Cloning and Expression Analysis of Two Wheat cDNAs Encoding Cinnamoyl-CoA Reductase

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.

Cloning and Bioinformatic Analysis of Cinnamoyl-CoA Reductase Gene (CCR) from Pennisetum purpureum

[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.

Aldo-keto reductase family member C3(AKR1C3)promotes hepatocellular carcinoma cell growth by producing prostaglandin F2α

Hepatocellular carcinoma(HCC)is a leading cause of death worldwide.Current therapies are effective for HCC patients with early disease,but many patients suffer recurrence after surgery and have a poor response to chemotherapy.Therefore,new therapeutic targets are needed.We analyzed gene expression profiles between HCC tissues and normal adjacent tissues from public databases and found that the expression of genes involved in lipid metabolism was significantly different.The analysis showed that AKR1C3 was upregulated in tumors,and high AKR1C3 expression was associated with a poorer prognosis in HCC patients.In vitro,assays demonstrated that the knockdown of AKR1C3 or the addition of the AKR1C3 inhibitor indomethacin suppressed the growth and colony formation of HCC cell lines.Knockdown of AKR1C3 in Huh7 cells reduced tumor growth in vivo.To explore the mechanism,we performed pathway enrichment analysis,and the results linked the expression of AKR1C3 with prostaglandin F2 alpha(PGF2a)downstream target genes.Suppression of AKR1C3 activity reduced the production of PGF2a,and supplementation with PGF2a restored the growth of indomethacin-treated Huh7 cells.Knockdown of the PGF receptor(PTGFR)and treatment with a PTGFR inhibitor significantly reduced HCC growth.We showed that indomethacin potentiated the sensitivity of Huh7 cells to sorafenib.In summary,our results indicate that AKR1C3 upregulation may promote HCC growth by promoting the production of PGF2α,and suppression of PTGFR limited HCC growth.Therefore,targeting the AKR1C3-PGF2a-PTGFR axis may be a new strategy for the treatment of HCC.

Aldo-keto reductases:Role in cancer development and theranostics

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.

Synthesis and Characterization of Naproxen-Salicylate Derivatives as Potential Dual-Targeted Inhibitors of Dihydrofolate Reductase

Dihydrofolate reductase (DHFR) is an enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). Chemotherapy drugs such as methotrexate help to slow the progression of cancer by limiting the ability of dividing cells to make nucleotides by competitively inhibiting DHFR. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been previously reported to exhibit competitive inhibition of DHFR, in addition to their primary action on cyclooxygenase enzymes. This interaction interferes with the enzymatic reduction of dihydrofolate to tetrahydrofolate, thereby impeding the folate metabolism pathway essential for nucleotide synthesis and cell proliferation. This activity stems from their structural resemblance to the p-aminobenzoyl-l-glutamate (pABG) moiety of folate, a substrate of DHFR. It has been established that NSAIDs containing a salicylate group (which has structural similarities to pABG), such as diflunisal, exhibit stronger DHFR-binding activity. In this study, we synthesized salicylate derivatives of naproxen with the aim of exploring their potential as inhibitors of DHFR. The interactions between these derivatives and human DHFR were characterized using a combination of biochemical, biophysical, and structural methods. Through polyacrylamide gel electrophoresis (PAGE) analysis, enzymatic assays, and quantitative ELISA, we investigated the binding affinity and inhibitory potency of the synthesized salicylate derivatives towards DHFR. The findings of this study suggest the potential of salicylate derivatives of naproxen as promising candidates for the inhibition of DHFR, thereby offering novel therapeutic opportunities for modulating the inflammatory process through multiple pathways. Further optimization of these derivatives could lead to the development of more efficacious dual-targeted analogs with enhanced therapeutic benefits.

Astragalin attenuates diabetic cataracts via inhibiting aldose reductase activity in rats

AIM:To investigate the aldose reductase(AR)inhibition capacity of astragalin(AST)against streptozoticin-induced diabetic cataracts(DCs)in rats.METHODS:Ex vivo investigations were conducted by treating the lens of a goat placed for 72h in artificial aqueous humor(AAH)of pH 7.8 at room temperature with cataract-causing substance(55 mmol/L of galactose)and in vivo studies were performed on rats via induction with streptozotocin.AST was administered at different dose levels and scrutinize for DC activity.RESULTS:In diabetic rats,AST improved the body weight,blood insulin,and glucose as well as the levels of galactitol in a dose-dependent way,other biochemical parameters i.e.inflammatory mediators and cytokines,and also suppress AR activity.The level of the antioxidant parameters such as superoxide dismutase(SOD),catalase(CAT),and glutathione(GSH)activity were also altered on a diabetic lens after the administration of the AST.CONCLUSION:AST protects against lens opacification to avoid cataracts and polyols formation,indicating that it could be used as a potential therapeutic agent for diabetes.

