insulin stimulated hepatic protein synthesis in yellow catfish through PI3K signaling pathway.

High Production of 2,3-butanediol by a Mutant Strain of the Newly Isolated Klebsiella pneumoniae SRP2 with Increased Tolerance Towards Glycerol.

Biodiesel, a renewable fuel produced by transesterification of animal fats and vegetable oils, generates about 10% (v/v) of crude glycerol as a core byproduct. The high volume of this non bio-degradable glycerol is becoming of a great environmental and economical concern due to its worldwide ever-growing surplus. Herein we report a high production of 2,3-butanediol (2,3-BD) from pure and biodiesel derived crude glycerol using a mutant K. pneumoniae SRM2 obtained from a newly isolated strain Klebsiella pneumoniae SRP2. The mutant strain SRM2 with standing high glycerol concentration (220 g L-1 of medium) could rapidly convert glycerol aerobically to 2,3-BD, a versatile product extensively used in chemical, pharmaceutical and fuel industries Our study revealed that an increased GDH activity led to a substantially enhanced production of 2,3-BD. The mutant strain exhibited 1.3-fold higher activity of GDH than that of parent strain (500.08 vs. 638.6 µmol min -1 mg -1 protein), yielding of 32.3 g L-1 and 77.5 g L-1 2,3-BD with glycerol in batch and fed-batch process respectively. However, in batch culture with crude glycerol, cell growth and glycerol consumption were expressively boosted, and 2,3-BD production was 27.7 g L-1 from 75.0 g/L crude glycerol. In this report, the optimal conditions for high production of 2,3-BD were defined in a completely aerobic process, and 0.59 g g-1 product yield of 2,3-BD was attained by the mutated strain K. pneumoniae SRM2, which is the highest amount obtained from batch biotransformation process of glycerol metabolism till today. These results indicated that our newly developed mutant can tolerate high concentration of glycerol, have a high glycerol utilization rate, and high product yield of 2,3-BD. It is demonstrated that the mutant strain K. pneumoniae SRM2 has an ability to produce fewer co-products at trace concentrations at higher glycerol concentrations, and could be a potential candidate for 2,3-DB production in an industrial bioconversion process.

Molecular cloning and functional analysis of the second gene encoding glutamate dehydrogenase in triticale

Glutamate dehydrogenase (GDH; EC 1.4.1.2) catalyzes the reductive amination of 2-oxoglutarate with ammonium and the oxidative deamination of glutamate. In some plants species, GDH is a hexamer and can be separated into seven isoenzymes that are composed of two distinct subunits: α and β. The large number of isoenzymes is the reason why GDH functions are still being intensively researched and widely studied. Until recently, the β subunit of GDH in triticale, a common Polish cereal, was thought to be encoded by the TsGDH1 gene, which can undergo posttranslational modifications and form a heterohexameric enzyme. Here, we report the cloning and molecular characterization of a second glutamate dehydrogenase gene—TsGDH2 (encoding α subunits). The TsGDH2 cDNA contains a 1236-bp open reading frame encoding a 411-amino-acid polypeptide with a calculated molecular mass of 44.5 kDa. To clarify the role of TsGDH2 in triticale, we used triticale GDH α-subunit cDNA to generate transgenic A. thaliana lines with increased and decreased GDH activity via alternation of α-subunit levels.

Structural Based Investigation of Artificial Electron Acceptor Interaction of Fungus Derived Flavin Adenine Dinucleotide Dependent Glucose Dehydrogenase

