Malondialdehyde and 4-Hydroxy-2-Nonenal in Plant Tissue Cultures: LC-MS Determination of2, 4-Dinitrophenylhydrazone Derivatives

The cytologically active secondary lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) have been detected as their2, 4-dinitro-phenylhydrazone (DNP) derivatives in plant tissue cultures using LC-MS. This paper reports, for the first time, the use of LC-MS methodology to definitively identify 4-hydroxy-2-nonenal in plants. Limits of detection for the two derivatives are approximately 5pmol (1.2 × 10^-9g; 1μM) and O.1pmol (3 × 10^-l1g; 20nM) respectively. Mass spectrometer response was linear in the range from 2-200μM DNP-MDA and 0.02-10μM DNP-HNE.

This methodology has been used to assess the formation of aldehydic secondary lipid peroxidation products in dedifferentiated callus cultures of Daucus carota. The finding that profiles of MDA and HNE can be correlated with embryogenic competence is of considerable interest as oxidative status has already been implicated as a regulatory factor in animal development.     

西洋参组织培养及MgSO4含量对培养结果的影响

本文报道了西洋参不同生长期、材料不同部位、生命活动代谢盛期愈伤组织的诱导及西洋参组织培养;细胞液体培养时,ＭｇＳＯ４含量对其生长状况、生长速度的影响。结果表明,当ＭｇＳＯ４含量为９２．５ｍｇ／ｌ时,与ＭＳ培养基中ＭｇＳＯ４含量３７０ｍｇ／ｌ相比,嫩茎愈伤组织生长速度提高１．３３倍,液培细胞生长速度提高１．２７倍。     

Induction of secondary metabolite production in transformed callus of Ammi majus L. grown after electromagnetic treatment of the culture medium

High-performance liquid chromatography analysis of crude chloroform and methanol extracts as well as methanol preparations subjected to enzymatic hydrolysis indicated two-fold higher accumulation of umbelliferone in transformed Ammi majus L. callus grown on medium exposed to ADR-4^® (energy stimulator plate) in comparison to the control cultures. Elicitation with ADR-4^® induced also two times higher accumulation of bergapten. Three different techniques: Soxhlet extraction, ultrasonication and accelerated solvent extraction (ASE) applied for the extraction of coumarins, furanocoumarins and corresponding glycosides from transformed callus of A. majus elicited by ADR-4^® plates were compared. ASE is shown to be the most efficient extraction method.     

Isolation of 4-acetyl-benzoxazolin-2-one (4-ABOA) and diferuloylputrescine from an extract of gibberella ear rot-resistant corn that blocks mycotoxin biosynthesis, and the insect toxicity of 4-ABOA and related compounds

Bioassay-directed fractionation of an extract of a gibberella ear rot-resistant corn hybrid resulted in the isolation of a fraction that inhibited mycotoxin production, but not growth, of Fusarium culmorum. The fraction contained diferuloylputrescine and a new allelochemical 4-acetyl-benzoxazolin-2-one (4-ABOA). This report describes the isolation of the bioactive fraction and tests of the effect of 4-ABOA on 3-acetyl deoxynivalenol (3ADON) and aflatoxin production. In addition, data are reported on the effects of 4-ABOA and related compounds on a number of insect species and some preliminary studies on the mechanism of insect toxicity. The effect of 4-ABOA on F. culmorum and Apergillus flavus was similar. Production of toxin was reduced without a concomitant reduction of growth. A 50% inhibition of 3ADON production occurred at ca. 4 μM and 0.12 mM for alfatoxin. 4-ABOA was slightly toxic to Spodoptera frugiperda.     

Toll样受体4(TLR4)基因剔除小鼠构建及初步表型分析

目的 利用CRISPR/Cas9技术构建Toll样受体4(TLR4)基因敲除小鼠模型,并观察突变小鼠对革兰氏阴性细菌脂多糖(LPS)刺激响应的变化。方法 针对TLR4基因外显子2设计并合成1对sgRNA片段,与编码Cas9的mRNA混合后通过受精卵显微注射方法,建立TLR4基因敲除小鼠,通过繁育获得基因敲除纯合子小鼠(TLR4^-/-小鼠);通过LPS刺激,分析TLR4^-/-小鼠对炎症应激的反应情况,并在分子和病理水平上和野生型对照(WT)进行比较。结果 PCR及测序检测表明TLR4基因外显子2在小鼠基因中被成功敲除;给予LPS刺激后,IL1β、IL6、MyD88、iNOS及TNFa等炎症因子的表达在野生型小鼠的心、肝和肺组织中显著上调,而在TLR4^-/-小鼠中则几乎没有变化;血生化指标显示LPS刺激后WT小鼠血清中的尿素(Urea)和肌酐(Cre)水平显著升高,而TLR4^-/-小鼠刺激前后无显著变化,病理分析同样发现TLR4^-/-小鼠能够抵抗LPS对肾组织的损伤。结论 利用CRISPR/Cas9技术成功构建了TLR4基因剔除小鼠模型,TLR4的缺失能够降低IL1β、IL6、MyD88、iNOS及TNFa炎症因子对LPS刺激的响应,抑制LPS引起的炎症反应及对组织的损伤。     