Two types of cinnamoyl-Co A reductase function divergently in accumulation of lignins,flavonoids and glucosinolates and enhance lodging resistance in Brassica napus

Brassica crops,which are of worldwide importance,provide various oil,vegetable and ornamental products,as well as feedstocks for animal husbandry and biofuel industry.Cinnamoyl-Co A reductase(CCR)is the entry point to the lignin pathway and a crucial locus in manipulation of associated traits,but CCRassociated metabolism and traits in Brassica crops have remained largely unstudied except in Arabidopsis thaliana.We report the identification of 16 CCR genes from Brassica napus and its parental species B.rapa and B.oleracea.The Bn CCR1 and Bn CCR2 subfamilies displayed divergent organ-specificity and participation in the yellow-seed trait.Their functions were dissected via overexpression of representative paralogs in B.napus.Bn CCR1 was expressed preferentially in G-and H-lignin biosynthesis and vascular development,while Bn CCR2 was expressed in S-lignin biosynthesis and interfascicular fiber development.Bn CCR1 showed stronger effects on lignification-related development,lodging resistance,phenylpropanoid flux control,and seed coat pigmentation,whereas Bn CCR2 showed a stronger effect on sinapate biosynthesis.Bn CCR1 upregulation delayed bolting and flowering time,while Bn CCR2 upregulation weakened the leaf vascular system in consequence of suppressed G-lignin accumulation.Bn CCR1 and Bn CCR2 were closely but almost oppositely linked with glucosinolate metabolism via inter-pathway crosstalk.We conclude that Bn CCR1 and Bn CCR2 subfamilies offer great but differing potential for manipulating traits associated with phenylpropanoids and glucosinolates.This study reveals the CCR1–CCR2 divergence in Brassicaceae and offers a resource for rapeseed breeding for lodging resistance,yellowseed traits,and glucosinolate traits.

多枝赖草Glutathione Reductase基因克隆及胁迫表达分析

为了获得完整的谷胱甘肽还原酶基因序列,根据已克隆到的谷胱甘肽还原酶cDNA片段设计引物,利用RACE扩增获得了基因全长序列,应用Southern印迹杂交法分析基因存在状态,Northern印迹杂交法研究基因表达情况.结果表明,该基因全长1580 bp,含一个1 140 bp的开放阅读框架,编码380个氨基酸,与其它植物谷胱甘肽还原酶氨基酸序列的同源性在77%-92%之间;Southern杂交表明该基因有一个拷贝;Northern杂交表明在逆境胁迫下GR基因表达加强.

Nitrate reductase activity and its diurnal variation rhythm for Camptotheca acuminata seedlings

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.

油菜素内酯合成酶(Steroid 5α-Reductase)基因的超量表达对毛白杨生长的影响

将棉花Steroid 5α-reductase基因(DET2)超量表达载体导入毛白杨中,观察其对毛白杨生长和芽休眠的影响。结果表明,超量表达DET2基因的毛白杨茎高度、直径生长和不定根的生长增强,芽的休眠破除提前。

Dynamic Changes of Nitrate Reductase Activity within 24 Hours

[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.

Single nucleotide polymorphism C677T in the methylenetetrahydrofolate reductase gene might be a genetic risk factor for infertility for Chinese men with azoospermia or severe oligozoospermia

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.

Influences of Mo on Nitrate Reductase, Glutamine Synthetase and Nitrogen Accumulation and Utilization in Mo-Efficient and Mo-Inefficient Winter Wheat Cultivars

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.

Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and gastric cancer susceptibility

AIM: To identify the association between methylenetetrahydrofolate reductase (MTHFR) polymorphisms and gastric cancer (GC) susceptibility.

Differential expression of 5-alpha reductase sozymes in the prostate and its clinical implications

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.

Inhibitory effect of two Indian medicinal plants on aldose reductase of rat lens in vitro

Objective:To assesse the inhibitor) effect of alcoholic extract of two Indian medicinal plants namely Ceasalpinia digyna Rottler and.Alangium lamarckii Thwaits on aldose reductase（AR） of rat lens.Methods:Rats lens were enucleated through posterior approach and their homogenate was prepared and centrifuged to obtain a clear supernatant for the determination of AR activity and protein content.Results:The alcoholic extract of Ceasalpirda digyna and Alangium lamarckii had a potent inhibitory effect on the lens AR enzyme.The IC50 values of alcoholic extract of the selected plants were calculated and were（46.29±11.17） and（106.00±5.11）μg/mL,respectively. Quercetin was used as a positive control and its IC50 value was（2.95±1.53）μg/mL.Conclusions:Thus,it is concluded that alcoholic extracts of the selected plant exhibit significant inhibitory effects on AR in the rat lens in vitro.

