Variation in 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Coding Sequences and Glyphosate Response among <i>Cyperus rotundus</i>L. Populations

The gene sequence encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzymatic target site of the herbicide glyphosate, was determined for several purple nutsedge (Cyperus rotundus L.) accessions from geographically distant locations and these were aligned to generate a consensus sequence. The EPSPS sequences each had single nucleotide polymorphisms (SNPs) only a few of which were predicted to cause an amino acid change in the EPSP synthase. None had the proline to serine substitution or other substitutions responsible for glyphosate resistance reported in other species. A dendrogram generated from the cluster analysis of the EPSPS gene sequences indicated similarities between accessions from Tanzania, Indonesia, California-2, Greece, Brazil, Argentina and Iran much like cluster analysis previously reported based on RAPD scores and morphological traits possibly indicating a common genetic background or origin. Considering the differences in EPSPS sequences, the response of these purple nutsedge accessions to 0.84 kg·ae·ha-1 of glyphosate was assessed to determine whether differential tolerance was present. At 7 days after the first application control ranged from 9% for the accession from Greece to 73% for the accession from Tanzania. Control of these accessions increased to 45% and 93% respectively by 14 days after the second application. The I50’s for glyphosate inhibition of growth for four accessions from geographically distant countries (Mississippi, Brazil, Indonesia and Tanzania) were 0.21, 0.10, 0.25 and 0.06 kg·ha-1, respectively, which represented a 4-fold difference. The difference in sensitivity to glyphosate may be a result of a non-target site mechanism such as differences in sequestration, translocation or cuticle thickness rather than alterations in EPSPS.

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.

Pyridoxal 5'-phosphate alleviates prenatal pyridaben exposureinduced anxiety-like behaviors in offspring

Pyridaben(PY)is a widely used organochlorine acaricide,which can be detected in the peripheral blood of pregnant women.Available evidence suggests that PY has reproductive toxicity.However,it remains uncertain whether prenatal PY exposure impacts neurobehavioral development in offspring.Here,we administered PY to pregnant mice at a dose of 0.5 and 5 mg kg^(-1)day^(-1)via gavage and observed anxietylike behaviors in PY offspring aged five weeks.We then integrated the metabolome and transcriptome of the offspring's brain to explore the underlying mechanism.Metabolome data indicated that the vitamin B6 metabolism pathway was significantly affected,and the pyridoxal 50-phosphate(PLP)concentration and the active form of vitamin B6 was significantly reduced.Moreover,the transcriptome data showed that both PLP generation-related Pdxk and anxiety-related Gad1 were significantly down-regulated.Meanwhile,there was a decreasing trend in the concentration of GABA in the hippocampal DG region.Next,we supplemented PLP at a dose of 20 mg kg^(-1)day^(-1)to the PY offspring via intraperitoneal injection at three weeks.We found up-regulated expression of Pdxk and Gad1 and restored anxiety-like behaviors.This study suggests that prenatal exposure to PY can disrupt vitamin B6 metabolism,reduce the concentration of PLP,down-regulate the expression levels of Pdxk and Gad1,inhibit the production of GABA,and ultimately lead to anxiety-like behaviors in offspring.

探讨丙氨酸氨基转移酶IFCC法检测试剂中加入5＇-磷酸吡哆醛对结果的影响

目的探讨丙氨酸氨基转移酶（ALT）IFCC法检测试剂中加入5’-磷酸吡哆醛对结果的影响。方法依据IFCC公布的酶学测定参考方法（37C）的SOPL]，使用该室建立的测定系统进行丙氨酸氨基转移酶测定，比较加入5’-磷酸吡哆醛和未加5’-磷酸吡哆醛两种试剂检测结果的相关性，并探讨加入5’-磷酸吡哆醛对结果的影响。结果在参考实验室用加5’-磷酸吡哆醛和未加5’-磷酸吡哆醛的ALT试剂分别用参考方法检测22份临床标本，对检测结果进行比较，回归方程为y=0．854X＋1．3059，R2=0．9758，两种试剂的检测结果相关性良好，但两种方法的捡测结果差异有统计学显著性意义（P〈0．05）。用两种试剂分别检测14份混合血清，加入5’-磷酸吡哆醛ALT试剂的检测结果：平均值（x）=104．1536U／L；标准差（s）-1．5820；变异系数（CV）=1．5％，而未加5’-磷酸吡哆醛ALT试剂的检测结果：平均值（i）=88．7231U／L；标准差0）-0．5542；变异系数（CV）-0．6％。结论加5’-磷酸吡哆醛ALT试剂与未加5’-磷酸吡哆醛ALT试剂有较好的相关性，但两组结果的差异有统计学意义。加5’-磷酸吡哆醛ALT试剂测试结果比未加5’-磷酸吡哆醛ALT试剂测试结果约高20％，加5’-磷酸吡哆醛ALT试剂测试结果的变异系数比未加5’-磷酸吡哆醛ALT试剂测试结果的变异系数高。

