Sennoside A(SA),a typical prodrug,exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases.Hydrolases have been identified,but reductases remain ...Sennoside A(SA),a typical prodrug,exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases.Hydrolases have been identified,but reductases remain unknown.By linking a photoreactive group to the SA scaffold,we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling(ABPP).From lysates of an active strain,Bifidobacterium pseudocatenulatum(B.pseudocatenulatum),397 proteins were enriched and subsequently identified using mass spectrometry(MS).Among these proteins,chromate reductase/nicotinamide adenine dinucleotide(NADH)phosphate(NADPH)-dependent flavin mononucleotide(FMN)reductase/oxygen-insensitive NADPH nitroreductase(nfrA)was identified as a potent SA reductase through further bioinformatic analysis and The Universal Protein Resource(UniProt)database screening.We also determined that recombinant nfrA could reduce SA.Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.展开更多
This letter critically comments on the article by Zheng et al investigating the role of aucubin in alleviating diabetic neuropathic pain(DNP).DNP arises from hyperglycaemia-induced nerve injury and microglial reprogra...This letter critically comments on the article by Zheng et al investigating the role of aucubin in alleviating diabetic neuropathic pain(DNP).DNP arises from hyperglycaemia-induced nerve injury and microglial reprogramming toward aerobic glycolysis.Aldose reductase(also known as AKR1B1)redirects excess glucose flux through the polyol pathway,thus increasing oxidative stress and inflammation.Zheng et al show that aucubin,a plant iridoid glycoside,reverses streptozotocin-induced mechanical and thermal hypersensitivity and anxiety-like behaviour in mice.Mechanistically,aucubin restores microglial morphology,reduces glycolytic flux,enhances oxidative phosphorylation and lowers tumour necrosis factor-α,interleukin(IL)-1βand IL-6 levels in spinal tissue and cultures of the BV-2 microglial cell line.Network pharmacology and molecular docking analyses identify AKR1B1 as a key target,confirmed by the fact that short hairpin RNA knockdown of AKR1B1 eliminates the effects of aucubin.Contrary to the other studies,this study uniquely implicates the polyol pathway in microglial immunometabolism.展开更多
BACKGROUND Treatment of diabetic neuropathy is often limited by side effects.Aucubin,an iridoid glycoside derived from natural plants,exhibits notable anti-inflammatory and antioxidant properties.AIM To investigate th...BACKGROUND Treatment of diabetic neuropathy is often limited by side effects.Aucubin,an iridoid glycoside derived from natural plants,exhibits notable anti-inflammatory and antioxidant properties.AIM To investigate the effects of aucubin on diabetic neuropathic pain(DNP)and glycolysis and inflammation in microglia.METHODS Streptozotocin(STZ)was used to establish a DNP animal model.Blood glucose levels and body weight of mice were measured following STZ administration.Paw withdrawal threshold was calculated for mechanical allodynia.Paw withdrawal latency was recorded for thermal hyperalgesia.The open field test and elevated plus maze was used to assess locomotor activity and anxiety-like behavior.Western blotting was utilized for analysis of protein expression.Immunofluorescence staining was measured for morphometric analysis of microglia.Glycolysis and ATP synthesis in BV-2 cell lines were detected by metabolic extracellular flux analysis.The SwissTargetPrediction and STRING databases were used for comprehensive screening to identify potential target proteins for aucubin.The molecular docking between the possible target proteins and aucubin was investigated using Auto Dock Tool.The BV-2 cell line was transfected with lentiviral AKR1B1-shRNA to further ascertain the function of AKR1B1 in the impact of aucubin on aerobic glycolysis and inflammation during high glucose stimulation.RESULTS Aucubin significantly improved pain and anxiety-like behavior in STZ-induced diabetic mice and restored microglial aerobic glycolysis and inflammation.Several public databases and molecular docking studies suggested that AKR1B1,MMP2 and MMP9 are involved in the effect of aucubin on DNP.Aucubin failed to restore aerobic glycolysis and inflammation in the context of AKR1B1 deficiency.CONCLUSION Aucubin has potential as a therapeutic agent for alleviating DNP by inhibiting expression of AKR1B1.展开更多
Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals,but their asymmetric synthesis remains challenging especially for those with multiple chiral centers.In ...Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals,but their asymmetric synthesis remains challenging especially for those with multiple chiral centers.In this study,IR104 from Streptomyces aureocirculatus was identified from 157 wild-type imine reductases for the synthesis of(S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide(antiepileptic drug Brivaracetam)via dynamic kinetic resolution reductive amination from ethyl 3-formylhexanoate and(S)-2-aminobutylamide with high diastereoselectivity.To further improve the catalytic efficiency of IR104,its mutant D191E/L195I/E253S/M258A(M3)was identified by saturation mutagenesis and iterative combinatorial mutagenesis,which exhibited a 102-fold increase in the catalytic efficiency relative to that of wild-type enzyme and high diastereoselectivity(98:2 d.r.).Crystal structural analysis and molecular dynamics simulations provided some insights into the molecular basis for the improved activity of the mutant enzyme.The imine reductase identified in this study could accept chiral amino amides/esters as amino donors for the dynamic kinetic resolution reductive amination of racemicα-substituted aldehydo-esters,expanding the substrate scope of imine reductases in the dynamic kinetic resolution-reductive amination.Finally,IR104-M3 was successfully used for the preparation of Brivaracetam at gram scale.Using this mutant,various N-substituted amino amides/esters with two chiral centers were also synthesized with up to 99:1 d.r.and 96%yields and subsequently converted intoγ-andδ-lactams,providing an efficient protocol for the synthesis of these important compounds via enzymatic dynamic kinetic resolution-reductive amination from simple building blocks.展开更多
