化学合成的萤火虫荧光素的理化性质

萤火虫荧光素酶(luciferase,简称荧光素酶)发光系统是各类生物发光中研究得最深入,最透彻的一类,也是生物发光分析中应用得最广泛的一种。在国外,有关生物发光分析试剂都已商品化,而国内则很少,只有粗荧光素酶。荧光素是有关生物发光分析中必不可少的有机试剂。为此,我们研制了荧光素,并与国外产品作了比较。     

木质素合成研究进展

木质素是植物体内一类重要的大分子有机物,具有支持、保护和运输等生物学功能。然而,木质素的存在对植物资源的利用有一些不利影响,主要体现在导致造纸污染严重及影响牲畜对饲草的消化和吸收等。通过生物工程手段调控木质素的合成具有重要的环境保护价值和经济价值,已经成为国际上生命科学研究的热点之一。近年来,这一领域发展十分迅速。本文介绍了木质素生物合成途径,重点综述了调控木质素合成的手段及限速酶研究方面的最新进展,并提出存在的问题及展望。     

链球菌合成透明质酸的研究进展

透明质酸是一种具有极大商业价值的线性、高分子粘多糖,微生物的生物合成是透明质酸来源的首选。主要综述链球菌合成透明质酸的研究概况,分析了目前存在的主要问题及解决途径,并对其前景进行了展望。     

脏器微血管对荧光素钠通透性的实验方法

大鼠颈动脉注射１％ＦｌＮａ,荧光显微镜下活体观察肠系膜微血管血流状态及ＦｌＮａ的渗出情况,并在不同时间点经股动脉采血,测定血浆内ＦｌＮａ浓度随时间的变化,利用组织匀浆测定不同脏器中ＦｌＮａ的分布,再辅以冰冻切片进行观察。活体观察发现,ＦｌＮａ注入体内后,经微血管迅速向周围组织渗出,最后汇集于淋巴管,血浆及组织匀浆ＦｌＮａ浓度的测定表明,ＦｌＮａ浓度随时间的变化呈指数衰减,各脏器ＦｌＮａ的分布极不相同。冰冻切片也显示了同样的分布差别。这些结果表明,我们所建立的方法可直观、定量地反映ＦｌＮａ在微血管的通透情况。     

荧光素酶研究进展

荧光素酶（Luciferase）可以分为萤火虫荧光素酶和细菌荧光素酶两大类。萤火虫荧光素酶是分子量为60－64kD的多肽链,在Mg^2 、ATP、O2存在时,催化D－荧光素（D－Luciferin）氧化脱羧,发出光（λ=550-580nm）。细菌荧光素酶是含α、β两个多肽亚基的加单氧酶,它催化长链脂肪醛、FMNH2和O2的氧化反应,发出绿蓝光（λ=490nm）。萤火虫荧光素酶和细菌荧光素酶可分别从萤火虫和发光细菌中直接提前,亦可用基因工程的方法进行生产。荧光素酶催化的发光反应能用生物发光检测仪进行灵敏、快速检测,因此该酶有多方面的途径,如应用于快速检测、报告基因分析、有毒有害物质分析等。     

荧光素二醋酸酯染色显示蓝藻细胞生活力

使用荧光素二醋酸酯染色鉴定蓝藻细胞生活力较其他染色方法更可靠。文中介绍了该方法的具体操作步骤和注意事项。     

荧光素二醋酸酯染色显示蓝藻细胞生活力

使用荧光素二醋酸酯染色鉴定蓝藻细胞生活力较其他染色方法更可靠。文中介绍了该方法的具体操作步骤和注意事项。     

羧基荧光素脂质体的制备及其与病原菌的响应行为

【背景】由于目前临床上检测细菌感染的方法既耗时又昂贵,所以开发快速简便的鉴别方法势在必行。【目的】通过在脂质体膜材料中添加不同的稳定剂,研究它们对于脂质体稳定性的影响,并探究荧光素脂质体与病原菌之间的响应情况。【方法】以磷脂酰胆碱和胆固醇为主要原料,1,2-棕榈酰磷脂酰甘油[1,2-Dipalmitoyl-sn-glycero-3-phospho-(1?-rac-glycerol),DPPG]、十八胺和10,12-二十三二炔酸(10,12-Tricosadiyonic acid,TCDA)为稳定剂,采用薄膜分散-超声法制备羧基荧光素脂质体。利用病原菌能够分泌毒力因子造成脂质体渗漏的原理,将病原菌菌液和上清液分别与荧光素脂质体孵育,检测从脂质体渗漏的羧基荧光素的荧光强度,反映病原菌的毒性程度。【结果】DPPG和TCDA都能增加脂质体的稳定性,而十八胺的添加则导致脂质体稳定性的降低。与金黄色葡萄球菌ATCC25923和铜绿假单胞菌PAO1菌液和上清液共同孵育的脂质体荧光强度大量增加,与大肠杆菌DH5α和PBS缓冲液共同孵育的脂质体荧光强度几乎不变。【结论】能够分泌外毒素的金黄色葡萄球菌和铜绿假单胞菌都能对脂质体产生响应,而不分泌外毒素的大肠杆菌则不会对脂质体产生响应。     

大肠杆菌高效合成L-苯甘氨酸的研究进展

自然界存在着多种氨基酸,除用于蛋白质合成的20种外,大量用于合成具有生物活性的物质,广泛应用于食品、医药等多个领域.其中,非天然芳香族氨基酸L-苯甘氨酸作为一种重要的组成单元广泛的应用于盘尼西林、维吉霉素S、原始霉素Ⅰ等β-内酰胺类抗生素的生物合成当中.目前L苯甘氨酸主要通过化学法合成,但该方法合成收率低、污染大,且不...     

