Transcriptional profile of gene clusters involved in the methylerythritol phosphate pathway in Bacillus subtilis 916

The methylerythritol phosphate pathway is responsible for the biosynthesis of terpenoids, the largest class of secondary metabolites. Although the structures and functions of the proteins involved in this pathway have been well studied in Bacillus subtilis, only a few studies have reported the transcriptional profile of the genes involved. Therefore, we analyzed methylerythritol phosphate pathway genes in the genome of B. subtilis 916, which has been developed as a biological control agent against some rice diseases in China. Our results showed that methylerythritol phosphate pathway genes were distributed throughout the genome of this strain. These genes were transcribed during both the exponential and stationary phases. We further confirmed the transcription units of dxs, dxr, ispD, ispF, ipK, ispG, ispH, idi, and ispA in B. subtilis 916 through reverse transcription-PCR analyses; the results showed that these nine genes were located in seven different operons. The transcript start sites of the seven different operons were determined by 5′-rapid amplification of cDNA ends-PCR. Thus, our study provides a molecular basis at the transcriptional level for investigating homoterpene synthesis in the methylerythritol phosphate pathway of B. subtilis 916.

Effects of GA3 and ABA on the respiratory pathways during the secondary bud burst in black currants

The effects of exogenous gibberellic acid (GA(3)) and abscisic acid (ABA) on the total respiratory rate, percentages of total respiratory rate contributed by respiratory pathways [Embden-Meyerhof- Parnas Pathway (EMP), Pentose Phosphate Pathway (PPP), and Tricarboxylic Acid Cycle (TCA)], and conversion of starch to soluble sugars in the buds of black currants during secondary bud burst were investigated to determine the relationship between respiratory rates and secondary bud burst. 'Adelinia', a black currant cultivar that is prone to secondary bud burst after the first harvest, was used in this study. Mature bushes of Adelinia were sprayed with 30 mg/L GA(3) and 50 mg/L ABA to manipulate bud burst. The results showed that exogenous applications of GA(3) and ABA had opposite effects on bud respiratory rate. Generally, GA(3) treatment increased the total respiratory rate and respiratory rate of the TCA and PPP, and the respiratory rates after GA(3) treatment were higher than those of control. While ABA treatment mostly decreased the total respiratory rate and the respiratory rate of TCA and PPP in buds in comparison to control. In terms of the percentage of the three respiratory rates in comparison to the total respiratory rate, GA(3) treatment significantly increased the percentage of TCA and PPP respiratory rate in comparison to the control (P < 0.01), whereas ABA decreased the rates. GA(3) significantly increased the content of soluble sugars and decreased the starch content, while the starch content in buds after ABA treatment was significantly higher than that of the control. All results showed that PPP is a critical process for the second bud burst in black currants. While the EMP-TCA pathway is still dominant in bud respiration, provides a series of basic materials and energy (ATP). The conversion of starch to soluble sugars is essential for bud burst. Thus, we conclude that an energy shortage is a main contributor in ABA inhibition of the secondary bud burst of black currants.

Nicotinamide adenine dinucleotide phosphate promote the rapid growth of the tumor

Nicotinamide adenine dinucleotide phosphate(NADPH) oxidase is the main source of ROS(intracellular reactive oxygen species), ROS plays an important role in a variety of tumor, the ROS mediated by NADPH oxidase increase the expression of hypoxia inducing factor alpha(HIF-α) through multiple signaling pathways in tumor, and HIFcould be regulated and controlled by downstream multiple targeted genes such as vascular endothelial growth factor(VEGF), glucose transporter(GLUT) to promote tumor angiogenesis, cell energy metabolism reprogram and tumor metastasis.HIF-α, meanwhile, also can regulate the expression of NADPH oxidase by ROS, thus further promote development of tumor.In this review, we will summarize the functions of NADPH in tumorigenesis and discuss their potential implications in cancer therapy.