Imine reductases(IREDs)have been extensively used for the imine reduction and reductive amination to access various amines.However,poor activity and severe substrate/product inhibition limit their widespread applicati...Imine reductases(IREDs)have been extensively used for the imine reduction and reductive amination to access various amines.However,poor activity and severe substrate/product inhibition limit their widespread application in industry.Herein,an engineered IRED from Streptomyces viridochromogenes was developed through four rounds of directed evolution.The engineered SvIRED displayed a significant increase in specific activity to 136.8 U mg^(-1),the highest reported for an IRED to date.Molecular dynamics simulations elucidated the surge in specific activity during mutations.The best mutant can also catalyse the reductive coupling of aldehyde homologs and primary amines with up to 66.9 U mg^(-1).Additionally,we established an in-situ product adsorption system using resin,which significantly alleviated substrate/product inhibition and enhanced substrate loading to 100 g L^(-1).Under optimal conditions,a wide range of chiral 2-aryl-pyrrolidines were successfully produced at high substrate loadings(50-100 g L^(-1))with enantiomeric excess over 99%.The usefulness of this biocatalytic system was further demonstrated by preparation of pharmaceutically relevant chiral 2-aryl pyrrolidines,particularly the decagram-scale synthesis of the key chiral aticaprant intermediate with 90%isolated yield,>99%ee,and 438 g L^(-1) d^(-1) space-time yield.展开更多
N-substituted furfurylamines(FAs)are valuable precursors for producing pharmacologically active compounds and polymers.However,enzymatic synthesis of the type of chemicals is still in its infancy.Here we report an imi...N-substituted furfurylamines(FAs)are valuable precursors for producing pharmacologically active compounds and polymers.However,enzymatic synthesis of the type of chemicals is still in its infancy.Here we report an imine reductase from Streptomyces albidoflavus(SaIRED)for the reductive amination of biobased furans.A simple,fast and interference-resistant high-throughput screening(HTS)method was developed,based on the coloration reaction of carbonyl compounds with 2,4-dinitrophenylhydrazine.The reductive amination activity of IREDs can be directly indicated by a colorimetric assay.With the reductive amination of furfural with allylamine as the model reaction,SaIRED with the activity of 4.8 U mg^(-1) was subjected to three rounds of protein engineering and screening by this HTS method,affording a high-activity tri-variant I127V/D241A/A242T(named M3,20.2 U mg^(-1)).The variant M3 showed broad substrate scope,and enabled efficient reductive amination of biobased furans with a variety of amines including small aliphatic amines and sterically hindered amines,giving the target FAs in yields up to>99%.In addition,other variants were identified for preparative-scale synthesis of commercially interesting amines such as N-2-(methylsulfonyl)ethyl-FA by the screen method,with isolated yields up to 87%and turnover numbers up to 9700 for enzyme.Gram-scale synthesis of N-allyl-FA,a valuable building block and potential polymer monomer,was implemented at 0.25 mol L^(-1) substrate loading by a whole-cell catalyst incorporating variant M3,with 4.7 g L^(-1) h^(-1) space-time yield and 91%isolated yield.展开更多
Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understandi...Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of the lion biosynthesis in wheat development, two cDNAs encoding CCR were identified from wheat (Triticum aestivum L. cv. H4564). DNA sequence analyses indicated that the two cDNAs represent two classes of CCR. RT-PCR and Northern blot hybridization demonstrated that one of them, W-cr6, was expressed actively in stem and leaf tissue, the other one, W-cr19, was expressed in root and stem tissue. The results suggested that there are at least two genes encoded for CCR existing in wheat genome.展开更多
[Objective] The aim was to clone the cDNA and DNA sequences of the CCR （Cinnamoyl-CoA reductase） gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these seq...[Objective] The aim was to clone the cDNA and DNA sequences of the CCR （Cinnamoyl-CoA reductase） gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these sequences. [Method] CCR sequences were cloned from P. purpureum by using conventional RT-PCR and RACE （Rapid Amplification of cDNA Ends） methods; and the bioinformatic analyses of the CCR were conducted by means of NCBI, ProtParam ProtScale, TMHMM, TargetP, SignalP, Pfam20.0, Prosite, Swiss-Model, ClustalW2, DNAman, DNAstar and MEGA5. [Result] The cloned PpCCR （P. purpureum CCR） cDNA sequence was 1 316 bp, including a 1 110 bp ORF and 206 bp 3’-UTR. The cloned DNA sequence from PpCCR was 6 133 bp in full-length, containing five exons and four introns. Bioinformatic analysis indicated that PpCCR encoded a polypeptide of 369 amino acids, the secondary structure of which was primarily composed of random coil and α-helix, belonging to NAD-dependent epimerase/dehydratase family, and its co-factor binding sites and substrate binding sites were highly conserved. [Conclusion] DNA and cDNA sequences of CCR gene were obtained from P. purpureum, which had the typical characteristics of other homologous genes. The obtained bioinformatic data provided theoretical references for the further analysis of CCR and better application of P. purpureum in the future.展开更多
A series of N5-substituted 8-deaza-5,6,7,8-tetrahydromethotrexate derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase（DHFR）.The results indicated that modification of the pteridine ri...A series of N5-substituted 8-deaza-5,6,7,8-tetrahydromethotrexate derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase（DHFR）.The results indicated that modification of the pteridine ring of methotrexate（MTX） rendered poor activity against human DHFR.展开更多
Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA...Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.展开更多
