Δ6-脂肪酸脱氢酶的分子生物学研究进展

包括γ-亚麻酸在内的多不饱和脂肪酸由于在人类健康中的重要作用而成为有价值的产品,目前市场上对γ-亚麻酸的需求持续增长,然而当前来源难以满足市场的需求,寻找合适的替代来源将有助于解决这一问题。Δ⁶-脂肪酸脱氢酶是多不饱和脂肪酸合成途径中的限速酶,这里从Δ⁶-脂肪酸脱氢酶的基因克隆、结构和功能的研究、系统进化和基因工程应用等方面探讨了Δ⁶-脂肪酸脱氢酶的研究进展。     

Purification and some properties of Δ2-isopentenylpyrophosphate:5′AMP Δ2-isopentenyltransferase from the cellular slime mold Dictyostelium discoideum

Δ²-Isopentenylpyrophosphate:5′AMP Δ²-isopentenyltransferase, which catalyzes the formation of isopentenyl-AMP from Δ²-isopentenylpyrophosphate and 5′AMP, was purified 6800-fold from the fruiting body of the cellular slime mold Dictyostelium discoideum using several separation procedures including 5′AMP^ox-redAH-Sepharose 4B affinity column chromatography. The final preparation was very unstable and lost its activity in a day. Various properties of the 1000-fold-purified enzyme preparation were examined. The molecular mass was 40,000 ± 2000 Da, as determined by Sephadex G-100 superfine gel filtration. The divalent metal ions Mn²⁺, Zn²⁺, and Mg²⁺ profoundly affected the enzymatic activity depending on their concentration, and also altered the optimum pH and temperature. Of the compounds tested, 5′AMP was the best acceptor of the isopentenyl group and, interestingly, ADP also served as a substrate, being 60–80% as effective as 5′AMP. Adenine, adenosine, and ATP were not substrates for this enzyme. Under the optimum assay conditions (pH 7.0, 1 mm Zn²⁺, and 25 °C) the K_m values for 5′AMP and Δ²-isopentenylpyrophosphate were 1.0 × 10^?7m and 2.2 × 10^?6m, respectively.     

Synthesis and antimicrobial activity of β-carboline derivatives with N2-alkyl modifications

β-Carbolines constitute a vast group of indole alkaloids and exhibit various biological actions. The objective of this study was to investigate the structure–activity relationships of β-carboline derivatives on in vitro inhibitory effects against clinically relevant microorganisms. A series of β-carboline dimers and their N²-alkylated analogues were therefore prepared and evaluated for their antimicrobial effects. Among these, a dimeric 6-chlorocarboline N²-benzylated salt exerted potent activity against Staphylococcus aureus at MICs of 0.01–0.05?μmol/mL. Our work highlights that N¹-N¹ dimerization and N²-benzylation significantly enhanced the antimicrobial effects of compounds.     

Investigations on the Δ23-, Δ24(28)- and Δ25-Sterols of zea mays

The sterols of Zea mays shoots were isolated and characterized by TLC, HPLC, GC/MS and ¹H NMR techniques. In all, 22 4-demethyl sterols were identified and they included trace amounts of the Δ²³-, Δ²⁴- and Δ²⁵-sterols, 24-methylcholesta-5,E-23-dien-3β-ol, 24-methylcholesta-5,Z-23-dien-3β-ol, 24-methylcholesta-5,25-dien-3β-ol, 24-ethylcholesta-5,25-dien-3β-ol and 24-ethylcholesta-5,24-dien-3β-ol. In the 4,4-dimethyl sterol fraction, cycloartenol and 24-methylenecycloartanol were the major sterol components but small amounts of the Δ²³-compound, cyclosadol, and the Δ²⁵-compound, cyclolaudenol, were recognized. These various Δ²³- and Δ²⁵-sterols may have some importance in alternative biosynthetic routes to the major sterols, particularly the 24β-methylcholest-5-en-3β-ol component of the C₂₈-sterols. Radioactivity from both [2-¹⁴C]MVA and [methyl-¹⁴C]methionine was incorporated by Z. mays shoots into the sterol mixture. Although 24-methylene and 24-ethylidene sterols were relatively highly labelled, the various Δ²³- and Δ²⁵-sterols contained much lower levels of radioactivity, which is possibly indicative of their participation in alternative sterol biosynthetic routes. (24R)-24-Ethylcholest-5-en-3β-ol (sitosterol) had a significantly higher specific activity than the 24-methylcholest-5-en-3β-ol indicating that the former is synthesized at a faster rate.     