Changes of Nitrate Reductase Activity in Cucumber Seedlings in Response to Nitrate Stress

In China, nitrogen fertilizer application rates in intensive agricultural systems have increased dramatically in recent years, especially in protected vegetable production systems. This excessive use of nitrogen fertilizer has resulted in soil secondary salinization, which has become a significant environmental stress for crops such as cucumber, in the protected farmland of China. So it is necessary to illuminate how crops respond to nitrate stress. The objective of this work was to examine the effects of increased nitrate concentration [14 （CK） and 140 mmol L^-1 （T）] on NO3- concentration, and in vitro and in vivo nitrate reductase activities in the roots and leaves of cucumber （Cucumis sativus L. cv. Xintaimici） seedlings with hydroponic culture. The results showed that the NO3- concentration in the roots and leaves of T seedlings significantly increased over treatment course, and at 12 d increased by 1.08 and 1.72 times with respect to CK seedlings, respectively; in vitro nitrate reductase activity of T was increased dramatically to 1.74 times of CK in the roots at 2 d and 1.56 times of CK in the leaves at 6 d, and then decreased. At 12 d, in vitro activity was still 24.3% higher in the roots and only 9.9% lower in the leaves than CK. Compared with in vitro nitrate reductase activity, in vivo activity responded differently to the increase of treatment time. At the beginning, in vivo nitrate reductase activity in the roots and leaves of T had no significant difference from CK, whereas with the increase of treatment duration, the activity decreased. At 12 d, in vivo activity in the roots and leaves of T lowered by 20.1 and 52.8% with respect to CK, respectively. This evidence suggests that posttranslational activation of nitrate reductase in cucumber seedlings may be seriously inhibited by nitrate stress.

Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.

Cytochrome b5 reductase 2 suppresses tumor formation in nasopharyngeal carcinoma by attenuating angiogenesis

Background:Cytochrome b5 reductase 2(CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma(NPC).Inactivation of CYB5R2 is associated with lymph node metastasis in NPC.This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2.Methods:Polymerase chain reaction(PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1.NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2,and data was validated by real-time PCR.A chick chorioallantoic membrane(CAM)embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis.An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor(VEGF).Results:In CYB5R2-transfected NPC cells,PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor(FAS),FBJ murine osteosarcoma viral oncogene homolog(FOS),phosphoinositide-3-kinase regulatory subunit 1(PIK3R1),integrin beta 3(ITGB3),metastasis suppressor 1(MTSSl),interferon beta 1(IFNB1),and cyclin-dependent kinase inhibitor 2A(CDKN2A) and down-regulated levels of integrin beta 5(ITGB5),insulin-like growth factor 1(IGF1),TEK tyrosine kinase(TEK),transforming growth factor beta receptor 1(TGFBRl),and VEGF.The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited.Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.Conclusion:CYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of VEGF to reduce angiogenesis,thereby suppressing tumor formation.

Evaluation of in vitro aldose reductase inhibitory potential of different fraction of Hybanthus enneaspermus Linn F.Muell

Objective:To evaluate the aldose reductase inhibitory(ARI)activity of different fractions of Hybanthus enneaspermus for potential use in diabetic cataract.Methods:Total phenol and flavonoid content of different fractions was determined.ARI activity of different fractions in rat lens was investigated in vitro.Results:The results showed significant level of phenolic and flavonoid content in ethyl acetate fraction[total phenol(212.15±0.79 mg/g),total flavonoid(39.11±2.27mg/g)]and aqueous fraction[total phenol(140.62±0.57mg/g),total flavonoid(26.07±1.49 mg/g)]as compared with the chloroform fraction[total phenol(68.56±0.51mg/g),total flavonoid(13.41±0.82mg/g)]and petrolium ether fraction[total phenol(36.68±0.43mg/g),total flavonoid(11.55±1.06mg/g)].There was a significant difference in the ARI activity of each fraction,and it was found to be the highest in ethyl acetate fraction[IC_(50)(49.26±1.76μg/mL)]followed by aqueous extract[IC_(50)(70.83±2.82μg/mL)]and it was least in the petroleum ether fraction[IC_(50)(118.89±0.71μg/mL)].Chloroform fraction showed moderate activity[IC_(50)(98.52±1.80μg/mL)].Conclusions:Different fractions showed significanct amount of ARI activity,where in ethyl acetate fraction it was found to be maximum which may be due to its high phenolic and flavonoid content.The extract after further evaluation may be used in the treatment of diabetic cataract.