Nitrate reductase activity (NRA) in different plant organs and leaves in different positions of Camptotheca acuminata seedlings was determined by an In vivo assay, the diurnal variation rhythm of NRA in leaves of diff...Nitrate reductase activity (NRA) in different plant organs and leaves in different positions of Camptotheca acuminata seedlings was determined by an In vivo assay, the diurnal variation rhythm of NRA in leaves of different positions was observed,and the correlations between leaf NRA, leaf area and lamina mass per unit area (LMA) were also examined. The results showed that NRA in the leaf was significantly highest, compared with that in other organs such as roots, stems and leaves. In this experiment, the 10 leaves were selected from the apex to the base of the seedlings in order. The different NRA occurred obviously in leaves of different positions of C. acuminata seedlings from the apex to the base, and NRA was higher in the 4th-6th leaves.The diurnal change rhythm of leaf NRA showed a one peak curve, and maximum NRA value appeared at about midday (at 12:30 or so). No obvious correlations between NRA and leaf area or lamina mass per unit area were observed. This study offered scientific foundation for the further research on nitrogen metabolism of C. acuminata.展开更多
The development of human benign or malignant prostatic diseases is closely associated with androgens, primarily testosterone （T） and dihydrotestosterone （DHT）. T is converted to DHT by 5-alpha reductase （5-AR） i...The development of human benign or malignant prostatic diseases is closely associated with androgens, primarily testosterone （T） and dihydrotestosterone （DHT）. T is converted to DHT by 5-alpha reductase （5-AR） isozymes. Differential expression of 5-AR isozymes is observed in both human benign and malignant prostatic tissues. 5-AR inhibitors （5-ARI） are commonly used for the treatment of benign prostatic hyperplasia （BPH） and were once promoted as chemopreventive agents for prostate cancer （PCa）. This review discusses the role of the differential expression of 5-AR in the normal development of the human prostate and in the pathogenesis and progression of BPH and PCa.展开更多
Aldo-keto reductase 1D1(AKR1D1) deficiency,a rare but life-threatening form of bile acid deficiency,has not been previously described in China.Here,we describe the first two primary 4-3-oxosteroid 5β-reductase defici...Aldo-keto reductase 1D1(AKR1D1) deficiency,a rare but life-threatening form of bile acid deficiency,has not been previously described in China.Here,we describe the first two primary 4-3-oxosteroid 5β-reductase deficiency patients in China's Mainland diagnosed by fast atom bombardment-mass spectroscopy of urinary bile acids and confirmed by genetic analysis.A high proportion of atypical 3-oxo-4-bile acids in the urine indicated a deficiency in 4-3-oxosteroid 5β-reductase.All of the coding exons and adjacent intronic sequence of the AKR1D1 gene were sequenced using peripheral lymphocyte genomic DNA of two patients and one of the patient's parents.One patient exhibited compound heterozygous mutations:c.396C>A and c.722A>T,while the other was heterozygous for the mutation c.797G>A.Based on these mutations,a diagnosis of primary 4-3-oxosteroid 5β-reductase deficiency could be confirmed.With ursodeoxycholic acid treatment and fat-soluble vitamin supplements,liver function tests normalized rapidly,and the degree of hepatomegaly was markedly reduced in both patients.展开更多
[Objective] The research aimed to study the circadian rhythm of nitrate re- ductase activity （NRA） in plant. [Method] The wheat plants at heading stage were used as the materials for the measurement of dynamic chang...[Objective] The research aimed to study the circadian rhythm of nitrate re- ductase activity （NRA） in plant. [Method] The wheat plants at heading stage were used as the materials for the measurement of dynamic changes of nitrate reductase activity （NRA） within 24 h under the conditions of constant high temperature. [Resulti The fluctuation of NRA in wheat changed greatly from 20：00 pm to 11：00 am. The enzyme activity remained constant, but at 14：00 the enzyme activity was the high- est, higher than all the other time points except the enzyme activity measured at11：00. The enzyme activity was the lowest of 17：00, which was lower than all the other time points except the enzyme activity measured at 2：00. [Conclusion] There were autonomous rhythm changes of NRA in wheat in a certain degree.展开更多
A series of 4-phenoxy quinoline-based mevalonolactone derivatives have been synthesized and evaluated as 3-hydroxy-3-methylglutaryl CoA reductase （HMG CoA reductase） inhibitors.One member of this series,（4R,6S）-6-...A series of 4-phenoxy quinoline-based mevalonolactone derivatives have been synthesized and evaluated as 3-hydroxy-3-methylglutaryl CoA reductase （HMG CoA reductase） inhibitors.One member of this series,（4R,6S）-6-{（E）-2-[6-fluoro-7-chloro-4-（4-fluorophenoxy-quinoline）-3-yl-]-ethenyl}-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one （3d）,showed more potent activity than rosuvastatin or pitavastatin to inhibit the rat HMG CoA reductase in vitro.This compound was selected for the extensive preclinical development as a potential hypocholesterolemic candidate.展开更多
Aldo-keto reductases(AKRs)are a superfamily of enzymes that play crucial roles in various cellular processes,including the metabolism of xenobiotics,steroids,and carbohydrates.A growing body of evidence has unveiled t...Aldo-keto reductases(AKRs)are a superfamily of enzymes that play crucial roles in various cellular processes,including the metabolism of xenobiotics,steroids,and carbohydrates.A growing body of evidence has unveiled the involvement of AKRs in the development and progression of various cancers.AKRs are aberrantly expressed in a wide range of malignant tumors.Dysregulated expression of AKRs enables the acquisition of hallmark traits of cancer by activating oncogenic signaling pathways and contributing to chemoresistance.AKRs have emerged as promising oncotherapeutic targets given their pivotal role in cancer development and progression.Inhibition of aldose reductase(AR),either alone or in combination with chemotherapeutic drugs,has evolved as a pragmatic therapeutic option for cancer.Several classes of synthetic aldo-keto reductase(AKR)inhibitors have been developed as potential anticancer agents,some of which have shown promise in clinical trials.Many AKR inhibitors from natural sources also exhibit anticancer effects.Small molecule inhibitors targeting specific AKR isoforms have shown promise in preclinical studies.These inhibitors disrupt the activation of oncogenic signaling by modulating transcription factors and kinases and sensitizing cancer cells to chemotherapy.In this review,we discuss the physiological functions of human AKRs,the aberrant expression of AKRs in malignancies,the involvement of AKRs in the acquisition of cancer hallmarks,and the role of AKRs in oncogenic signaling,and drug resistance.Finally,the potential of aldose reductase inhibitors(ARIs)as anticancer drugs is summarized.展开更多