[Introduction]Self-monitoring of blood glucose (SMBG) is inevitable for the glycemic control of the diabetic patients, and plays an indispensable role in the current diabetes management. The current most popular principle for the sensor for SMBG is based on the so-called “second generation” type that are the use of the dye mediated dehydrogenase reactions. The principle employs the artificial electron acceptor and monitoring the concentration/quantity of thus reacted (reduced) artificial electron acceptor on the electrode. Fungi-derived flavin adenine dinucleotide dependent glucose dehydrogenases (FADGDHs) have recently been paid significant attention, focusing its narrow specificity, especially as the enzyme not active toward maltose. However, interaction between FADGDHs and artificial electron acceptor has not yet been investigated. Recently, we have reported on the first 3D structures of FADGDH, using Aspergillus flavus derived FADGDH (AfGDH). Here we report the investigation of interaction between mediator and enzyme by elucidating the 3D structure of AfGDH complex with potassium ferricyanide. Mutagenesis studies were also carried out to investigate the role of residues elucidated to interact with ferricyanide. [Methods]The structural gene of AfGDH was inserted into an expression vector and the enzyme was recombinantly expressed using E. coli BL21 (DE3). Soluble AfGDH was harvested and purified by hydrophobic interaction chromatography and anion exchange chromatography. Purified protein was concentrated, and crystallized by sitting-drop vapor-diffusion method. In order to obtain a ternary complex structure with FAD and ferricyanide, a single crystal was soaked in the potassium ferricyanide solution. X-ray diffraction data were collected and structure was determined. Based on the 3D structure, several mutants were constructed by site-directed mutagenesis and evaluated GDH activity, which was determined using either phenazine methosulphate/2,6-dichlorophenol indophenol (PMS/DCIP) or potassium ferricyanide as the electron acceptors. [Results & Discussion] A ternary complex structure of AfGDH with FAD and ferricyanide complex was obtained. Two potassium ferricyanide molecules were observed in the protein structure. One potassium ferricyanide interacted with one lysine residue, whereas the other molecules interacted with two lysine residues. In order to elucidate the role of these lysine residues, mutant AfGDHs were constructed by substituting lysine residues with alanine. The structural gene of AfGDH mutants were inserted into an expression vector and the enzyme was recombinantly prepared using E. coli BL21 (DE3). All mutants were expressed at soluble fraction. These mutants were subjected for anion exchange chromatography for purification. Interestingly, the mutant enzymes were eluted at different salt concentration than those eluted in the purification of wild type enzyme, suggesting that surface net charge was significantly changed by these mutations. Some of the mutant AfGDH showed unique property in the specificity toward electron acceptors. The GDH activities were compared by using either PMS/DCIP or ferricyanide as the electron acceptor, for both wild type and mutant enzymes. Although the alanine substitutions to these lysine residues did not significantly influenced PMS/DCIP based dye mediated GDH activity, but some mutation affected the ratio of dye mediated GDH activities of those using PMS/DCIP and of using ferricyanide. These results, together with the results of the change in the elution properties during anion exchange chromatography, suggested the lysine residues, we focused, play important role in the affinity toward ferricyanide during oxidative half reaction. (1) K. Mori, et al., Biotechnol Lett. 33(11) 2255-63(2011) (2) G. Sakai, et al., Biotechnol Lett. 37(5) 1091-99(2015) (3) H. Yoshida, et al. Sci. Rep. 5:13498. doi:10.1038/srep13498 (2015)

정신분열증 치료제에 의한 사람 글루탐산염 탈수소효소 동종효소의 억제효과

Glutamate is one of the major excitatory neurotransmitters in the central nervous system of vertebrates. Human GDH (hGDH) is the enzyme that regulates the glutamate metabolism and its expression is higher in the brains of schizophrenia patients than in normal subjects. This study examined the changes in the hGDH enzymatic activity caused by antipsychotic drugs (haloperidol, risperidone, (±)-sulpride, chlopromazine hydrochloride, melperone, (±)butaclamol, domperidone, clozapine) related to schizophrenia. First of all, hGDH isozymes (hGDH1, hGDH2) were synthesized by genetic recombination. As a result of the enzyme assay, haloperidol, (±)-sulpride, melperone and clozapine had an inhibitory effect on the hGDH isozymes. In addition, haloperidol showed a non-competitive inhibition against the substrate, 2-oxoglutarate. In contrast, it showed an uncompetitive inhibition against another substrate, NADH. The inhibitory effect of haloperidol on hGDH2 was abolished by the presence of L-leucine, an allosteric effector of hGDH, but by not other antipsychotic drugs. These results revealed the inhibition of enzyme activity by psychotropic drugs in hGDH isoenzymes (hGDH1 and hGDH2) and the possibility that haloperidol may be used to regulate the GDH activity and glutamate concentration in the central nervous system.