A conformal titanyl phosphate amorphous overlayer for enhancing photoelectrochemical hydrogen peroxide production

Photoelectrochemical(PEC)H_(2)O_(2)production through water oxidation reaction(WOR)is a promising strategy,however,designing highly efficient and selective photoanode materials remains challenging due to competitive reaction pathways.Here,for highly enhanced PEC H_(2)O_(2)production,we present a conformal amorphous titanyl phosphate(a-TP)overlayer on nanoparticulate TiO_(2)surfaces,achieved via lysozyme-molded in-situ surface reforming.The a-TP overlayer modulates surface adsorption energies for reaction intermediates,favoring WOR for H_(2)O_(2)production over the competing O_(2)evolution reaction.Our density functional theory calculations reveal that a-TP/TiO_(2)exhibits a substantial energy uphill for the O·*formation pathway,which disfavors O_(2)evolution but promotes H_(2)O_(2)production.Additionally,the a-TP overlayer strengthens the built-in electric field,resulting in favorable kinetics.Consequently,a-TP/TiO_(2)exhibits 3.7-fold higher Faraday efficiency(FE)of 63%at 1.76 V vs.reversible hydrogen electrode(RHE)under 1 sun illumination,compared to bare TiO_(2)(17%),representing the highest FE among TiO_(2)-based WOR H_(2)O_(2)production systems.Employing the a-TP overlayer constitutes a promising strategy for controlling reaction pathways and achieving efficient solar-to-chemical energy conversion.

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.

CT23通过调节H6PD表达影响磷酸戊糖途径调控肝细胞癌细胞凋亡

目的:探讨癌-睾丸抗原23(CT23)参与磷酸戊糖途径(PPP)调控,促进肝细胞癌(HCC)细胞凋亡的分子机制。方法:通过全转录组测序、葡萄糖消耗检测、乳酸生成分析、还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)生成检测、活性氧(ROS)生成分析和线粒体示踪等方法探讨CT23与PPP的关系;蛋白质印记法(western blotting)及实时荧光定量PCR(RT-qPCR)检测敲低CT23的HCC细胞中已糖-6-磷酸脱氢酶(H6PD)表达量变化,TUNEL法分析细胞凋亡。结果:CT23与PPP有关;与control组相比,shCT23组HCC细胞葡萄糖消耗减少,乳酸生成水平降低,NADPH生成水平降低,ROS水平升高,细胞凋亡增加,H6PD mRNA水平降低,H6PD蛋白水平降低(均P<0.05);电镜下细胞形态发生变化并伴随线粒体损伤;与shCT23组相比,shCT23+H6PDOE组HCC细胞H6PD蛋白水平升高,葡萄糖消耗增多,乳酸生成水平升高,NADPH生成水平升高,细胞凋亡减少(均P<0.05)。结论:CT23通过H6PD增强PPP促进HCC细胞凋亡。

Regulation of the pentose phosphate pathway in cancer

Energy metabolism is significantly reprogrammed in many human cancers, and these alterations confer many advantages to cancer cells, including the pro- motion of biosynthesis, ATP generation, detoxification and support of rapid proliferation. The pentose phos- phate pathway （PPP） is a major pathway for glucose catabolism. The PPP directs glucose flux to its oxi- dative branch and produces a reduced form of nico- tinamide adenine dinucleotide phosphate （NADPH）, an essential reductant in anabolic processes. It has become clear that the PPP plays a critical role in regulating cancer cell growth by supplying cells with not only ribose-5-phosphate but also NADPH for detoxification of intracellular reactive oxygen species, reductive biosynthesis and ribose biogenesis. Thus, alteration of the PPP contributes directly to cell pro- liferation, survival and senescence. Furthermore, recent studies have shown that the PPP is regulated oncogenically and/or metabolically by numerous fac- tors, including tumor suppressors, oncoproteins and intracellular metabolites. Dysregulation of PPP flux dramatically impacts cancer growth and survival. Therefore, a better understanding of how the PPP is reprogrammed and the mechanism underlying the balance between glycolysis and PPP flux in cancer will be valuable in developing therapeutic strategies targeting this pathway.