3-Hydroxy-3-methylglutaryl coenzyme A （HMG CoA） reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant...3-Hydroxy-3-methylglutaryl coenzyme A （HMG CoA） reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism. Here, I will discuss recent advances that shed light on one mechanism for control of reductase, which involves rapid degradation of the enzyme. Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum （ER） membrane proteins called Insig-1 and Insig-2. Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78, which initiates ubiquitination of reductase. This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes. Thus, sterol-accelerated degradation of reductase represents an example of how a general cellular process （ER-associated degradation） is used to control an important metabolic pathway （cholesterol synthesis）.展开更多
The objective is to study whether the accumulation and utilization of plant N are controlled by Mo status in winter wheat cultivars. Mo-efficient cultivar 97003 （eft） and Mo-inefficient cultivar 97014 （ineff） were...The objective is to study whether the accumulation and utilization of plant N are controlled by Mo status in winter wheat cultivars. Mo-efficient cultivar 97003 （eft） and Mo-inefficient cultivar 97014 （ineff） were grown in severely Mo-deficient acidic soil （Tamm-reagent-extractable Mo 0.112 mg kg^-1） with （＋Mo） and without （-Mo） the application of 0.13 mg kg^-1 Mo. The accumulation and use efficiency of plant total N were significantly higher in ＋Mo than that in -Mo and in eft than that in ineff under Mo deficiency. N use efficiency was remarkably higher in maturity but it was forwarded to jointing stage after Mo supply, thus indicating that Mo supply promoted the N use efficiency besides N uptake and eff was efficient in N uptake and utilization. The overall activity of nitrate reductase （NR, EC 1.6.6.1） was significantly higher in ＋Mo than in -Mo and ratio of ＋Mo/-Mo was even to 14.8 at filleting stage for ineff. Activity of glutamine synthetase （GS, EC 6.3.1.2） was significantly lower in ＋Mo than in -Mo. Concentration of nitrate and glutamate were also significantly lower in ＋Mo than in -Mo, thus provided evidences for enhancing N use efficiency by Mo supply. Activities of NR and GS were significantly higher and concentrations of nitrate and glutamate were significantly lower in eff than ineff under Mo deficiency, thus indicated eff was more efficient in N reduction and utilization. It is therefore concluded that Mo could promote N accumulation and utilization in winter wheat which was directly related to NR and feedback regulated by GS. Higher Mo status also results in higher accumulation and utilization of plant N in eft.展开更多
Aim： To analyze the distribution of the single nucleotide polymorphism （SNP） C677T in the methylenetetrahydrofolate reductase （MTHFR） gene in 355 infertile Chinese patients with idiopathic azoospermia or severe o...Aim： To analyze the distribution of the single nucleotide polymorphism （SNP） C677T in the methylenetetrahydrofolate reductase （MTHFR） gene in 355 infertile Chinese patients with idiopathic azoospermia or severe oligozoospermia and 252 fertile Chinese men as controls to explore the possible association of the SNP and male infertility. Methods： Using the polymerase chain reaction （PCR）-restriction fragment length polymorphism technique, the allele and genotype distribution of SNP C677T in the MTHFR gene were investigated in both patients and controls. Results： The frequencies of allele T （40.9% vs 30.4%, P = 0.002, odds ration [OR] = 1.58, 95% confidence interval [CI]： 1.24-2.02） and mutant homozygote （TT） （18.3% vs. 11.5%, P = 0.023, OR = 1.72, 95% CI： 1.07-2.76） as well as carrier with allele （TT ＋ CT） （63.4% vs. 49.2%, P = 0.0005, OR = 1.79, 95% CI： 1.29-2.48） in infertile patients were significantly higher than those in controls. After patient stratification, the significant differences in distribution of the SNP between each patient subgroup and control group still remained. Conclusion： Our findings indicate that there is an association of SNP C677T in the MTHFR gene with male infertility, suggesting that this polymorphism might be a genetic risk factor for male infertility in Chinese men.展开更多
Real-time polymerase chain reaction analysis was used to compare the effect of NO3^- on the activities of nitrate reductase （NR） and glutamine synthetase （GS）, and the transcript levels of two NR genes, OsNial and...Real-time polymerase chain reaction analysis was used to compare the effect of NO3^- on the activities of nitrate reductase （NR） and glutamine synthetase （GS）, and the transcript levels of two NR genes, OsNial and OsNia2, two cytosolic GS1 genes, OsGln1;1 and OsGln1;2, and one plastid GS2 gene OsGln2, in two rice （Oryza sativa L.） cultivars Nanguang （NG） and Yunjing （Y J）. Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH4^＋ nutrition. Supply of NO3^- increased NR activity in both leaves and roots. Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNial. NO3 also caused an increase in GS activity, but had a complex effect on the expression of the three GS genes. In roots, the OsGln1;1 transcript increased, but OsGln1;2 decreased. In leaves, NO3^- had no effect on the GS1 expression, but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N. These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes. In addition, the NO3-induced increase of biomass, NR activity, GS activity, and the transcript levels of NR and GS genes were proportionally higher in NG than in Y J, indicating a stronger response of NG to NO3^- nutrition than YJ.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.:U21A20407 and 81973467)Beijing Natural Science Foundation,China(Grant No.:7222276).