5′-磷酸吡哆醛高分子衍生物的辅酶功效

5′-磷酸吡哆醛(PLP)与经β-硫酸酯乙砜基苯胺活化的聚乙二醇(PEG)或右旋糖酐(Dx)共价结合,得可溶性5′-磷酸吡哆醛衍生物。用PLP-PEG和PLP-Dx衍生物代替PLP作为谷氨酸脱羧酶的辅酶,谷氨酸脱羧酶活力回复率达18%左右;用甘氨酸封闭的PLP-PEG代替PLP作为辅酶,谷氨酸脱羧酶活力回复率达44%。

5’-磷酸吡哆醛的合成研究

本文对5’-磷酸吡哆醛的合成工艺进行研究,以离子液体为溶剂合成5’-磷酸吡哆醛,确定了最佳生产工艺参数,并对离子液体进行重复利用5次,该工艺具有操作简单、产率高、后处理方便等特点。

速率法检测加5’-磷酸吡哆醛血清丙氨酸氨基转移正常参考范围的研究

目的研究血清丙氨酸氨基转移酶（ALT）正常参考范围。方法正常人群360例，男、女各180例，仪器选用BT224半自动生化分析仪。结果37℃时，男性ALT参考值为31±24U／L，女性ALT参考值为36±24U／L。结论加磷酸吡哆醛的ALT试剂做出的正常参考值是理想的。

5’-磷酸吡哆醛影响血清丙氨酸氨基转移酶测定的研究

目的研究丙氨酸氨基转移酶(Alanine Aminotraasferase,ALT)检测试剂中5’-磷酸吡哆醛(Pyrldoxal-5’-phos-phate,PLP)的添加对血清ALT活力测定的影响。方法按IFCC推荐方法,自配ALT检测试剂(含PLP)和相应不添加PLP的检测试剂,分别用这两种试剂测定相同的新鲜临床标本,并对检测结果进行统计学比较。结果使用含PLP的IFCC配方试剂较不含PLP试剂的检测结果明显增高。结论健康人群和不同疾病人群由于个体差异,体内PLP含量不一,各组数据表明不含PLP配方试剂较IFCC配方检测结果明显偏低,因此两种配方的检测结果不能通过简单系数实现转换。

高效液相色谱分析法测定磷酸-5'-吡哆醛质量分数

建立了磷酸-5＇-吡哆醛的高效液相色谱定量检测方法。实验采用C18反相色谱柱（5μm,250 mm×4.6mm）;流动相为乙腈-水,二者体积比为（10-30）∶（70-90）;流速为1.0 m L/min;柱温为30℃;检测波长为388 nm;进样量为10.0μL（将测定主成分质量分数的进样样品用水溶解并稀释成0.2 g/L）。实验结果表明,磷酸-5＇-吡哆醛的线性方程为y=7 575.6x＋4 801.8,r=0.999 95,检出限为0.2 g/L。

Fingolimod protects against neurovascular unit injury in a rat model of focal cerebral ischemia/reperfusion injury

Recent research on the underlying mechanisms of cerebral ischemia indicates that the neurovascular unit can be used as a novel subject for general surveys of neuronal damage and protein mechanisms.Fingolimod(FTY-720)is a newly developed immunosuppressant isolated from Cordyceps sinensis that exhibits a wide range of biological activities,and has recently attracted much attention for the treatment of ischemic cerebrovascular diseases.In the current research,the role of FTY-720 and its possible mechanisms were assessed from an neurovascular unit perspective using a rat cerebral ischemia model.Our results revealed that FTY-720 markedly decreased infarct volume,promoted neurological function recovery,and weakened the blood-brain barrier permeability of ischemic rats.The protective roles of FTY-720 in ischemic stroke are ascribed to a combination of sphingosin-1-phosphate receptor-1 and reduced expression of sphingosin-1-phosphate receptor-1 in microvessels and reduction of interleukin-17A protein levels.These findings indicate that FTY-720 has promise as a new therapy for neurovascular protection and functional recovery after ischemic stroke.