An expeditious synthesis of 4-hydroxy-2E-nonenal (4-HNE), its dimethyl acetal and of related compounds

The facile one step synthesis of 4-hydroxy-2(E)-nonenal and its dimethyl acetal via a cross-metathesis reaction between commercially available octen-3-ol and acrolein or its dimethyl acetal is reported. The method was extended to the synthesis of C₆ and C₁₂ 4-hydroxy-2(E)-enals, their dimethyl acetal and of the 4-hydroxy-2(E)-nonenoic acid (4-HNA).     

New 4-O-substituted xylosyl units in the xyloglucan from leaves of Hymenaea courbaril

A homogeneous fucogalactoxyloglucan, isolated from the leaves of Hymenaea courbaril, was analysed by methylation-GC–MS. These procedures involved derived partially O-methylated alditol acetates and acetylated aldononitriles, which demonstrated the presence of both 2-O- and 4-O-substituted Xylp units in the side-chains. The presence of the unusual, latter structure was confirmed by 2D NMR spectroscopy with a correlated HMQC C-4/H-4 signal at δ 77.8/3.73. A similar 4-O-substituted xylosyl structure was present in a decasaccharide Glc₄Xyl₃Gal₂Fuc obtained via endo-glucanase treatment of the polysaccharide, which gave rise to a molecular ion with m/z 1555 (ESI-MS, Na⁺ form).     

84K杨4CL3/4CL5基因克隆及生物信息学分析

木质素是木材的重要组成成分,主要起机械支持、水分运输及防御病虫害等作用。4CL酶是控制木质素合成途径中的关键酶之一。利用PCR技术从84K杨幼叶cDNA中克隆得到Pag4CL3/4CL5基因,GenBank 登录号分别为MK183033（Pag4CL3）及MK183034（Pag4CL5）。利用生物信息学软件,对这两个基因的功能和特征进行分析。结果发现84K杨中4CL3/4CL5蛋白与毛果杨中4CL3/4CL5蛋白相似度高达97%;此外,均含有SSGTTGLPKGV和GEICIRG两个保守基序;亚细胞定位预测显示Pag4CL3/4CL5蛋白主要定位在内质网中,推测可能是一种膜蛋白;蛋白的亲水性预测Pag4CL3/4CL5均为亲水性蛋白。通过qRT-PCR分析Pag4CL3/4CL5在不同组织部位中的表达差异,结果发现Pag4CL3在叶及茎中表达量较高,在根和顶芽中表达量较低;Pag4CL5在叶及根中表达量较高,在茎和顶芽中表达量较低,两个同源基因表达具有组织部位的差异性。Pag4CL3和Pag4CL5可能在叶中共同行使功能,Pag4CL5主要在根中行使功能,Pag4CL3主要在茎中行使功能。     

马铃薯StNPR4基因的克隆与功能分析

为探究StNPR4基因在马铃薯（Solanum tuberosum）中应对生物胁迫和非生物胁迫的功能,本研究通过克隆StNPR4的CDS序列和启动子序列,进行生物信息学分析;利用qRT-PCR进行组织表达特异性分析;同时构建了由其自身启动子驱动的StNPR4双元表达载体,转化马铃薯获得了转基因马铃薯,研究转基因马铃薯对水杨酸、致病疫霉和高盐胁迫的响应。结果显示：StNPR4具有典型的NPR1家族的功能结构域,启动子上具有响应于生物胁迫和非生物胁迫的顺式作用元件。StNPR4在叶中的表达量最高;StNPR4受SA诱导表达,且在转基因植株中的诱导表达程度高于对照;转基因马铃薯增强了对致病疫霉的抗性,在高盐胁迫下生根率更高。说明StNPR4不仅在马铃薯生物胁迫中发挥重要作用,而且在非生物胁迫中也扮演着重要角色。     

Synthesis and HMG CoA reductase inhibition of 4-thiophenyl quinolines as potential hypocholesterolemic agents