文摘Sennoside A(SA),a typical prodrug,exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases.Hydrolases have been identified,but reductases remain unknown.By linking a photoreactive group to the SA scaffold,we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling(ABPP).From lysates of an active strain,Bifidobacterium pseudocatenulatum(B.pseudocatenulatum),397 proteins were enriched and subsequently identified using mass spectrometry(MS).Among these proteins,chromate reductase/nicotinamide adenine dinucleotide(NADH)phosphate(NADPH)-dependent flavin mononucleotide(FMN)reductase/oxygen-insensitive NADPH nitroreductase(nfrA)was identified as a potent SA reductase through further bioinformatic analysis and The Universal Protein Resource(UniProt)database screening.We also determined that recombinant nfrA could reduce SA.Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.
基金Supported by the Top-level Talents Support Program of Yangzhou University“Lv Yang Jin Feng”Outstanding Doctor of Yangzhou,No.YZLYJFJH2023YXBS169Natural Science Foundation of Jiangsu Province,No.BK20240907.
文摘This letter critically comments on the article by Zheng et al investigating the role of aucubin in alleviating diabetic neuropathic pain(DNP).DNP arises from hyperglycaemia-induced nerve injury and microglial reprogramming toward aerobic glycolysis.Aldose reductase(also known as AKR1B1)redirects excess glucose flux through the polyol pathway,thus increasing oxidative stress and inflammation.Zheng et al show that aucubin,a plant iridoid glycoside,reverses streptozotocin-induced mechanical and thermal hypersensitivity and anxiety-like behaviour in mice.Mechanistically,aucubin restores microglial morphology,reduces glycolytic flux,enhances oxidative phosphorylation and lowers tumour necrosis factor-α,interleukin(IL)-1βand IL-6 levels in spinal tissue and cultures of the BV-2 microglial cell line.Network pharmacology and molecular docking analyses identify AKR1B1 as a key target,confirmed by the fact that short hairpin RNA knockdown of AKR1B1 eliminates the effects of aucubin.Contrary to the other studies,this study uniquely implicates the polyol pathway in microglial immunometabolism.
基金Supported by National Natural Science Foundation of China,No.82001424.
文摘BACKGROUND Treatment of diabetic neuropathy is often limited by side effects.Aucubin,an iridoid glycoside derived from natural plants,exhibits notable anti-inflammatory and antioxidant properties.AIM To investigate the effects of aucubin on diabetic neuropathic pain(DNP)and glycolysis and inflammation in microglia.METHODS Streptozotocin(STZ)was used to establish a DNP animal model.Blood glucose levels and body weight of mice were measured following STZ administration.Paw withdrawal threshold was calculated for mechanical allodynia.Paw withdrawal latency was recorded for thermal hyperalgesia.The open field test and elevated plus maze was used to assess locomotor activity and anxiety-like behavior.Western blotting was utilized for analysis of protein expression.Immunofluorescence staining was measured for morphometric analysis of microglia.Glycolysis and ATP synthesis in BV-2 cell lines were detected by metabolic extracellular flux analysis.The SwissTargetPrediction and STRING databases were used for comprehensive screening to identify potential target proteins for aucubin.The molecular docking between the possible target proteins and aucubin was investigated using Auto Dock Tool.The BV-2 cell line was transfected with lentiviral AKR1B1-shRNA to further ascertain the function of AKR1B1 in the impact of aucubin on aerobic glycolysis and inflammation during high glucose stimulation.RESULTS Aucubin significantly improved pain and anxiety-like behavior in STZ-induced diabetic mice and restored microglial aerobic glycolysis and inflammation.Several public databases and molecular docking studies suggested that AKR1B1,MMP2 and MMP9 are involved in the effect of aucubin on DNP.Aucubin failed to restore aerobic glycolysis and inflammation in the context of AKR1B1 deficiency.CONCLUSION Aucubin has potential as a therapeutic agent for alleviating DNP by inhibiting expression of AKR1B1.
文摘Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals,but their asymmetric synthesis remains challenging especially for those with multiple chiral centers.In this study,IR104 from Streptomyces aureocirculatus was identified from 157 wild-type imine reductases for the synthesis of(S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide(antiepileptic drug Brivaracetam)via dynamic kinetic resolution reductive amination from ethyl 3-formylhexanoate and(S)-2-aminobutylamide with high diastereoselectivity.To further improve the catalytic efficiency of IR104,its mutant D191E/L195I/E253S/M258A(M3)was identified by saturation mutagenesis and iterative combinatorial mutagenesis,which exhibited a 102-fold increase in the catalytic efficiency relative to that of wild-type enzyme and high diastereoselectivity(98:2 d.r.).Crystal structural analysis and molecular dynamics simulations provided some insights into the molecular basis for the improved activity of the mutant enzyme.The imine reductase identified in this study could accept chiral amino amides/esters as amino donors for the dynamic kinetic resolution reductive amination of racemicα-substituted aldehydo-esters,expanding the substrate scope of imine reductases in the dynamic kinetic resolution-reductive amination.Finally,IR104-M3 was successfully used for the preparation of Brivaracetam at gram scale.Using this mutant,various N-substituted amino amides/esters with two chiral centers were also synthesized with up to 99:1 d.r.and 96%yields and subsequently converted intoγ-andδ-lactams,providing an efficient protocol for the synthesis of these important compounds via enzymatic dynamic kinetic resolution-reductive amination from simple building blocks.
