Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.     

天然氨基酸诱导野油菜黄单胞菌降解DSF-家族群体感应信号活性分析

【背景】野油菜黄单胞菌(Xanthomonas campestris pv. campestris,Xcc)是十字花科植物黑腐病的致病菌。Xcc中DSF (Diffusible signal factor)信号依赖的群体感应系统和RpfB介导的群体感应退出机制均与其致病性密切相关。【目的】分别检测18种氨基酸对DSF-家族群体感应信号分子合成的影响,为研发新型生物防治方法提供思路。【方法】添加不同浓度的氨基酸到ΔrpfC菌株XYS培养体系中,接种后不同时间点取样提取DSF信号分子,利用高效液相色谱法(High performance liquid chromatography,HPLC)分析DSF和BDSF浓度。【结果】18种氨基酸中,甲硫氨酸、色氨酸和胱氨酸能有效降低ΔrpfC菌株培养体系中DSF和BDSF水平,抑制效果与氨基酸浓度密切相关;3种氨基酸对DSF信号分子的抑制作用存在叠加效应;甲硫氨酸、色氨酸或胱氨酸不影响ΔrpfCΔrpfB双突变体菌株中DSF和BDSF水平。【结论】首次发现了甲硫氨酸、色氨酸和胱氨酸通过RpfB诱导Xcc退出群体感应状态。     

Molecular cloning,expression and characterization of three distinctive genes encoding methionine aminopeptidases in cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC6803

Methionine aminopeptidase, known to be encoded by single genes in prokaryotes, is a cobalt-dependent enzyme that catalyzes the removal of N-terminal methionine residues from nascent polypeptides. Three ORFs encoding putative methionine aminopeptidases from the genome of cyanobacterium Synechocystis sp. strain PCC6803, designated as slr0786 (map-1), slr0918 (map-2) and sll0555 (map-3) were cloned and expressed in Escherichia coli. The purified recombinant proteins encoded by map-1 and map-3 had much higher methionine aminopeptidase activity than the recombinant protein encoded by map-2. Comparative analysis revealed that the three recombinant enzymes differed in their substrate specificity, divalent ion requirement, pH, and temperature optima. The broad activities of the iso-enzymes are discussed in light of the structural similarities with other peptidase families and their levels of specificity in the cell. Potential application of cyanobacterial MetAPs in the production of recombinant proteins used in medicine is proposed. This is the first report of a prokaryote harboring multiple methionine aminopeptidases.Abbreviations map Gene encoding methionine aminopeptidase - MetAP Methionine aminopeptidase - eMetAP-Ia Escherichia coli methionine aminopeptidase type Ia - yMetAP-Ib Yeast methionine aminopeptidase type Ib - yMetAP-IIa Yeast methionine aminopeptidase type IIa - hMetAP-IIb Human methionine aminopeptidase type IIb - pfMetAP–IIa Pyrococcus furiosis methionine aminopeptidase type Ia - bst MetAP-Ia Bacillus stearothermophilus methionine aminopeptidase type Ia - c1MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-1 - c2MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-2 - c3MetAP-Ib Cyanobacterial methionine aminopeptidase type Ib, ncoded by map-3     

蛋氨酸脑啡肽对疟疾感染鼠一氧化氮产生的研究

探讨蛋氨酸-脑啡肽(M-ENK)对约氏疟原虫感染早期小鼠脾细胞分泌一氧化氮(NO)的免疫调节效应及相关机制。以不同浓度的M-ENK(单独或与LPS合用)与疟原虫感染后第3dBALB/c或DBA/2小鼠的脾细胞共同培养,通过Griess反应和ELISA法分别检测其上清中NO和IFN-γ的水平。脾细胞与浓度为10-10或10-11mol/L的M-ENK培养后,NO的分泌水平明显升高,并且与LPS联合刺激后其NO的产生进一步增强,但IFN-γ水平均未出现有意义的变化。提示浓度为10-10或10-11mol/L的M-ENK能够促进约氏疟原虫感染小鼠脾细胞NO的产生,该作用与IFN-γ活化巨噬细胞路径可能无关,同时M-ENK对LPS的刺激效应也具有强化作用。     

甲硫氨酸限制对骨肉瘤细胞增殖、凋亡及铁死亡的影响

摘要 目的：探讨甲硫氨酸限制对骨肉瘤细胞增殖,凋亡和铁死亡的影响。方法：使用细胞计数检测剥夺甲硫氨酸对三株不同的骨肉瘤细胞系增殖的影响;高通量测序（RNA-seq）分析甲硫氨酸限制后骨肉瘤细胞的转录组学变化;流式细胞术检测细胞周期,凋亡以及ROS,脂质ROS水平;Western blot检测铁死亡关键蛋白谷胱甘肽过氧化物GPX4以及铁死亡标志蛋白前列腺素内过氧化物合成Cox-2的表达。结果：甲硫氨酸限制显著抑制骨肉瘤细胞的增殖（P<0.001）;RNA-seq分析筛选出1719个差异表达基因,基因富集分析发现甲硫氨酸限制可显著激活铁死亡通路并显著抑制细胞周期通路;细胞实验证实甲硫氨酸限制将骨肉瘤细胞阻滞在G2M期,显著诱导凋亡细胞比例增加;同时,细胞内Cox-2的表达增加,GPX4的活性降低,活性氧和脂质活性氧积累,最终导致细胞死亡,且这种作用可以被铁死亡抑制剂Fer-1部分挽救。结论：本研究的结果为甲硫氨酸限制治疗骨肉瘤提供了一定的科学依据。     