转酮醇酶在糖尿病及其并发症发生和治疗中作用的研究进展

转酮醇酶(TKT)是催化磷酸戊糖途径(PPP)非氧化阶段基团转移反应的重要酶,参与机体各类能源物质,如葡萄糖、核糖、核苷酸以及脂质的代谢。TKT可以通过抑制高糖环境下蛋白质和脂质非酶糖基化(NEG)产生的晚期糖基化终末产物(AGEs),减少氧化应激、炎性反应、动脉粥样硬化、内皮功能障碍以及Tau蛋白磷酸化等,进而改善血糖、糖耐量和β细胞功能,达到预防和治疗糖尿病及其并发症的作用。

p53 promotes AKT and SP1-dependent metabolism through the pentose phosphate pathway that inhibits apoptosis in response to Nutlin-3a

Nutlin-3a is a MDM2 antagonist and preclinical drug that activates p53. Cells with MDM2 gene amplification are especially prone to Nutlin-3a-induced apoptosis, though the basis for this is unclear. Glucose metabolism can inhibit apoptosis in response to Nutlin-3a through mechanisms that are incompletely understood. Glucose metabolism through the pentose phosphate pathway （PPP） produces NADPH that can protect cells from potentially lethal reactive oxygen species （ROS）. We compared apoptosis and glucose metabolism in cancer cells with and without MDM2 gene amplification treated with Nutlin-3a. Apoptosis in MDM2-amplified cells was associated with a reduction in glycolysis and the PPP, reduced NADPH, increased ROS, and depletion of the transcription factor SP1, which normally promotes PPP gene expression. In contrast, glycolysis and the PPP were maintained or increased in MDM2 non-amplified cells treated with Nutlin-3a. This was dependent on p53-mediated AKT activation and was associated with maintenance of SP1 and continued expression of PPP genes. Knockdown or inhibition of AKT, SP1, or the PPP sensitized MDM2-non-amplified cells to apoptosis. The data indicate that p53 promotes AKT and SP1-dependent activation of the PPP that protects cells from Nutlin-3a-induced apoptosis. These findings provide insight into how glucose metabolism reduces Nutlin-3a-induced apoptosis, and also provide a mechanism for the heightened sensitivity of MDM2-amplified cells to apoptosis in response to Nutlin-3a.

磷酸戊糖途径抑制感光细胞凋亡及分子机制的研究进展

感光细胞是一种特殊的神经上皮细胞,在视觉信号的产生和传导中具有重要作用,其主要通过大量摄取葡萄糖进行糖代谢以满足生理需求。而感光细胞凋亡,则是视网膜疾病导致患者盲的共同原因,该过程伴有氧化应激和合成代谢的改变。近期研究发现,糖代谢的重要分支——磷酸戊糖途径,在上述病理发展中起重要作用。其产物烟酰胺腺嘌呤二核苷酸磷酸(NADPH)作为感光细胞中重要的供氢体,参与物质合成代谢,对抗氧化应激,进而抑制感光细胞的凋亡。本文将从代谢活动和分子通路的角度,针对磷酸戊糖途径抑制感光细胞凋亡的作用机制进行综述,以期为临床科研工作提供参考。

Biodegradation pathway and mechanism of tri (2-chloropropyl) phosphate by Providencia rettgeri

Tri(2-chloropropyl)phosphate(TCPP)was an emerging contaminant of global concern because of its frequent occurrence,potential toxic effects,and persistence in the environment.Microbial degradation might be an efficient and safe removal method,but limited information was available.In this study,Providencia rettgeri was isolated from contaminated sediment and showed it could use TCPP as unique phosphorus source to promote growth,and decompose 34.7%of TCPP(1 mg/L)within 5 days.The microbial inoculation and the initial concentration of TCPP could affect the biodegradation efficient.Further study results indicated that TCPP decomposition by Providencia rettgeri was mainly via phosphoester bond hydrolysis,evidenced by the production of bis(2-chloropropyl)phosphate(C_(6)H_(13)Cl_(2)PO_(4))and mono-chloropropyl phosphate(C_(3)H_(8)ClPO_(4)).Both intracellular and extracellular enzymes could degrade TCPP,but intracellular degradation was dominant in the later reaction stage,and the presence of Cu^(2+) ions had a promoting effect.These findings developed novel insights into the potential mechanism of TCPP microbial degradation.