A comparison of the disposition of 14C-Δ9-tetrahydrocannabinol and 3H-Δ9-tetrahydrocannabinol

Preliminary experiments suggested that total levels of radioactivity disappeared from the blood of male, Fischer rats much more rapidly following intragastric administration of ¹⁴C-Δ⁹-tetrahydrocannabinol (¹⁴C-THC) than ³H-THC. Collaborative experiments at the Stanford Research Institute (SRI) and the Research Institute on Alcoholism (RIA) verified and characterized the initial observations. In rats that had food available throughout the experiments, the concentrations of total ³H and ¹⁴C in fresh plasma reached a maximum at 2 – 4 hours after treatment with ³H-THC plus ¹⁴C-THC. Thereafter, ¹⁴C levels fell while ³H levels decreased very slowly or not at all. In fasted rats, peak plasma concentrations of both isotopes were not attained until about 8 hours following drug administration. The concentrations of ¹⁴C then decreased more rapidly than ³H. The differences between the plasma disappearance curves for ¹⁴C and ³H were not dependent upon the method of blood collection or the techniques of isotope counting. However, when plasma or whole blood samples were dried before radioisotope analysis, the difference between ¹⁴C and ³H concentrations was virtually abolished in fed and fasted rats. Experiments suggest that tritiated water, produced during the metabolism of ³H-THC, may be responsible for the prolonged maintenance of high ³H levels in the blood.     

A simple method for preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

Inhibition of cholesterol esterases by Δ1-tetrahydrocannabinol

Δ¹-Tetrahydrocannabinol was found to inhibit the action of esterases derived from rat adrenal and luteinized ovary on exogenous cholesteryl palmitate. The drug was effective at a dose of 3.2μM causing greater than 30% inhibition; at 16μM almost complete inhibition occured. These findings are similar to those we have recently reported with mouse Leydig cells (1) showing that this is an effect common to steroidogenic tissues and raising the possibility that a variety of endocrine effects of this drug may be due to direct action on these tissues.     

Δ12-脂肪酸脱氢酶基因在大肠杆菌中的表达*

把高山被孢霉 (Mortierellaalpina)和深黄被孢霉 (Mortierellaisabellina)的Δ¹²-脂肪酸脱氢酶基因亚克隆到大肠杆菌表达载体pET21a中 ,获得重组表达载体pMACL12和pMI CL12 ,并用氯化钙方法将重组表达载体转化到大肠杆菌BL21 (DE3)中。筛选阳性克隆进行培养 ,然后分离其细胞膜蛋白 ,并构建体外表达体系 ,同时加入外源性底物油酸进行表达。经气相色谱 (GC)分析表明 ,分别有 17.87%和 17     

Δ6-脂肪酸脱氢酶对n-6和n-3途径中脂肪酸底物的偏好*

添加α 亚麻酸作为底物 ,经半乳糖诱导 ,在含有少根根霉Δ⁶-脂肪酸脱氢酶基因的酿酒酵母总脂肪酸中检测到十八碳四烯酸的生成 ;同时添加亚油酸和α-亚麻酸时 ,检测到γ 亚麻酸和十八碳四烯酸生成 ,而且十八碳四烯酸的含量是γ 亚麻酸含量的3.81倍 ,表明在酿酒酵母中少根根霉Δ⁶-脂肪酸脱氢酶不仅能催化α-亚麻酸生成十八碳四烯酸 ,而且偏好n 3途径中的底物α 亚麻酸。同样 ,在改变少根根霉Δ⁶-脂肪酸脱氢酶基因的转译起始密码子周边序列后所构     