Betaine lipids

Diacylglyceryltrimethylhomoserine (DGTS) and diacylglyceryl hydroxymethyltrimethyl-β-alanine (DGTA) belong to a new type of glycerolipids called betaine lipids, which are composed of diacylglycerol andN-permethylated hydroxyamino acids, that are linked by an ether linkage. Betaine lipids are widely distributed in lower plants and algae as well as in some non-photosynthetic microorganisms. They are no longer regarded as “unusual” lipids but are important constituents of membranes of lower organisms. The present state of knowledge of the phylogenetic distribution, the fatty acid composition, the thermal properties, and the biosynthesis of the bataine lipids is briefly summarized in this review in a perspective of a future search for the biological roles and activities of betaine lipids. Recipient of the Botanical Society Award of Young Scientists, 1991.     

Ammonia assimilation and metabolism byBeggiatoa alba

Beggiatoa alba B18LD was investigated for its pathways of ammonia assimilation. The increase in growth yields ofB. alba with excess acetate was linear from 0.1 to 2.0 mM ammonia.B. alba had strong glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, irrespective of the ammonia concentration in the medium. Glutamate dehydrogenase activity was not found, and alanine dehydrogenase (aminating) was observed only whenB. alba was grown at high (2.0 mM) ammonia. Methionine sulfoximine, an inhibitor of GS, inhibited growth ofB. alba irrespective of the ammonia concentration in the medium. Thus it appears the primary pathway for ammonia assimilation inB. alba is via the GS-GOGAT pathway at both low and high ammonia concentrations. Preliminary experiments were unable to discern if theB. alba GS is modified by covalent modification.Non-standard abbreviations GS Glutamine synthetase - GOGAT glutamate-oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - MSX methionine sulfoximine - GOT glutamate-oxaloacetate aminotransferase - GPT glutamate-pyruvate aminotransferase     

Radiation chemical studies of Gly-Met-Gly in aqueous solution

Abstract

Important biological consequences are related to the reaction of HO^? radicals with methionine (Met). Several fundamental aspects remain to be defined when Met is an amino acid residue incorporated in the interior of peptides and proteins. The present study focuses on Gly-Met-Gly, the simplest peptide where Met is not a terminal residue. The reactions of HO^? with Gly-Met-Gly and its N-acetyl derivative were studied by pulse radiolysis technique. The transient absorption spectra were resolved into contributions from specific components of radical intermediates. Moreover, a detailed product analysis is provided for the first time for Met-containing peptides in radiolytic studies to support the mechanistic proposal. By parallel radiolytical and electrochemical reactions and consequent product identification, the formation of sulfoxide attributed to the direct HO^? radical attack on the sulfide functionality of the Met residue could be excluded, with the in situ generated hydrogen peroxide responsible for this oxidation. LC–MS and high resolution MS/MS were powerful analytical tools to envisage the structures of five products, thus allowing to complete the mechanistic picture of the overall Met-containing peptide reactivity.     

Role of Glial Cells for the Basal and Ca2+-Dependent K+-Evoked Release of Transmitter Amino Acids Investigated by Microdialysis

The role of glial cells for the inactivation and synthesis of precursors for amino acid transmitters was studied in the brains of anesthetized rats in vivo using the microdialysis technique. The dialysis probes were inserted stereotactically into each neostriatum. One neostriatum was treated with the gliotoxin fluorocitrate, whereas the contralateral side served as a control. The basal efflux of amino acids, reflecting the extracellular level, was measured as well as the efflux during depolarization with 100 mM K+ in the dialysis stream. The potassium-evoked efflux of transmitter amino acids was calcium dependent and thus considered to reflect release from the transmitter pool. gamma-Aminobutyric acid (GABA) and glutamate release from the treated side was higher than the control value during the first 2-3 h, a result indicating an important role of glial cells in the inactivation of released transmitter. After 6-7 h with fluorocitrate, the release of glutamate was lower than the control value, a result indicating an important role of glial cells in the synthesis of precursors for the releasable pool of glutamate. The role of glutamine for the production of transmitter glutamate and GABA in vivo was further investigated by inhibiting glutamine synthetase with intrastriatally administered methionine sulfoximine. The release of gluatamate into the dialysis probe decreased to 54% of the control value, whereas the release of GABA decreased to 22% of the control value, a result indicating that glutamine may be more important for transmitter GABA than for transmitter glutamate.     

硫对水稻幼苗乙烯产生的影响

水稻幼苗缺硫培养12—15天,叶和根中的乙烯产生量明显降低,缺硫培养时间延长,根的乙烯产生量继续降低,叶的乙烯产生量升高。在缺硫幼苗中供给 SO_4~(2-)和0.1mmol/l 的半胱氨酸,乙烯产生量分别在36和12小时后升高到加硫幼苗的水平。供给0.1mmol/l 蛋氨酸,6小时后的乙烯产生量已高于加硫的幼苗。测定培养13天的幼苗叶片中游离氨基酸表明,缺硫幼苗其蛋氨酸和胱氨酸的含量降低。