重组大肠杆菌合成聚(3-巯基丙酸)和聚(3-羟基丁酸-3-巯基丙酸)的研究

利用Clostridium acetobutylicum的丁酸激酶基因 (buk) 和磷酸转丁酰基酶基因(ptb),以及Thiocapsa pfennigii的PHA合成酶基因,设计了一条能够合成多种聚羟基烷酸的代谢途径,用构建的质粒转化大肠杆菌,获得了重组大肠杆菌菌株.前期的研究表明,在合适的前体物条件下,该重组大肠杆菌能够合成包括聚羟基丁酸、聚(羟基丁酸-戊酸)等多种生物聚酯[Liu and Steinbüchel, Appl. Environ. Microbiol. 66739-743].利用该重组大肠杆菌,通过生物催化作用合成了3-巯基丙酸的同型共聚酯,同时利用该重组大肠杆菌还获得了含3-巯基丙酸单体的多种异型共聚物.实验首先研究了3-巯基丙酸对大肠杆菌生长的影响,在此基础上优化了培养过程中添加3-巯基丙酸的时机和浓度,结果表明,在实验的条件下,细胞合成聚(3-巯基丙酸)可达6.7%(占细胞干重),合成聚(3-羟基丁酸-3-巯基丙酸)(分子中3-巯基丙酸3-羟基丁酸=31)可达24.3%.实验进一步研究了同时或分别表达以上3个基因的重组大肠杆菌合成聚合物的能力,结果表明只有当3个基因同时表达时才能合成聚合物,说明3个基因对合成过程是必须的,从而表明了合成途径是按照设计的路线进行的.还通过GC/MS、GPC、IR等手段对合成的化合物进行了定性的研究.聚(3-巯基丙酸)或聚(3-羟基丁酸-3-巯基丙酸)等聚酯属于一类新型生物聚合物,它在分子骨架中含有硫酯键,不同于聚羟基烷酸酯的氧酯键,从而具有显著不同的物理、化学、光学等性质和具有重要的潜在应用价值.     

14-3-3? and 14-3-3σ Inhibit Toll-like Receptor (TLR)-mediated Proinflammatory Cytokine Induction

Toll-like receptors (TLRs) are a group of pattern recognition receptors that play a crucial role in the induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral double-stranded RNA. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Limited studies have applied proteomics toward understanding the dynamics of TLR signaling. Herein, a proteomics approach identified 14-3-3ϵ and 14-3-3σ proteins as new members of the TLR signaling complex. Toward the functional characterization of 14-3-3ϵ and 14-3-3σ in TLR signaling, we have shown that both of these proteins impair TLR2, TLR3, TLR4, TLR7/8, and TLR9 ligand-induced IL-6, TNFα, and IFN-β production. We also show that 14-3-3ϵ and 14-3-3σ impair TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-mediated NF-κB and IFN-β reporter gene activity. Interestingly, although the 14-3-3 proteins inhibit poly(I:C)-mediated RANTES production, 14-3-3 proteins augment Pam₃CSK₄, LPS, R848, and CpG-mediated production of RANTES (regulated on activation normal T cell expressed and secreted) in a Mal (MyD88 adaptor-like)/MyD88-dependent manner. 14-3-3ϵ and 14-3-3σ also bind to the TLR adaptors and to both TRAF3 and TRAF6. Our study conclusively shows that 14-3-3ϵ and 14-3-3σ play a major regulatory role in balancing the host inflammatory response to viral and bacterial infections through modulation of the TLR signaling pathway. Thus, manipulation of 14-3-3 proteins may represent novel therapeutic targets for inflammatory conditions and infections.     