1,3-Bis(α-aminoisopropyl)benzene, meta-C6H4(CMe2NH2)2: An N,N-bridging and N,C,N-cyclometalating ligand

Saponification of the bis(carbamic acid ester) 1,3-C₆H₄(CMe₂NHCO₂Me)₂ (1), made by the addition of methanol to commercial 1,3-C₆H₄(CMe₂NCO)₂, yielded the meta-phenylene-based bis(tertiary carbinamine) 1,3-C₆H₄(CMe₂NH₂)₂ (2). Dinuclear [{(η⁴-1,5-C₈H₁₂)RhCl}₂{μ-1,3-C₆H₄(CMe₂NH₂)₂}] (3) resulted from the action of 2 on [{(η⁴-1,5-C₈H₁₂)Rh(μ-Cl)}₂] in toluene. Combination of 2 with PdCl₂ or K₂[PdCl₄] gave the dipalladium macrocycle trans,trans-[{μ-1,3-C₆H₄(CMe₂NH₂)₂}₂(PdCl₂)₂] (4) along with cyclometalated [{2,6-C₆H₃(CMe₂NH₂)₂-κN,κC¹,κN′}PdCl] (5). Substitution of PEt₃ for the labile chlorido ligand of 5 afforded [{2,6-C₆H₃(CMe₂NH₂)₂-κN,κC¹, κN′}Pd(PEt₃)]Cl (6). The crystal structures of the following compounds were determined: bis(carbamic acid ester) 1, ligand 2 as its bis(trifluoroacetate) salt [1,3-C₆H₄(CMe₂NH₃)₂](O₂CCF₃)₂, 2 · (HAc_f)₂, complexes 3 and 6, as well as 1,3-C₆H₄(CMe₂OH)₂ (the diol analogue of 2).     

少根根霉Δ6-脂肪酸脱氢酶基因在毕赤酵母中的表达

Δ⁶-脂肪酸脱氢酶是一种膜整合蛋白,也是多不饱和脂肪酸合成途径中的限速酶。在前期工作中,通过RT-PCR和RACE技术,从少根根霉NK300037中克隆到一个潜在编码Δ⁶-脂肪酸脱氢酶的序列,序列和功能分析结果表明该序列具有一个长度为1377bp、编码由458个氨基酸组成、大小为52kD的新的Δ⁶-脂肪酸脱氢酶基因。把少根根霉Δ⁶-脂肪酸脱氢酶基因(RAD6)亚克隆到表达载体pPIC3.5K,构建重组表达载体pPICRAD6,并转化到毕赤酵母菌株GS115进行表达。提取酵母细胞总脂肪酸和进行甲酯化,经气相色谱和气相色谱质谱连用分析表明,目的基因的编码产物能将C16∶1、C17∶1、C18∶1、亚油酸和α-亚麻酸在Δ⁶和7位间特异性脱氢而引入一个新的双键,生成更高不饱和的脂肪酸,该催化反应没有链长特异性,只有键位特异性。此外,按Kozak序列特点,改变目的基因转译起始密码子周边序列结构,并把改变后序列导入毕赤酵母GS115中进行功能表达分析,结果表明在毕赤酵母中这种改变同样能提高目的基因的表达水平。综合所有分析结果表明,巴斯德毕赤酵母更适合用来综合分析Δ⁶-脂肪酸脱氢酶基因的功能。     