The Isogene 1-Deoxy-D-Xylulose 5-Phosphate Synthase 2 Controls Isoprenoid Profiles, Precursor Pathway Allocation, and Density of Tomato Trichomes

Plant isoprenoids are formed from precursors synthesized by the mevalonate （MVA） pathway in the cytosol or by the methyl-D-erythritol 4-phosphate （MEP） pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms （DXS1, DXS2）. In tomato （Solanum lycopersicum）, the SIDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SIDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SIDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SIDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ^13C to ^12C natural isotope ratios. A compensatory up-regulation of SIDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.

Arginine methylation of ribose-5-phosphate isomerase A senses glucose to promote human colorectal cancer cell survival

Cancer cells remodel their metabolic network to adapt to variable nutrient availability. Pentose phosphate pathway(PPP) plays protective and biosynthetic roles by oxidizing glucose to generate reducing power and ribose. How cancer cells modulate PPP activity in response to glucose supply remains unclear. Here we show that ribose-5-phosphate isomerase A(RPIA), an enzyme in PPP, directly interacts with co-activator associated arginine methyltransferase 1(CARM1) and is methylated at arginine 42(R42). R42 methylation up-regulates the catalytic activity of RPIA. Furthermore, glucose deprivation strengthens the binding of CARM1 with RPIA to induce R42 hypermethylation. Insufficient glucose supply links to RPIA hypermethylation at R42, which increases oxidative PPP flux. RPIA methylation supports ROS clearance by enhancing NADPH production and fuels nucleic acid synthesis by increasing ribose supply. Importantly, RPIA methylation at R42 significantly potentiates colorectal cancer cell survival under glucose starvation. Collectively, RPIA methylation connects glucose availability to nucleotide synthesis and redox homeostasis.

心脏瓣膜置换术对5′-磷酸吡哆醛依赖的血清ALT、AST活力检测的影响

目的探讨心脏瓣膜置换术(HVR)对5′-磷酸吡哆醛依赖的血清ALT、AST活力检测的影响。方法收集接受单个瓣膜HVR的22例患者术前、术后即刻、术后第1天、术后第2天和术后第3天的血清标本,不同时间点血清ALT和AST活力均分别采用VITROS 5600干生化分析仪(干片法)和Beckman Coulter AU5800型全自动生化分析仪(试剂法)检测。计算两种方法检测ALT和AST活力的测量偏倚。测量不同时间点的血磷浓度。结果22例HVR患者术前肝功能及肾功能指标均在正常范围内。术后第2天和第3天ALT活力测量偏倚高于术后第1天(P<0.05)。术后第3天的AST活力测量偏倚高于术后即刻和术后第1天(P<0.05)。女性患者术后第3天ALT活力测量偏倚高于术后第1天(P<0.05)。术后各时间点血磷浓度均低于术前(P<0.05);术后第1天血磷浓度低于其余时间点(P<0.05)。结论HVR患者术后ALT活力测量偏倚增大可能与低浓度血磷有关。HVR患者术后采用含5′-磷酸吡哆醛的干片法检测可得到更准确的血清ALT活力,有助于病情变化的临床监测。

健康人群丙氨酸氨基转移酶及天门冬氨酸氨基转移酶参考区间初步调查

目的建立含5′-磷酸吡哆醛(PLP)的试剂测定血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)催化活性浓度的参考区间,并与不含PLP试剂方法的参考区间作比较。方法采用国际临床化学联合会(IFCC)推荐方法测定3 249名健康人血清ALT和AST,试剂采用罗氏公司试剂,分别用罗氏公司cfas定标液中加PLP及不加PLP的标准液进行定标。结果加入PLP的试剂测定健康人血清ALT、AST的结果与不加PLP试剂的测定结果有明显差异(P<0.000 1)。健康人群血清ALT、AST测定值男、女性之间有明显差异(P<0.000 1)。如以含PLP试剂检测值的95%分位作为参考值上限时,ALT为男性55 IU/L、女性36 IU/L;AST为男性42 IU/L、女性33 IU/L。如以不含PLP试剂检测值的95%分位作为参考值上限时,ALT为男性50 IU/L、女性32 IU/L;AST为男性35 IU/L、女性28 IU/L。二者均有随年龄升高的趋势。结论含PLP的ALT和AST试剂测定健康人ALT和AST活性升高。PLP能有效地激活ALT和AST活性,用含PLP的试剂测定ALT、AST活性可准确反映患者血液中ALT、AST的实际含量。推荐临床用含PLP的试剂作为测定ALT、AST的常规方法 。