文摘Imine reductases(IREDs)have been extensively used for the imine reduction and reductive amination to access various amines.However,poor activity and severe substrate/product inhibition limit their widespread application in industry.Herein,an engineered IRED from Streptomyces viridochromogenes was developed through four rounds of directed evolution.The engineered SvIRED displayed a significant increase in specific activity to 136.8 U mg^(-1),the highest reported for an IRED to date.Molecular dynamics simulations elucidated the surge in specific activity during mutations.The best mutant can also catalyse the reductive coupling of aldehyde homologs and primary amines with up to 66.9 U mg^(-1).Additionally,we established an in-situ product adsorption system using resin,which significantly alleviated substrate/product inhibition and enhanced substrate loading to 100 g L^(-1).Under optimal conditions,a wide range of chiral 2-aryl-pyrrolidines were successfully produced at high substrate loadings(50-100 g L^(-1))with enantiomeric excess over 99%.The usefulness of this biocatalytic system was further demonstrated by preparation of pharmaceutically relevant chiral 2-aryl pyrrolidines,particularly the decagram-scale synthesis of the key chiral aticaprant intermediate with 90%isolated yield,>99%ee,and 438 g L^(-1) d^(-1) space-time yield.
文摘N-substituted furfurylamines(FAs)are valuable precursors for producing pharmacologically active compounds and polymers.However,enzymatic synthesis of the type of chemicals is still in its infancy.Here we report an imine reductase from Streptomyces albidoflavus(SaIRED)for the reductive amination of biobased furans.A simple,fast and interference-resistant high-throughput screening(HTS)method was developed,based on the coloration reaction of carbonyl compounds with 2,4-dinitrophenylhydrazine.The reductive amination activity of IREDs can be directly indicated by a colorimetric assay.With the reductive amination of furfural with allylamine as the model reaction,SaIRED with the activity of 4.8 U mg^(-1) was subjected to three rounds of protein engineering and screening by this HTS method,affording a high-activity tri-variant I127V/D241A/A242T(named M3,20.2 U mg^(-1)).The variant M3 showed broad substrate scope,and enabled efficient reductive amination of biobased furans with a variety of amines including small aliphatic amines and sterically hindered amines,giving the target FAs in yields up to>99%.In addition,other variants were identified for preparative-scale synthesis of commercially interesting amines such as N-2-(methylsulfonyl)ethyl-FA by the screen method,with isolated yields up to 87%and turnover numbers up to 9700 for enzyme.Gram-scale synthesis of N-allyl-FA,a valuable building block and potential polymer monomer,was implemented at 0.25 mol L^(-1) substrate loading by a whole-cell catalyst incorporating variant M3,with 4.7 g L^(-1) h^(-1) space-time yield and 91%isolated yield.
文摘Cinnamoyl-CoA reductase (CCR) is responsible for the first committed reaction in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of the lion biosynthesis in wheat development, two cDNAs encoding CCR were identified from wheat (Triticum aestivum L. cv. H4564). DNA sequence analyses indicated that the two cDNAs represent two classes of CCR. RT-PCR and Northern blot hybridization demonstrated that one of them, W-cr6, was expressed actively in stem and leaf tissue, the other one, W-cr19, was expressed in root and stem tissue. The results suggested that there are at least two genes encoded for CCR existing in wheat genome.
基金Supported by the National Natural Science Foundation of China(30972138)the Guangdong Natural Science Foundation(9451064201003804)~~
文摘[Objective] The aim was to clone the cDNA and DNA sequences of the CCR （Cinnamoyl-CoA reductase） gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these sequences. [Method] CCR sequences were cloned from P. purpureum by using conventional RT-PCR and RACE （Rapid Amplification of cDNA Ends） methods; and the bioinformatic analyses of the CCR were conducted by means of NCBI, ProtParam ProtScale, TMHMM, TargetP, SignalP, Pfam20.0, Prosite, Swiss-Model, ClustalW2, DNAman, DNAstar and MEGA5. [Result] The cloned PpCCR （P. purpureum CCR） cDNA sequence was 1 316 bp, including a 1 110 bp ORF and 206 bp 3’-UTR. The cloned DNA sequence from PpCCR was 6 133 bp in full-length, containing five exons and four introns. Bioinformatic analysis indicated that PpCCR encoded a polypeptide of 369 amino acids, the secondary structure of which was primarily composed of random coil and α-helix, belonging to NAD-dependent epimerase/dehydratase family, and its co-factor binding sites and substrate binding sites were highly conserved. [Conclusion] DNA and cDNA sequences of CCR gene were obtained from P. purpureum, which had the typical characteristics of other homologous genes. The obtained bioinformatic data provided theoretical references for the further analysis of CCR and better application of P. purpureum in the future.
基金National Natural Science Foundation of China(Grant No.20972011)
文摘A series of N5-substituted 8-deaza-5,6,7,8-tetrahydromethotrexate derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase（DHFR）.The results indicated that modification of the pteridine ring of methotrexate（MTX） rendered poor activity against human DHFR.
基金supported by grants from China Na-tional Basic Research Program (NO. 2004CB117302)National Natural Science Foundation of China (No.30470171)the Sigrid Jusélius Foundation Finland and the Academy of Finland
文摘Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.