深黄被孢霉Δ6-脂肪酸脱氢酶基因在转基因烟草中的表达

γ-亚麻酸(GLA)是人体和动物饮食中具有营养作用的重要的多烯不饱和脂肪酸,在大多数油料作物种子中不含有GLA,而只含有其前体物亚油酸,只有少数油料植物种子中含有GLA,如夜来香（Oenothera spp）,琉璃苣（Borago officinalis）等。Δ⁶脂肪酸脱氢酶可将亚油酸转化为γ亚麻酸,为了能够在传统的油料作物种子中产生GLA,我们将从深黄被孢霉中克隆的Δ⁶脂肪酸脱氢酶基因,与植物表达载体pGA643连接,构建了重组质粒pGAMICL6,将其通过农杆菌介导法,导入模式植物烟草中。经PCR和Southern杂交分析表明该基因已导入并整合到烟草的基因组中,Northern杂交结果表明该基因在转基因烟草的mRNA水平上获得表达。对转基因植株进行脂肪酸分析,结果显示,GLA和十八碳四烯酸(OTA)分别占总脂肪酸含量的19.7%和3.5%。     

高山被孢霉ATCC16266Δ6-脂肪酸脱氢酶基因在酿酒酵母中的表达

Δ⁶-脂肪酸脱氢酶是形成γ-亚麻酸的关键酶。从含有高山被孢霉Δ⁶-脂肪酸脱氢酶基因的重组质粒pTMACL6中,酶切出14kb的目的片段,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒pYMAD6,用醋酸锂方法转化到酿酒酵母的缺陷型菌株INCSc1中,在SC-Ura合成培养基中,选择得到酿酒酵母工程株YMAD6。在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体。通过GC-MS对酵母工程株进行脂肪酸色谱分析,结果表明,产生了31.6%的γ-亚麻酸。这是迄今为止,国内外Δ⁶-脂肪酸脱氢酶基因在酿酒酵母中表达量最高的报道。     

Omega chain methylated analogs of PGF2α and PGE2

Our previously published prostaglandin (PG) synthesis route, in which the ω-chain is added in the penultimate step, provides facile access to a wide variety of ω-chain variant PG analogs. Each series requires only the synthesis of the appropriate methylated acylphosphonate for the Emmons' condensation. The syntheses of analogs bearing the following methylation pattern are detailed: 15-Me; 17, 17-(Me)₂; 17, 17, 20-(Me)₃; 18, 18, 20-(Me)₃; 15, 18, 18, 20-(Me)₄; and 15-Ome-18, 18, 20-(Me)₃. The well-known 16, 16-dimethyl prostaglandins have also been prepared by this sequence. The synthesis of 16, 16-tetramethylene-PG analogs is also described.     

Synthesis and structural characterization of the molecular loop [cis-Mo2(N,N-di-p-anisylformamidinate)2]2(O2C-CHCCH-CO2)2

A new molecular loop composed of two quadruply bonded Mo₂(DAniF)₂ units (DAniF=N,N^′-di-p-anisylformamidinate) linked by two chiral allene-1,3-dicarboxylate anions has been prepared from the reaction of [cis-Mo₂(DAniF)₂(MeCN)₄](BF₄)₂ with the bis(tetraethylammonium) salt of allene-1,3-dicarboxylic acid. This compound, [cis-Mo₂(DAniF)₂]₂(O₂C-CHCCH-CO₂)₂ (1), has been characterized by X-ray crystallography and by ¹H NMR and UV-Vis spectroscopy. The molecule possesses a center of inversion and hence is meso. There is only weak electronic coupling between the two Mo₂ ⁴⁺ units as revealed by electrochemical measurements.     

Enzymic synthesis of glutamic acid γ-semialdehyde (Δ1-pyrroline-5-carboxylate) and N-acetyl-l-glutamic acid γ-semialdehyde: Isolation and characterization of their 2,4-dinitrophenylhydrazones

A protocol is given for the synthesis, by a simple enzyme preparation, of the l-isomers of glutamic acid γ-semialdehyde and N-acetylglutamic acid γ-semialdehyde. The isolation and physical and chemical properties of these products as the 2,4-dinitrophenylhydrazone derivatives are described. Spectral characteristics, NMR, ir, and visible, and melting points for these compounds are given.     