离子液体中壳聚糖磷酸吡哆醛席夫碱衍生物的合成与表征

引言壳聚糖是自然界中唯一含有氨基的碱性多糖,具有多样的生物活性、极好的生物相容性、生物可降解性以及无毒性等特性,是一种新兴的生物功能材料[1-3]。磷酸吡哆醛是维生素B6参与多种代谢反应的一种活性形式。磷酸吡哆醛作为一种辅酶参与所有转氨基反应及一些氨基酸的脱羧及脱氢反应。研究表明,磷酸吡哆醛不但可以用于预防治疗高血压、心血管疾病及糖尿病等,还可用于治疗迟发性运动障碍和治疗难治性儿童期癫痫[4-5]。王涛等[6]以壳聚糖微球为载体,然后固定磷酸吡哆醛等制备得到了低密度脂蛋白亲和吸附剂。

烟草磷酸吡哆醛水解酶的分离纯化与表征

采用硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换、Sephadex G-100凝胶过滤和SP Sephadex C-25阳离子交换柱层析等步骤,对烟草磷酸吡哆醛水解酶进行了分离纯化。结果表明:该酶被纯化了119.6倍,得率为28.49%,经凝胶过滤和SDS-PAGE测得该酶的全分子量为49.6kDa,亚基分子量约为25kDa;该酶最适温度为50℃,最适反应pH为5.5;Mg2+、Ca2+、Mn2+等对该酶有激活作用,金属离子螯合剂EDTA对酶有抑制作用,加入Mg2+后抑制作用得到解除;在最适反应条件下,测得反应底物磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)的Km值分别为0.23mmol/L和0.56mmol/L。

常规试剂加入磷酸吡哆醛测定丙氨酸转氨酶催化活性浓度的研究

目的探讨在常规试剂中加入磷酸吡哆醛(PPA)测定丙氨酸转氨酶(ALT)催化活性浓度的可行性。方法用加与不加PPA两种常规方法分别比较肝脏患者、心脏患者、透析患者及其表观健康人群血清ALT活性。同时对两种常规方法在参考范围、线性范围、干扰物、精密度、试剂稳定性方面进行比较。结果加入PPA,健康人、急性肝炎、慢性肝炎、肝硬化、肝癌患者、透析后患者、心绞痛患者和AMI患者的ALT分别升高3.2%、13.2%、23.1%、26.3%、15.2%、35.0%、5.2%和4.0%。结论无论男性还是女性,加入PPA其ALT参考范围仍然在40U/L以内,与目前常规方法的参考范围无差别,临床检验中可以推广加PPA的常规方法。

脂质体催化其表面甘氨酸与磷酸吡哆醛形成希夫碱

利用脂质体的疏水微环境,完成了甘氨酸与磷酸吡哆醛的希夫碱反应。甘氨酸进行双烷基化修饰后,组装到脂质体中。利用肽脂质构建带正电荷的脂质体(pH=7.00),吸附带负电荷的磷酸吡哆醛,增强磷酸吡哆醛与脂质体上甘氨酸的伯胺基团作用,并通过脂质体疏水微环境促进脱水缩合过程。紫外光谱检测结果表明,单独的磷酸吡哆醛吸收峰为387 nm,吸附到脂质体后吸收峰红移到395 nm;当PLP与脂质体上甘氨酸反应转变为希夫碱后,最大吸收峰蓝移到336 nm;希夫碱结合铜离子后,最大吸收峰从336 nm再次红移,出现383 nm吸收峰。脂质体上新形成的希夫碱与乳酸脱氢酶竞争铜离子,解除铜离子对乳酸脱氢酶活性的抑制。

利用小麦胚芽制备GABA

探讨以小麦胚芽为原料,通过添加谷氨酸和吡哆素来实现γ-氨基丁酸(GABA)的生物转化,为研发食品来源的高安全性的GABA提供理论基础。以单因素实验(谷氨酸添加量、反应温度、反应时间和反应液p H)为基础,采用响应面分析法对制备GABA工艺参数进行优化,结果表明,最优反应条件为,谷氨酸添加量为80 g/L,反应液p H为5.6时,小麦胚芽中的谷氨酸脱羧酶酶活最高,并在反应温度为40℃反应4 h,最终可以生成GABA(35.42±2.19)mg/L(RSD=1.94%)。本研究建立的小麦胚芽制备GABA二次线性回归模型准确有效,优化制备工艺参数是可行的。