文摘Nitrate reductase activity (NRA) in different plant organs and leaves in different positions of Camptotheca acuminata seedlings was determined by an In vivo assay, the diurnal variation rhythm of NRA in leaves of different positions was observed,and the correlations between leaf NRA, leaf area and lamina mass per unit area (LMA) were also examined. The results showed that NRA in the leaf was significantly highest, compared with that in other organs such as roots, stems and leaves. In this experiment, the 10 leaves were selected from the apex to the base of the seedlings in order. The different NRA occurred obviously in leaves of different positions of C. acuminata seedlings from the apex to the base, and NRA was higher in the 4th-6th leaves.The diurnal change rhythm of leaf NRA showed a one peak curve, and maximum NRA value appeared at about midday (at 12:30 or so). No obvious correlations between NRA and leaf area or lamina mass per unit area were observed. This study offered scientific foundation for the further research on nitrogen metabolism of C. acuminata.
基金This work was supported by the National Natural Science Foundation of China （No. 30973015） and the Beijing Natural Science Foundation （No. 7122074） at Beijing Chaoyang Hospital, Capital Medical University to YNN.
文摘The development of human benign or malignant prostatic diseases is closely associated with androgens, primarily testosterone （T） and dihydrotestosterone （DHT）. T is converted to DHT by 5-alpha reductase （5-AR） isozymes. Differential expression of 5-AR isozymes is observed in both human benign and malignant prostatic tissues. 5-AR inhibitors （5-ARI） are commonly used for the treatment of benign prostatic hyperplasia （BPH） and were once promoted as chemopreventive agents for prostate cancer （PCa）. This review discusses the role of the differential expression of 5-AR in the normal development of the human prostate and in the pathogenesis and progression of BPH and PCa.
基金Supported by National Natural Science Foundation of China, No.81070281
文摘Aldo-keto reductase 1D1(AKR1D1) deficiency,a rare but life-threatening form of bile acid deficiency,has not been previously described in China.Here,we describe the first two primary 4-3-oxosteroid 5β-reductase deficiency patients in China's Mainland diagnosed by fast atom bombardment-mass spectroscopy of urinary bile acids and confirmed by genetic analysis.A high proportion of atypical 3-oxo-4-bile acids in the urine indicated a deficiency in 4-3-oxosteroid 5β-reductase.All of the coding exons and adjacent intronic sequence of the AKR1D1 gene were sequenced using peripheral lymphocyte genomic DNA of two patients and one of the patient's parents.One patient exhibited compound heterozygous mutations:c.396C>A and c.722A>T,while the other was heterozygous for the mutation c.797G>A.Based on these mutations,a diagnosis of primary 4-3-oxosteroid 5β-reductase deficiency could be confirmed.With ursodeoxycholic acid treatment and fat-soluble vitamin supplements,liver function tests normalized rapidly,and the degree of hepatomegaly was markedly reduced in both patients.
基金Supported by the National Natural Science Foundation of China(31160254)~~
文摘[Objective] The research aimed to study the circadian rhythm of nitrate re- ductase activity （NRA） in plant. [Method] The wheat plants at heading stage were used as the materials for the measurement of dynamic changes of nitrate reductase activity （NRA） within 24 h under the conditions of constant high temperature. [Resulti The fluctuation of NRA in wheat changed greatly from 20：00 pm to 11：00 am. The enzyme activity remained constant, but at 14：00 the enzyme activity was the high- est, higher than all the other time points except the enzyme activity measured at11：00. The enzyme activity was the lowest of 17：00, which was lower than all the other time points except the enzyme activity measured at 2：00. [Conclusion] There were autonomous rhythm changes of NRA in wheat in a certain degree.
基金The State New Drug Innovation (Grant No. 2009ZX09301-007)Shanghai Rising-Star Program (Grant No. 08QB1403800)Shanghai Innovation Action Project (Grant No. 09431901300)
文摘A series of 4-phenoxy quinoline-based mevalonolactone derivatives have been synthesized and evaluated as 3-hydroxy-3-methylglutaryl CoA reductase （HMG CoA reductase） inhibitors.One member of this series,（4R,6S）-6-{（E）-2-[6-fluoro-7-chloro-4-（4-fluorophenoxy-quinoline）-3-yl-]-ethenyl}-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one （3d）,showed more potent activity than rosuvastatin or pitavastatin to inhibit the rat HMG CoA reductase in vitro.This compound was selected for the extensive preclinical development as a potential hypocholesterolemic candidate.
基金SN and GBR are supported by grants from the Science and Engineering Research Board,Government of India(EMR/2016/001984)Indian Council of Medical Research.
文摘Aldo-keto reductases(AKRs)are a superfamily of enzymes that play crucial roles in various cellular processes,including the metabolism of xenobiotics,steroids,and carbohydrates.A growing body of evidence has unveiled the involvement of AKRs in the development and progression of various cancers.AKRs are aberrantly expressed in a wide range of malignant tumors.Dysregulated expression of AKRs enables the acquisition of hallmark traits of cancer by activating oncogenic signaling pathways and contributing to chemoresistance.AKRs have emerged as promising oncotherapeutic targets given their pivotal role in cancer development and progression.Inhibition of aldose reductase(AR),either alone or in combination with chemotherapeutic drugs,has evolved as a pragmatic therapeutic option for cancer.Several classes of synthetic aldo-keto reductase(AKR)inhibitors have been developed as potential anticancer agents,some of which have shown promise in clinical trials.Many AKR inhibitors from natural sources also exhibit anticancer effects.Small molecule inhibitors targeting specific AKR isoforms have shown promise in preclinical studies.These inhibitors disrupt the activation of oncogenic signaling by modulating transcription factors and kinases and sensitizing cancer cells to chemotherapy.In this review,we discuss the physiological functions of human AKRs,the aberrant expression of AKRs in malignancies,the involvement of AKRs in the acquisition of cancer hallmarks,and the role of AKRs in oncogenic signaling,and drug resistance.Finally,the potential of aldose reductase inhibitors(ARIs)as anticancer drugs is summarized.