淋巴细胞活化基因-3敲除(Lag-3~(-/-))小鼠构建及初步表型分析

目的构建淋巴细胞活化基因-3(Lag-3)基因敲除小鼠,初步分析Lag-3基因敲除对小鼠表型影响,为后续Lag-3体内功能研究提供动物模型。方法利用CRISPR/Cas9技术结合受精卵显微注射构建敲除小鼠,通过分子鉴定筛选基因敲除阳性小鼠,利用RT-PCR和流式检测方法进一步验证Lag-3基因敲除小鼠体内Lag-3基因敲除效果;通过建立Con A诱导的肝损伤模型研究Lag-3基因在体内的功能。结果 PCR和产物测序结果显示,获得了exon 2缺失的Lag-3基因敲除小鼠;该基因敲除纯合子小鼠(Lag-3~(-/-))经刀豆蛋白(Con A)刺激后,小鼠心脏、脾、肺、巨噬细胞和淋巴结中仅能检测到Lag-3 mRNA本底表达信号,小鼠巨噬细胞、脾细胞和淋巴结中仅能检测到非常低数量的Lag-3阳性细胞。表型分析发现,Lag-3基因的缺失显著减少了Con A诱导的小鼠外周血、骨髓和脾CD3+T细胞的数量,减轻了Con A诱发的急性肝损伤情况。结论成功构建Lag-3基因敲除小鼠模型,肝损伤过程中的体内功能研究显示,Lag-3缺失可以显著缓解Con A引发的肝损伤的发生。     

RNA Modified Uridines VI: Conformations of 3-[3-(S)-Amino-3-Carboxypropyl]Uridine (acp3U) from tRNA and 1-Methyl-3-[3-(S)-Amino-3-Carboxypropyl]Pseudouridine (m1acp3Ψ) from rRNA

Abstract

The conformation of chemically synthesized acp³U is 60& 3′-endo, gauche⁺, whereas that of m¹acp³Ψ is 60& 2′-endo, gauche⁺. We conclude that the difference in conformation probably imparts important local structures to their respective tRNA and rRNA.     

14-3-3 蛋白

介绍了14－3－3蛋白的基本结构和功能,并简要概述了14－3－3蛋白在信号转导,细胞周期调控以及前体蛋白的折叠与运输过程中的作用机理。     

Microbial production of (R)-3-halolactic acid from (±)-3-halo-1,2-propanediol

Microbial oxidation of (±)-3-halo-1,2-propanediol was studied and it was found that several microorganisms accumulated (R)-3-halolactic acid. Geotrichum loubieri CBS 252.61 produced the most and gave optically pure (R)-3-chlorolactic acid and (R)-3-bromolactic acid from the corresponding diols.     

Bacillus megaterium WZ009催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯

以外消旋4-氯-3-羟基丁酸乙酯为唯一C源的富集培养筛选得到一株菌株WZ009,经16S rDNA测序鉴定为巨大芽胞杆菌(Bacillus megaterium)。B.megaterium WZ009静息细胞可以立体选择性催化(S)-4-氯-3-羟基丁酸乙酯水解和脱氯反应得到光学纯的(R)-4-氯-3-羟基丁酸乙酯(e.e.≥99%)和(S)-3-羟基-γ-丁内酯(e.e.≥95%)。笔者对B.megaterium WZ009不对称催化反应影响因素(温度、pH、中和剂、底物浓度、时间进程以及细胞重复利用)进行优化研究,确定了该反应体系最优条件:底物浓度200 mmol/L,中和剂氨水,pH 7.2,40℃反应12 h,转化率达到50.6%,底物对映体过量值为99.6%。该生物催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯过程具有良好的工业化应用前景。     

固定化天冬氨酸酶制备(R)-3-氨基丁酸

从芽孢杆菌Bacillus sp.YM55-1基因组中克隆得到天冬氨酸酶基因,以pET-28a(+)为载体构建天冬氨酸酶基因的表达载体pET-28a(+)-Asp,将天冬氨酸酶基因进行定点突变,在E.coli BL21(DE3)系统中实现了天冬氨酸酶的异源表达。利用重组的天冬氨酸酶,以氨水、(NH_4)_2SO_4为辅料,将底物巴豆酸转化为(R)-3-氨基丁酸。将天冬氨酸酶的工程菌制备成固定化细胞,通过反应条件的优化研究,提高底物的转化率。结果表明:天冬氨酸酶最适pH为9.0,最适反应温度为40℃。在此反应条件下,加入30 g/L固定化细胞,转化22 h,(R)-3-氨基丁酸质量浓度达到220 g/L,对映体过量值e.e._s≥99.95%,底物转化率达到98%,固定化细胞重复使用次数不低于24次。     

14-3-3蛋白研究进展

14-3-3蛋白是高度保守的、所有真核生物细胞中都普遍存在的、在大多数生物物种中由一个基因家族编码的一类蛋白调控家族。它几乎参与生命体所有的生理反应过程,人们在各种组织细胞中发现了各种不同的14-3-3蛋白。作为与磷酸丝氨酸／苏氨酸结合的第一信号分子,14-3-3蛋白在细胞的信号转导中起着至关重要的作用,尤其是它直接参与调节蛋白激酶和蛋白磷酸化酶的活性,被称为蛋白质与蛋白质相互作用的”桥梁蛋白”;它可以与转录因子结合形成复合体,调节相关基因的表达。一些研究表明,14-3-3蛋白调控机制的紊乱可以直接导致疾病的发生,在临床上14-3-3蛋白常常可以作为诊断的标志物。     

Quercetin 3-O-[6″-(3-hydroxy-3-methylglutaroyl)-β-galactoside] from blackberries

Quercetin 3-[6″-(3-hydroxy-3-methylglutaroyl)-galactoside] was isolated and identified from blackberries.     