植物戊糖磷酸途径及其两个关键酶的研究进展

戊糖磷酸途径是植物体中糖代谢的重要途径,主要生理功能是产生供还原性生物合成需要的NADPH,可供核酸代谢的磷酸戊糖以及一些中间产物可参与氨基酸合成和脂肪酸合成等。葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶是戊糖磷酸途径的两个关键酶,广泛的分布于高等植物的胞质和质体中。本文综述了植物戊糖磷酸途径及其两个关键酶的分子生物学的研究进展,讨论了该途径在植物生长发育和环境胁迫应答中的作用。

敲低及过表达G6PD对肝癌细胞增殖和迁移的影响

目的研究在人肝癌细胞株PLC/PRF/5中,敲低及过表达G6PD对细胞株增殖、生长及迁移能力的影响。方法包装慢病毒颗粒,选用人肝癌细胞株PLC/PRF/5建立G6PD敲低及过表达稳转细胞株,通过PCR及Western blot检验过表达及敲低效果,利用建立好的细胞株进行功能实验:通过实时细胞分析系统(RTCA)记录细胞增殖及迁移曲线,EDU实验可直观显示处于DNA复制阶段的细胞比例,以克隆形成实验反映细胞的生长能力。结果在G6PD敲低株中,细胞的倍增时间较对照组延长,生长速率明显下降,EDU实验中处于增殖期细胞的比例较对照组下降43.2%,克隆形成率明显下调(P<0.05),敲低组的迁移速率亦明显降低,曲线分离显著;而在过表达株及其对照组两株细胞中,增殖、生长及迁移实验结果无明显差异。结论在肝癌细胞中降低G6PD的表达将抑制肝癌的增殖生长,为进一步研究肝癌的发生机制及治疗方案打下了基础。

涝渍逆境对玉米根系磷酸戊糖途径的影响

涝渍胁迫下玉米幼苗根系磷酸戊糖途径占总呼吸的百分率增加,葡萄糖-6-磷酸脱氢酶活性升高;次生根的增幅大于初生根。耐涝品种与不耐涝品种相比根内磷酸戊糖途径的增强没有明显差异,但玉米与水稻相比,上述增值与二者的耐涝性一致。

磷酸戊糖途径的调节对红豆杉细胞生长及紫杉醇合成的影响

在正常的红豆杉细胞悬浮培养过程 ,葡萄糖 - 6-磷酸脱氢酶 (G6PDH)活性的变化趋势与生物量的基本相似。而在chitosan处理的细胞中G6PDH活性升高而生物量下降。 1 0 0mg .L- 1 chitosan和 5 0 0mg .L- 1 chitosan均对细胞G6PDH具有诱导作用 ,且后者的诱导强度较前者的高。乙二醇双 2 氨基乙基醚四乙酸 (EGTA)的加入降低chitosan对细胞G6PDH的诱导程度 ,显示chitosan对G6PDH的诱导需要Ca2 +的参与。谷胱甘肽 (GHS)的处理可反馈抑制chitosan对细胞G6PDH的诱导。通过分析调节后G6PDH的各种活性与细胞中紫杉醇产量的关系 ,认为采用合适的处理方法调节磷酸戊糖途径 。