A series of novel 4-thiophenyl quinoline-based mevalonolactone derivatives were synthesized from ethyl 6,7,8-trisubstituted-4-chloro-quinoline-3-carboxylates by several reactions and evaluated for their ability to inhibit the rat HMG CoA reductase in vitro. It was found that substitution with a variety of thiophenyl groups at position 4 in quinoline resulted in retention or enhancement of the inhibition and the preferable groups were 4-isopropyl-thiophenyl and 3-methoxy-thiophenyl. (4R,6S)-6-[(E)-2-(6,7,8-trifluoro-4-isopropylthiophenyl-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (A16) and (4R, 6S)-6-[(E)-2-(6-fluoro-4,7-di-(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (A23) were approximately three times more potent than rosuvastatin or pitavastatin in inhibiting HMG CoA reductase and selected as the hypocholesterolemic candidates for further evaluation.     

不同浓度新型分裂素PBU诱导的尾巨桉愈伤组织中miRNA396及CKX基因表达差异研究

尾巨桉愈伤组织的生长分化受内源激素影响,而miRNA396是一个调控植物叶片与根系生长发育的小RNA,与细胞分裂素的合成相关,CKX是负责调控细胞分裂素的氧化酶基因。为探讨miRNA396与CKX基因对尾巨桉愈伤组织生长发育的调控作用,以尾巨桉基因组为模板,进行PCR扩增及测序分析尾巨桉基因中的miRNA396序列,用不同PBU细胞分裂素浓度培养下的尾巨桉愈伤组织RNA逆转录的cDNA为模板,通过荧光定量PCR,测定不同PBU浓度处理的尾巨桉愈伤组织中miRNA396及CKX的表达差异。结果表明,相对于0.5 mg·^L(-1)PBU处理的桉树愈伤组织,1 mg·^L(-1)PBU处理的桉树愈伤组织miRNA396及CKXA、CKXB和CKXF表达量显著下调,差异达到极显著水平,CKXC、CKXD和CKXE均上调,但只有CKXC相对表达量达到极显著水平;2 mg·^L(-1)PBU处理的尾巨桉愈伤组织miRNA396A、CKXD、CKXE和CKXF表达量均下调,差异达到极显著水平,其他CKX表达量均上调,CKXA相对表达量差异...     

白桦BpZFP4基因启动子克隆和逆境响应元件功能分析

前期研究发现白桦锌指蛋白BpZFP4基因响应多种非生物逆境胁迫。为了深入研究其调控机制,本研究利用染色体步移技术克隆了该基因上游1 360 bp的启动子区域,序列分析结果表明该区域含有多个逆境响应顺式作用元件、激素响应元件、光响应以及病原菌和损伤响应元件等。在此基础上,构建了BpZFP4启动子5'-端一系列缺失片段融合GUS报告基因的表达载体。通过对系列缺失突变体在正常条件以及渗透胁迫、盐胁迫和脱落酸（ABA）处理条件下的GUS基因表达分析,鉴定得到BpZFP4启动子对干旱、盐和ABA应答响应的主要调节区域及可能的顺式作用元件。本研究结果为进一步探究BpZFP4基因应答非生物胁迫和信号分子刺激的调控机理提供了重要的理论依据。     

Photosynthetic carbon metabolism in Panicum milioides, a C3-C4 intermediate species: Evidence for a limited C4 dicarboxylic acid pathway of photosynthesis

Panicum milioides, a naturally occurring species with C₄-like Kranz leaf anatomy, is intermediate between C₃ and C₄ plants with respect to photorespiration and the associated oxygen inhibition of photosynthesis. This paper presents direct evidence for a limited degree of C₄ photosynthesis in this C₃-C₄ intermediate species based on:

1. (a) the appearance of 24% of the total ¹⁴C fixed following 4 s photosynthesis in ¹⁴CO₂-air by excised leaves in malate and aspartate and the complete transfer of label from the C₄ acids to Calvin cycle intermediates within a 15 s chase in ¹²CO₂-air;

2. (b) pyruvate- or alanine-enhanced light-dependent CO₂ fixation and pyruvate stimulation of oxaloacetate- or 3-phosphoglycerate-dependent O₂ evolution by illuminated mesophyll protoplasts, but not bundle sheath strands; and

3. (c) NAD-malic enzyme-dependent decarboxylation of C₄ acids at the C-4 carboxyl position, C₄ acid-dependent O₂ evolution, and ¹⁴CO₂ donation from [4-¹⁴C]C₄ acids to Calvin cycle intermediates during photosynthesis by bundle sheath strands, but not mesophyll protoplasts.