Modulation of brain glutamate dehydrogenase as a tool for controlling seizures.

Glutamate (Glu) is a major excitatory neurotransmitter involved in epilepsy. Glu is synthesized by glutamate dehydrogenase (GDH, E.C. 1.4.1.3) and dysfunction of the enzymatic activity of GDH is associated with brain pathologies. The main goal of this work is to establish the role of GDH in the effects of antiepileptic drugs (AEDs) such as valproate (VALP), diazepam (DIAZ) and diphenylhydantoin (DPH) and its repercussions on oxygen consumption. Oxidative deamination of Glu and reductive amination of αketoglutarate (αK) in mice brain were investigated. Our results show that AEDs decrease GDH activity and oxygen consumption in vitro. In ex vivo experiments, AEDs increased GDH activity but decreased oxygen consumption during Glu oxidative deamination. VALP and DPH reversed the increase in reductive amination of αK caused by the chemoconvulsant pentylenetetrazol. These results suggest that AEDs act by modulating brain GDH activity, which in turn decreased oxygen consumption. GDH represents an important regulation point of neuronal excitability, and modulation of its activity represents a potential target for metabolic treatment of epilepsy and for the development of new AEDs.

The impact of GABA in harpin-elicited biotic stress responses in Nicotiana tabaccum

Harpin is a bacterial elicitor protein that was first isolated from Erwinia amylovora. Infiltration of this elicitor into the leaves of plants activates systemic acquired resistance against a variety of plant pathogens via the salicyclic acid defense pathway. The non-protein amino acid, neurotransmission inhibitor molecule of mammals—GABA- is found in all organisms and is known to be an important component of stress responses in plants. We hypothesized a possible interaction between harpin-induced defense responses and GABA shunt. Therefore, we conducted experiments on harpin-infiltrated tobacco and analyzed the components of GABA shunt in relation to growth, photosynthesis and H2O2 levels. RGR, RWC and photosynthetic efficiency were all affected in harpin-infiltrated tobacco leaves, but the rate of decline was more remarkable on RGR. H2O2 levels showed significant difference on 7 days after harpin infiltration when the necrotic lesions were also visible. GABA accumulation was increased and glutamate levels were decreased parallel to the differences in GDH and GAD enzyme activities, especially on days 5 and 7 of harpin infiltration. Transcript abundance of GDH and GAD encoding genes were differentially regulated in harpin-infiltrated leaves as compared to that of control and mock groups. In the present study, for the first time we showed a relationship between harpin-elicited responses and GABA in tobacco that was not mediated by H2O2 accumulation. Harpin infiltration significantly induced the first components of the GABA shunt such as GDH, GAD, glutamate and GABA in tobacco.

The relationship between resistance to tobacco brown spot and nitrogen metabolism in mature tobacco (Nicotiana tabacum) plants

Differences in nitrogen metabolism were analyzed in mature leaves of three tobacco cultivars with different levels of resistance to brown spot disease (Alternaria alternata) including one resistant (JYH) and two susceptible cultivars (NC89 and CBH). The results were as follows: All three cultivars had low levels of both total nitrogen and leaf tissue NH4 +; the activity of glutamine synthetase (GS) declined sharply in all cultivars, but that of JYH was significantly higher than that of NC89 and CBH; levels of NAD(H)-glutamate dehydrogenase (NAD(H)-GDH) activity in JYH increased until 75 days after germination, and then decreased. In contrast, NAD(H)-GDH activities in NC89 and CBH increased gradually at maturity; apoplastic NH4 + concentrations in the three cultivars gradually increased, but their apoplastic pHs and NH3 compensation points decreased, and these apoplastic parameters were significantly higher in NC89 and CBH than in JYH. These results indicated that the nitrogen metabolism in mature leaves differed remarkably among tobacco cultivars with different levels of resistance to brown spot disease. The resistant cultivar has a greater capacity to re-use nitrogen, while the susceptible cultivar loses more nitrogen via ammonia volatilization, which plays an important role in the initiation of tobacco brown spot. Our data suggest that the characteristics of leaf nitrogen metabolism of tobacco cultivars affect the level of resistance to tobacco brown spot.