Identification of (η6-arene)ruthenium(II) protein binding sites in E. coli cells by combined multidimensional liquid chromatography and ESI tandem mass spectrometry: specific binding of [(η6- p -cymene)RuCl2(DMSO)] to stress-regulated proteins and to helicases

An automated multidimensional protein identification technology, which combines biphasic liquid chromatography with electrospray ionisation tandem mass spectrometry (MS/MS), was employed to analyse tryptic peptides from Escherichia coli cells treated with the antiproliferation agent [(η⁶-p-cymene)RuCl₂(DMSO)], where DMSO is dimethyl sulfoxide. MS/MS spectra were recorded for molecular ions generated by neutral loss of p-cymene from intensive peptide ions coordinated by the (η⁶-p-cymene)Ru^II fragment. Matching of the MS/MS spectra of the ruthenated peptides to spectra of proteins in the E. coli database enabled the identification of five protein targets for [(η⁶-p-cymene)RuCl₂(DMSO)]. One of these is the constitutive cold-shock protein cspC, which regulates the expression of genes encoding stress-response proteins, and three of the other targets, ppiD, osmY and sucC, are proteins of the latter type. The DNA damage-inducible helicase dinG was likewise established as a protein target. Aspartate carboxylate functions were identified as the probable Ru binding sites in cspC, ppiD and dinG, and threonine and lysine side chains in osmY and sucC, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The photodynamic activity of 131-[2′-(2-pyridyl)ethylamine] chlorin e6 photosensitizer in human esophageal cancer

A novel 13¹-pyridine substituted chlorin e₆ derivative (Chlorin A) was synthesized. It has characteristic long wavelength absorption at 664?nm and the emission wavelength at 667?nm. The generation rate of singlet oxygen of this compound is higher than Temoporfin. In vitro, Chlorin A showed higher phototoxicity against the human esophageal cancer cells than Temoporfin while with lower dark-toxicity. Its accumulation effect in mitochondria, lysosomes and endoplasmic reticulum was traced in subcellular localization tests. In flow cytometry obvious apoptosis cells were observed after 2?h irradiation. Significant in vivo photodynamic anti-tumor efficacy was also exhibited on mice bearing esophageal cancer. So Chlorin A could be suggested as a promising anti-tumor drug candidate in photodynamic therapy.     

Synthesis, X-ray crystal structure and magnetic study of the 1D {[Cu2(N,N,N′,N′-tetramethyl-ethylenediamine)2(μ-1,5-dca)2(dca)2]}n complex

Reaction of Cu(NO₃)₂ · 3H₂O, N,N,N′,N′-tetramethyl-ethylenediamine (L) and sodium dicyanamide (Nadca) in aqueous medium yields a complex the {[Cu₂L₂(μ-1,5-dca)₂(dca)₂]}_n complex, 1. Single crystal X-ray analysis reveals that complex 1 has a 1D infinite chain structure in which copper(II) ions are bridged by single dicyanamide anions in an end-to-end fashion. The coordination environment around copper(II) is distorted square pyramidal. Two among the four coordination sites of the basal plane are occupied by the nitrogen atoms of the diamine and two remaining sites are occupied by the terminal nitrogen atom of a bridging and of a monodentate dca anions. The fifth coordination site (apical) is occupied by a nitrogen atom from a bridging dca anion of an adjacent CuL(dca)₂ moiety, yielding the [Cu₂L₂(μ-1,5-dca)₂(dca)₂] dinuclear unit. Dimeric units are connected to each other by single μ-1,5-dicyanamido group to form infinite 1D chains which propagate parallel to the crystallographic c-axis. The variable temperature magnetic susceptibility measurements evidenced weakly antiferromagnetic interactions (J = −0.26 cm⁻¹) in {[Cu₂L₂(μ-1,5-dca)₂(dca)₂]}_n, 1.