人参皂苷Rg3通过抑制mTOR通路介导的磷酸戊糖途径对肺癌细胞的放射增敏作用

目的探讨人参皂苷Rg3通过抑制哺乳动物雷帕霉素靶蛋白(mTOR)通路介导的磷酸戊糖途径(PPP),对肺癌细胞放射敏感性的影响。方法以0、10、20、40、60、80 mg/L的人参皂苷Rg3处理肺癌细胞A549;MTT法检测细胞增殖情况;将细胞分为对照组(正常培养,不照射)、放射组(X射线照射处理)、人参皂苷Rg3组(60 mg/L人参皂苷Rg3,不照射)、联合组(X射线照射+60 mg/L人参皂苷Rg3)、激活剂组(X射线照射+60 mg/L人参皂苷Rg3+100 nmol/L mTOR通路激活剂MHY1485);均为加入相对应药物培养48 h后放射组、联合组和激活剂组采用8 Gy X射线照射。平板克隆实验检测各组细胞克隆形成率;酶联免疫吸附试验(ELISA)测定各组细胞葡萄糖-6-磷酸脱氢酶(G6PD)、还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)水平;DCFH-DA荧光探针法检测细胞内活性氧(ROS)水平;流式细胞仪检测细胞凋亡;γ-H2AX免疫荧光染色分析DNA损伤修复情况;Western blot检测细胞中mTOR、p-mTOR、增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(caspase-3)、γ-H2AX蛋白的表达。结果人参皂苷Rg3以剂量依赖性的方式抑制A549细胞的增殖(P<0.05);与对照组比较,放射组、人参皂苷Rg3组细胞克隆形成率、G6PD、ROS、NADPH水平下降,p-mTOR/mTOR、PCNA蛋白表达水平降低,细胞凋亡率、γ-H2AX焦点数、Bax、caspase-3、γ-H2AX蛋白表达水平升高(P<0.05);与放射组、人参皂苷Rg3组比较,联合组细胞克隆形成率、G6PD、ROS、NADPH水平下降,p-mTOR/mTOR、PCNA蛋白表达水平降低,细胞凋亡率、γ-H2AX焦点数、Bax、caspase-3、γ-H2AX蛋白表达升高(P<0.05);与联合组比较,激活剂组细胞克隆形成率、G6PD、ROS、NADPH水平升高,p-mTOR/mTOR、PCNA蛋白表达水平升高,细胞凋亡率、γ-H2AX焦点数、Bax、caspase-3、γ-H2AX蛋白表达水平降低(P<0.05)。结论人参皂苷Rg3发挥抑制肺癌作用可能是通过抑制mTOR介导的PPP实现的。

D-核糖生产菌的选育

将枯草芽胞杆菌通过紫外线诱变得到了莽草酸缺陷突变株，在２８株突变株中有１０株积累Ｄ－核糖。这些菌株均属戊糖磷酸途径的非氧化支路缺失突变株。对这些菌株的产核糖能力进行了验证。

脑胶质瘤中G6PD蛋白的表达及与肿瘤分级的相关性

目的观察胶质瘤中G6PD蛋白的表达情况,探讨G6PD在胶质瘤中的表达特点、与胶质瘤分级的关系及意义。方法用免疫组化方法检测41例胶质瘤和8例正常脑组织标本中G6PD蛋白的表达并将胶质瘤分级,比较各级之间阳性表达率的差异。结果41例胶质瘤标本中G6PD蛋白阳性表达率为56.1%(23/41),而8例正常脑组织中G6PD蛋白阳性表达率为0,二者相比有显著性差异(P<0.05)。G6PD蛋白在胶质瘤各病理分级中阳性表达率分别为Ⅰ级+Ⅱ级42.3%(11/26),Ⅲ级+Ⅳ级80.0%(12/15),二者之间有显著性差异(P<0.05)。结论G6PD蛋白在胶质瘤中的表达上调,且与胶质瘤病理分级相关。

转酮酶在癌症中的作用及其机制研究进展

肿瘤细胞的一大重要特征是代谢水平的改变。戊糖磷酸途径作为细胞产生NADPH和五碳糖的主要通路,在肿瘤发生发展过程中发挥着重要功能。转酮酶是戊糖磷酸途径中的关键酶之一,越来越多的研究表明其与癌症病人预后具有显著相关性。作为肿瘤诊断、治疗的潜在靶标,转酮酶具有重要的研究价值。我们就目前癌症研究中对转酮酶的研究进展做简要综述。

脑胶质瘤中G6PD蛋白的表达及与肿瘤分级的相关性

目的观察胶质瘤中G6PD蛋白的表达情况,探讨G6PD在胶质瘤中的表达特点与胶质瘤分级的关系及意义。方法用免疫组化方法检测41例胶质瘤和8例正常脑组织标本中G6PD蛋白的表达并将胶质瘤分级,比较各级之间阳性表达率的差异。结果41例胶质瘤标本中,G6PD蛋白阳性表达率为56.1%(23/41),而8例正常脑组织中,G6PD蛋白阳性表达率为0%,两者相比有显著性差异(P<0.05)。G6PD蛋白在胶质瘤各病理分级中,阳性表达率分别为Ⅰ+Ⅱ级42.3%(11/26),Ⅲ+Ⅳ级80.0%(12/15),两者之间有显著性差异(P<0.05)。结论G6PD蛋白在胶质瘤中的表达上调,且与胶质瘤病理分级相关。