However, P. milioides differs from C₄ plants in that the activity of the C₄ cycle enzymes is only 15 to 30% of a C₄ Panicum species and the Calvin cycle and phosphoenolpyruvate carboxylase are present in both cell types. From these and related studies (Rathnam, C.K.M. and Chollet, R. (1979) Arch. Biochem. Biophys. 193, 346–354; (1978) Biochem. Biophys. Res. Commun. 85, 801–808) we conclude that reduced photorespiration in P. milioides is due to a limited degree of NAD-malic enzyme-type C₄ photosynthesis permitting an increase in pCO₂ at the site of bundle sheath, but not mesophyll, ribulosebisphosphate carboxylase-oxygenase.     

A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli. In this study, a rad4 mutant that can be grown in E. coli was isolated. This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E. coli when expressed alone. The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair. The smallest Rad4 fragment that is toxic to E. coli consists of 336 amino acids with a calculated pI = 9.99.     

Inhibition of cytokinin-induced plant growth by jasmonic acid and its methyl ester

Effects of jasmonic acid, methyl jasmonate and ABA on cytokinin-induced soybean ( Glycine max Merrill cv. Kingen No. 1) callus growth and radish ( Raphanus sativus L. cv. Comet) cotyledon growth were studied. Jasmonic acid and methyl jasmonate were powerful inhibitors of kinetin- or N-phenyl-N'-(2-chloro-4-pyridyl)urea-induced soybean callus growth, the former being more effective than the latter, especially at low concentrations (0.45 to 4.5 μ M ). These compounds could completely eliminate kinetin- or N-phenyl-N'-(2-chloro-4-pyridyl)urea-induced soybean callus growth at 45 μ M . At low concentrations ABA had no effect but at 450 μ M it completely eliminated callus growth induced by kinetin. Of the compounds tested ABA was the most powerful inhibitor of radish cotyledon growth in the presence or absence of kinetin. Jasmonic acid and methyl jasmonate were much less effective than ABA in this assay system.     

6-(2, 3, 4-Trihydroxy-3-methylbutylamino)purine,a cytokinin with high biological activity

6-(2, 3, 4-Trihydroxy-3-methylbutylamino)purine, isolated from the oxidation of cis- zeatin with potassium permanganate, has been identified by ¹H NMR and high resolution mass spectrometry. Its activity as a cell division factor, when examined by the soybean callus assay in the concentration range 10^?11–10^?5 M, equalled that of the parent compound.     

进境美国大豆中值得关注的真菌Diaporthe novem

采用常规平板分离法,从一批进境的美国大豆样品中获得1株可疑的间座壳属菌株MDD57.经形态学观察发现,该菌株在PDA培养基上产生分生孢子器,且同时产生大量α型和β型分生孢子,未见有性阶段.经ITS和tef1α基因扩增、核酸序列比对分析,发现该菌株同GenBank中2株Diaporthe novem菌株的基因序列同源性达...     

An improved cytokinin bioassay using cultured soybean hypocotyl sections

This paper describes a modified soybean (Glycine max) tissue culture bioassay for cytokinins. Soybean hypocotyls were grown under sterile conditions and sliced into 1-mm sections. Sections were cultured for 5,9,13, or 22 days on a callus medium with zeatin or other cytokinins. The fresh weight of sections increased with the cytokinin concentration from 0.0005 to 1 mum zeatin; 2-fold concentration differences were readily distinguishable at 9 days. The assay should prove to have several advantages over the conventional soybean callus bioassay including convenience, lower variability between tissue samples, and improved resolution. Its specificity is comparable to that of the soybean callus bioassay.     

Comparison of kinetic properties between MSS4 and Rab3A GRF GDP/GTP exchange proteins

The kinetic properties of MSS4 are studied in comparison with those of Rab3A GRF. MSS4 stimulates the dissociation of [³H]GDP from the lipid-modified and lipid-unmodified forms of Rab3A to the same extent, although Rab3A GRF is more effective on the lipid-modified form than on the lipid-unmodified form. Both MSS4 and Rab3A GRF are inactive on other Rab/Sec/Ypt family members including at least Rab2, Rab5, and Rab11. Rab GDI inhibits the MSS4 and Rab3A GRF effects on the lipid-modified form of Rab3A, but the doses of Rab GDI necessary for this inhibitory effect on Rab3A GRF are lower than those on MSS4. Moreover, Rab GDI slightly inhibits the Rab3A GRF effect on the lipid-unmodified form of Rab3A, but does not affect the MSS4 effect on the lipid-unmodified form of Rab3A. These results suggest that MSS4 and Rab3A GRF are different GDP/GTP exchange proteins for Rab3A.