TYRO3~(-/-)AXL~(-/-)MER~(-/-)小鼠骨髓细胞mRNA的差异表达谱

TYRO3、AXL和MER受体是受体酪氨酸激酶亚家族之一,广泛地表达在哺乳动物神经、免疫、生殖、血液等系统多种细胞中,促进细胞存活、增殖和分化。本研究利用基因芯片技术筛选基因敲除的TYRO3~(~(-/-))AXL~(-/-)MER~(-/-)小鼠骨髓细胞中差异性表达的m RNA,并进行生物信息学分析。与正常小鼠骨髓细胞相比,TYRO3~(-/-)AXL~(-/-)MER~(-/-)小鼠骨髓细胞中差异表达基因探针共1 363条,其中表达上调的基因探针499条、下调的基因探针864条。GO功能富集分析发现上调的基因主要参与免疫反应,下调的基因主要参与造血。KEGG Pathway富集分析发现上调的基因主要参与细胞粘附分子和抗原呈递等信号通路。Real-time PCR验证5个差异基因,与芯片中表达变化趋势一致。因此TYRO3、AXL和MER受体共同参与调控机体的免疫反应和血细胞分化。     

Discovery of (3S,4S)-3-methyl-3-(4-fluorophenyl)-4-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxyprop-2-yl)phenyl)pyrrolidines as novel RORγt inverse agonists

A novel series of cis-3,4-diphenylpyrrolidines were designed as RORγt inverse agonists based on the binding conformation of previously reported bicyclic sulfonamide 1. Preliminary synthesis and structure–activity relationship (SAR) study established (3S,4S)-3-methyl-3-(4-fluorophenyl)-4-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxyprop-2-yl)phenyl)pyrrolidine as the most effective scaffold. Subsequent SAR optimization led to identification of a piperidinyl carboxamide 31, which was potent against RORγt (EC₅₀ of 61 nM in an inverse agonist assay), selective relative to RORα, RORβ, LXRα and LXRβ, and stable in human and mouse liver microsomes. Furthermore, compound 31 exhibited considerably lower PXR Y_max (46%) and emerged as a promising lead. The binding mode of the diphenylpyrrolidine series was established with an X-ray co-crystal structure of 10A/RORγt.     

Stereocontrolled Facile Synthesis and Biological Evaluation of (3′S) and (3′R)-3′-Amino (and Azido)-3′-Deoxy Pyranonucleosides

This article describes the synthesis of (3 ′S) and (3 ′R)-3 ′-amino-3 ′-deoxy pyranonucleosides and their precursors (3 ′S) and (3 ′R)-3 ′-azido-3 ′-deoxy pyranonucleosides. Azidation of 1,2:5,6-di-O-isopropylidene-3-O-toluenesulfonyl-α-D-allofuranose followed by hydrolysis and subsequent acetylation afforded 3-azido-3-deoxy-1,2,4,6-tetra-O-acetyl-D-glucopyranose, which upon coupling with the proper silylated bases, deacetylation, and catalytic hydrogenation, obtained the target 3 ′-amino-3 ′-deoxy-β-D-glucopyranonucleosides. The desired 1-(3 ′-amino-3 ′-deoxy-β-D-allopyranosyl)5-fluorouracil was readily prepared from the suitable imidazylate sugar after azidation followed by a protection/deprotection sequence and reduction of the unprotected azido precursor. No antiviral activity was observed for the novel nucleosides. Moderate cytostatic activity was recorded for the 5-fluorouracil derivatives.     