文摘3-Hydroxy-3-methylglutaryl coenzyme A （HMG CoA） reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism. Here, I will discuss recent advances that shed light on one mechanism for control of reductase, which involves rapid degradation of the enzyme. Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum （ER） membrane proteins called Insig-1 and Insig-2. Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78, which initiates ubiquitination of reductase. This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes. Thus, sterol-accelerated degradation of reductase represents an example of how a general cellular process （ER-associated degradation） is used to control an important metabolic pathway （cholesterol synthesis）.
基金Financial supports by the National Natural Science Foun-dation of China (30070431)the Key Technologies R&D Program of China during the 9th Five-Year Plan period(95-Agric-18-04)+1 种基金the Doctoral Fund of Ministry of Edu-cation of China (200805041061)the Earmarked Fund for Modern Agro-Industry Technology Research System, China
文摘The objective is to study whether the accumulation and utilization of plant N are controlled by Mo status in winter wheat cultivars. Mo-efficient cultivar 97003 （eft） and Mo-inefficient cultivar 97014 （ineff） were grown in severely Mo-deficient acidic soil （Tamm-reagent-extractable Mo 0.112 mg kg^-1） with （＋Mo） and without （-Mo） the application of 0.13 mg kg^-1 Mo. The accumulation and use efficiency of plant total N were significantly higher in ＋Mo than that in -Mo and in eft than that in ineff under Mo deficiency. N use efficiency was remarkably higher in maturity but it was forwarded to jointing stage after Mo supply, thus indicating that Mo supply promoted the N use efficiency besides N uptake and eff was efficient in N uptake and utilization. The overall activity of nitrate reductase （NR, EC 1.6.6.1） was significantly higher in ＋Mo than in -Mo and ratio of ＋Mo/-Mo was even to 14.8 at filleting stage for ineff. Activity of glutamine synthetase （GS, EC 6.3.1.2） was significantly lower in ＋Mo than in -Mo. Concentration of nitrate and glutamate were also significantly lower in ＋Mo than in -Mo, thus provided evidences for enhancing N use efficiency by Mo supply. Activities of NR and GS were significantly higher and concentrations of nitrate and glutamate were significantly lower in eff than ineff under Mo deficiency, thus indicated eff was more efficient in N reduction and utilization. It is therefore concluded that Mo could promote N accumulation and utilization in winter wheat which was directly related to NR and feedback regulated by GS. Higher Mo status also results in higher accumulation and utilization of plant N in eft.
基金Acknowledgment This work was supported by the National High Tech- nology Research and Development Program of China （Grants 2004AA216090 and 2002BA711A08）, National Basic Research Program of China （Grant 2004Cb518805）, the Natural National Science Foundation of China （Grant 30470960） and the China Medical Board of New York.
文摘Aim： To analyze the distribution of the single nucleotide polymorphism （SNP） C677T in the methylenetetrahydrofolate reductase （MTHFR） gene in 355 infertile Chinese patients with idiopathic azoospermia or severe oligozoospermia and 252 fertile Chinese men as controls to explore the possible association of the SNP and male infertility. Methods： Using the polymerase chain reaction （PCR）-restriction fragment length polymorphism technique, the allele and genotype distribution of SNP C677T in the MTHFR gene were investigated in both patients and controls. Results： The frequencies of allele T （40.9% vs 30.4%, P = 0.002, odds ration [OR] = 1.58, 95% confidence interval [CI]： 1.24-2.02） and mutant homozygote （TT） （18.3% vs. 11.5%, P = 0.023, OR = 1.72, 95% CI： 1.07-2.76） as well as carrier with allele （TT ＋ CT） （63.4% vs. 49.2%, P = 0.0005, OR = 1.79, 95% CI： 1.29-2.48） in infertile patients were significantly higher than those in controls. After patient stratification, the significant differences in distribution of the SNP between each patient subgroup and control group still remained. Conclusion： Our findings indicate that there is an association of SNP C677T in the MTHFR gene with male infertility, suggesting that this polymorphism might be a genetic risk factor for male infertility in Chinese men.
基金the National Natural Science Foundation of China (Nos.30390082 and 40471074).
文摘Real-time polymerase chain reaction analysis was used to compare the effect of NO3^- on the activities of nitrate reductase （NR） and glutamine synthetase （GS）, and the transcript levels of two NR genes, OsNial and OsNia2, two cytosolic GS1 genes, OsGln1;1 and OsGln1;2, and one plastid GS2 gene OsGln2, in two rice （Oryza sativa L.） cultivars Nanguang （NG） and Yunjing （Y J）. Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH4^＋ nutrition. Supply of NO3^- increased NR activity in both leaves and roots. Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNial. NO3 also caused an increase in GS activity, but had a complex effect on the expression of the three GS genes. In roots, the OsGln1;1 transcript increased, but OsGln1;2 decreased. In leaves, NO3^- had no effect on the GS1 expression, but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N. These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes. In addition, the NO3-induced increase of biomass, NR activity, GS activity, and the transcript levels of NR and GS genes were proportionally higher in NG than in Y J, indicating a stronger response of NG to NO3^- nutrition than YJ.