Nitrogen Source Differently Regulates Barley (Hordeum vulgare) Response to NaCl Stress At Seed Germination and Early Seedling Development Stages

Nitrogen (N) acts as nutrient and signaling molecule in plants all over their development stages. The involvement of various N forms in the regulation of seed germination response to salt stress was assessed in the present work. Nitrogen sources (NO, NO2−, NO3−, NH4+, glutamine and glutamate) were added at 1mMto the germination medium of barley (Hordeum vulgare, cv Ardhaoui) in combination or not with NaCl stress (14 g.L−1). The application of nitrogen monoxide (NO) alleviated by about 20% the NaCl-induced germination capacity decrease. However, the addition of ammonium ions (NH4+) and glutamic acid (Glu) accentuated the inhibitory effects of NaCl, decreasing germination capacity by about 50% compared to the control. The levels of malondialdehyde (MDA), which is an indicator of membrane lipid peroxidation by stresses, were increased by salinity in seeds treated with nitrite (NO2−), NO3−, Glu and Gln. In N-free medium, NaCl stress induced a severe nitrate reductase activity (NR, EC 1.6.1.6) inhibition. Such an effect was alleviated by the application of N treatments. Glutamate dehydrogenase (GDH, EC 1.4.1.2) aminating activity (NADH-GDH) of seedlings was inhibited by NaCl stress in the presence of NO, Glu and Gln. Conversely, there was stimulation by salt stress of NADH-GDH activity in seedlings treated with NaCl and NH4+. Deaminating GDH activity (NAD-GDH) was found to be enhanced by salt stress in NO2− and NO3− treatments. The differential effects of applied N forms on germination and early seedling development processes in this grass probably underlines different regulatory actions within N mobilization and assimilation.

Screening, morphological and molecular characterization of fungi producing cystathionine γ-lyase.

The potency for production of cystathionine γ-lyase (CGL) by the fungal isolates was screened. Among the tested twenty-two isolates, Aspergillus carneus was the potent CGL producer (6.29 U/mg), followed by A. ochraceous (6.03 U/mg), A. versicolor (2.51 U/mg), A. candidus (2.12 U/mg), A. niveus and Penicillium notatum (2.0 U/mg). The potent six isolates producing CGL was characterized morphologically, A. carneus KF723837 was further molecularly characterized based on the sequence of 18S-28S rDNA. Upon sulfur starvation, the yield of A. carneus extracellular CGL was increased by about 1.7- and 4.1-fold comparing to non-sulfur starved and L-methionine free medium, respectively. Also, the uptake of L-methionine was duplicated upon sulfur starvation, assuming the activation of specific transporters for L-methionine and efflux of CGL. Also, the intracellular thiols and GDH activity of A. carneus was strongly increased by S starvation, revealing the activation of in vivo metabolic antioxidant systems. Upon irradiation of A. carneus by 2.0 kGy of γ-rays, the activity of CGL was increased by two-fold, regarding to control, with an obvious decreases on its yield upon further doses. Practically, CGL activity from the solid A. carneus cultures, using rice bran as substrate, was increased by 1.2-fold, comparing to submerged cultures, under optimum conditions.