Synthesis and biological evaluation of 3-(azolylmethyl)-1H-indoles and 3-(α-azolylbenzyl)-1H-indoles as selective aromatase inhibitors

This present study identifies a number of azolyl-substituted indoles as potent inhibitors of aromatase. In the sub-series of 3-(azolylmethyl)-1H-indoles, four imidazole derivatives and their triazole analogues were tested. Imidazole derivatives 11 and 14 in which the benzyl moiety was substituted by 2-chloro and 4-cyano groups, respectively, were the most active, with IC₅₀ values ranging between 0.054 and 0.050 μM. In the other sub-series, eight 3-(α-azolylbenzyl)-1H-indoles were prepared and tested. Compound 30, the N-ethyl imidazole derivative, proved to be an aromatase inhibitor, showing an IC₅₀ value of 0.052 μM. All target compounds were further evaluated against 17α-hydroxylase/C17,20-lyase to determine their selectivity profile.     

3-(2-萘基)-D-丙氨酸的制备

<正> 在生物界已发现40多种D-氨基酸,其生理作用正为人们逐渐认识。例如D-A.1-a—D-A.1a二肽是各种致病菌细胞壁肽聚糖的组成部分,D-丙氨酸是由丙氨酸消旋酶对L-丙氨酸消旋而来。如果丙氨酸消旋酶的活性被仰制,断绝了D-丙氨酸的来源,细菌     

Synthesis of methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-β-d-galactopyranoside and methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-O-β-d-galactopyranosyl-(1→6)-β-d-galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α-d-galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β-d-galactopyranoside (4) gave a fully acetylated (1→6)-β-d-galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α-d-galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β-d-galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β-d-galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Antiangiogenic properties of substituted (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol/one analogs and their derivatives

In the past half century research efforts have defined a critical role for angiogenesis in tumor growth and metastasis. We previously reported that inhibition of a novel target, ENOX1, by a (Z)-2-benzylindol-3-ylmethylene) quinuclidin-3-ol, suppressed tumor angiogenesis. The present study was undertaken in order to establish structure-activity relationships for quinuclidine analogs. The angiogenesis inhibiting activity of a series of substituted (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ols (1a-1k), (Z)-2-benzylindol-3-ylmethylene)quinuclidin-3-ones (2a-2h), (Z)-(±)-2-(1H/N-methyl-indol-3-ylmethylene)quinuclidin-3-ols (3a-3b), and substituted (Z)-(±)-2-(N-benzenesulfonylindol-3-yl-methylene)quinuclidin-3-ols and their derivatives (4a-4d) that incorporate a variety of substituents in both the indole and N-benzyl moieties was evaluated using Human Umbilical Vein Endothelial Cells (HUVECs) subjected to in vitro cell migration scratch assays, tubule formation in Matrigel, cell viability and proliferation assays. In total, 25 different analogs were evaluated. Based on in vitro cell migration scratch assays, eight analogs were identified as potent angiogenesis inhibitors at 10 μM, a concentration that was determined to be nontoxic by colony formation assay. In addition, this approach identified a potent antiangiogenic ENOX1 inhibitor, analog 4b.     

Arachidonic Acid Binds 14-3-3ζ, Releases 14-3-3ζ from Phosphorylated BAD and Induces Aggregation of 14-3-3ζ

Polyunsaturated fatty acids, like arachidonic acid, can bind proteins and affect their function. The 14-3-3 proteins bind phosphorylated sites on a diverse array of client proteins and, in this way, are involved in many intracellular signaling pathways. In this study, we used a novel approach to discover that 14-3-3ζ is able to directly bind arachidonic acid. Furthermore, arachidonic acid, at physiological concentrations, reduced the binding of 14-3-3ζ to phosphorylated BAD, an interaction that is important in regulating apoptosis. In addition, high concentrations of arachidonic acid caused the polymerization of 14-3-3ζ, an event observed in neurodegenerative disorders. Taken together, these results indicate that arachidonic acid directly interacts with 14-3-3ζ and that this interaction may be important in both normal and pathological cellular events. If so, then factors that mediate the release, metabolism and reacylation of arachidonic acid into membranes represent key points of regulation.     

植物14-3-3蛋白研究进展

14-3-3蛋白是真核生物中许多信号传导级联反应的主要调节分子,易于与具有磷酸化的丝氨酸和苏氨酸残基的靶蛋白互作进而调节碳氮代谢、三羧酸循环、莽草酸合成等多种生理过程中的多种酶活性。该文根据近年来国内外对14-3-3蛋白的研究进展,对植物中14-3-3蛋白的发现、基因鉴定、结构和功能以及14-3-3蛋白与其靶蛋白的互作机制进行综述,并对14-3-3蛋白的研究提出了进一步的展望。