Isoenzyme pattern of glutamate dehydrogenase as a reflection of nitrogen metabolism in Lupinus albus

Glutamate dehydrogenase (L-glutamate: NAD dehydrogenase, EC 1.4. 1.2; GDH) activity and electrophoretic separation pattern of the enzyme were studied. The enzyme was extracted from embryos of <i>Lupinus albus</i> decotyledonized and cultured for 24, 48 and 72 hours in media containing various combinations of saccharose, ammonium chloride, nitrate as well as amino acids: glutamate, aspartate, glutamine and asparagine. The absence of sugar in the medium resulted in an increase of specific activity of GDH, measured by the rate of NADH + H<sup>+</sup> oxidation, and induced formation of new isoenzymes of NAD<sup>+</sup> - dependent GDH. Most significant increase in GDH specific activity and most evident appearance of new isoenzymes in the embryos were noted when sugar was substituted in the medium by any of the mentioned amino acids. Induction of new isoenzymes could also be seen when ammonium salts were pre-sent in the medium. GHD isoenzymatic patterns were obtained in various trophic conditions. It is suggested that the GDH isoenzyme patterns may serve as a nitrogen metabolism index of a tissue from which the enzyme has been extracted.

Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol.

The yeast Arxula adeninivorans was used for the overexpression of an ADH gene of Lactobacillus brevis coding for (R)-specific alcohol dehydrogenase (LbADH) to synthesise enantiomerically pure 1-(R)-phenylethanol. Glucose dehydrogenase gene from Bacillus megaterium (BmGDH) or glucose 6-phosphate dehydrogenase of Bacillus pumilus (BpG6PDH) were coexpressed in Arxula to regenerate the cofactor NADPH by oxidising glucose or glucose 6-phosphate. The yeast strain expressing LbADH and BpG6PDH produced 5200U l(-1) ADH and 370U l(-1) G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700U l(-1) ADH and 170U l(-1) GDH activity. However, the crude extract of both strains reduced 40mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99% in 60min without detectable by-products. An increase in yield was achieved using immobilised crude extracts (IEs), Triton X-100 permeabilised cells (PCs) and permeabilised immobilised cells (PICs) with PICs being most stable with GDH regeneration over 52 cycles. Even though the activity and synthesis rate of 1-(R)-phenylethanol with the BpG6PDH and LbADH coexpressing strain was higher, the BmGDH-LbADH strain was more stable over successive reaction cycles. This, combined with its higher total turnover number (TTN) of 391 mol product per mole NADP(+), makes it the preferred strain for continuous reaction systems. The initial non-optimised semi-continuous reaction produced 9.74gl(-1)day(-1) or 406gkg(-1) dry cell weight (dcw) day(-1) isolated 1-(R)-phenylethanol with an ee of 100% and a TTN of 206mol product per mole NADP(+). In conclusion, A. adeninivorans is a promising host for LbADH and BpG6PDH or BmGDH production and offers a simple method for the production of enantiomerically pure alcohols.

Over-expression of a glutamate dehydrogenase gene, MgGDH, from Magnaporthe grisea confers tolerance to dehydration stress in transgenic rice.

Heterologous expression of a fungal NADP(H)-GDH gene ( MgGDH ) from Magnaporthe grisea can improve dehydration stress tolerance in rice by preventing toxic accumulation of ammonium. Glutamate dehydrogenase (GDH; EC 1.4.1.2 and EC 1.4.1.4) may act as a stress-responsive enzyme in detoxification of high intracellular ammonia and production of glutamate for proline synthesis under stress conditions. In present study, a fungal NADP(H)-GDH gene (MgGDH) from Magnaporthe grisea was over-expressed in rice (Oryza sativa L. cv. 'kitaake'), and the transgenic plants showed the improvement of tolerance to dehydration stress. The kinetic analysis showed that His-TF-MgGDH preferentially utilizes ammonium to produce L-glutamate. Moreover, the affinity of His-TF-MgGDH for ammonium was dramatically higher than that of His-TF-OsGDH for ammonium. Over-expressing MgGDH transgenic rice plants showed lower water-loss rate and higher completely close stomata than the wild-type plants under dehydration stress conditions. In transgenic plants, the NADP(H)-GDH activities were markedly higher than those in wild-type plants and the amination activity was significantly higher than the deamination activity. Compared with wild-type plants, the transgenic plants accumulated much less NH4 (+) but higher amounts of glutamate, proline and soluble sugar under dehydration stress conditions. These results indicate that heterologous expression of MgGDH can prevent toxic accumulation of ammonium and in return improve dehydration stress tolerance in rice.