喷司他丁的合成及其生物合成机制研究进展

喷司他丁是一种核苷类抗生素,对腺苷脱氨酶有极强的抑制效果,在临床治疗恶性肿瘤方面具有广泛应用.但其生产成本高、市售价格昂贵,难以满足需求.近10年来,关于生物合成喷司他丁的研究主要集中在菌种选育、优化培养基组分与发酵工艺等方面.目前,尽管喷司他丁的生物合成机制得到了阐明,但生物合成喷司他丁方面的综述尚无.对此,文中综述...     

Luciferin derivatives in bioluminescence-enhanced enzyme immunoassays

Ultrasensitive bioluminescence immunoassays for the determination of peptides and proteins (illustrated with human urinary kallikrein, bradykinin and the determination of human urinary kallikrein antibody titres) have been developed. The usable ranges of the standard curves are from 5 pg to 5000 pg per litre. The relative intra-assay coefficients of variation of the tests were between 2% and 6%, and the inter-assay coefficients of variation between 4% and 12%.     

Emerging tools for bioluminescence imaging

Bioluminescence (BL) relies on the enzymatic reaction between luciferase, a substrate conventionally named luciferin, and various cofactors. BL imaging has become a widely used technique to interrogate gene expression and cell fate, both in small and large animal models of research. Recent developments include the generation of improved luciferase–luciferin systems for deeper and more sensitive imaging as well as new caged luciferins to report on enzymatic activity and other intracellular functions. Here, we critically evaluate the emerging tools for BL imaging aiming to provide the reader with an updated compendium of the latest developments (2018–2020) and their notable applications.     

Optimization of Fungal Luciferin Synthesis

Russian Journal of Bioorganic Chemistry - Current studies of fungal bioluminescent systems, including the purification of luciferase, require large quantities of the synthetic substrate luciferin...     

Measurements of ATP in mammalian cells

Levels of phosphorylated adenosine nucleotides, including the universal energy carrier adenosine 5(')-triphosphate (ATP) and its metabolites adenosine 5(')-diphosphate (ADP) and adenosine 5(')-monophosphate (AMP), define the energy state in living cells and are dependent mainly on mitochondrial function. In this article, we describe a method based on the luciferase-luciferin system used to measure mitochondrial ATP synthesis continuously in permeabilized mammalian cells and mitochondria isolated from animal tissues. We also describe a technique that uses the expression of recombinant targeted luciferase to report ATP content in different cell compartments. Finally, we describe an HPLC-based method for accurate measurement of ATP, ADP, and AMP in cultured cells and animal tissues.     

2-S-cysteinylhydroquinone is an intermediate for the firefly luciferin biosynthesis that occurs in the pupal stage of the Japanese firefly,Luciola lateralis

Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.     

Sensitive luciferin derived probes for selective carboxypeptidase activity

Highly selective luminescent probes, QLUC-TYR and LUC-GLU, for detection of carboxypeptidase activity were synthesized. Caged substrates were first cleaved by corresponding carboxypeptidases, and then they were activated by luciferase to emit light. Enzymatic activities of biologically important carboxypeptidases can be determined using this technology.     

Role of molecular oxygen in the bioluminescence of the firefly squid, Watasenia scintillans

The bioluminescence system in the "firefly squid," Watasenia scintillans, is described. The light-emitting components consist of luciferin (coelenterazine disulfate), a membrane-bound luciferase, ATP, Mg2+, and molecular oxygen. A hypothetical scheme is proposed for the light-emitting reaction.     

Synthesis and characterization of valyloxy methoxy luciferin for the detection of valacyclovirase and peptide transporter

An amino acid ester derivative of luciferin (valoluc) was synthesized to mimic the transport and activation of valacyclovir. This molecule was characterized in vitro for specificity and enzymatic constants, and then assayed in two different, physiologically-relevant conditions. It was demonstrated that valoluc activation is sensitive to the same cellular factors as valacyclovir and thus has the potential to elucidate the dynamics of amino acid ester prodrug therapies in a functional, high-throughput manner.     

Bioluminescence expression during the transition from mycelium to mushroom in three North American Armillaria and Desarmillaria species

Unlike most bioluminescent fungi, mycelia of Armillaria and Desarmillaria are constitutively bioluminescent while mature mushrooms are not. The absence of the luciferin, 3-hydroxyhispidin, and its precursor hispidin in mature mushrooms have been proposed to explain the lack of bioluminescence from Armillaria mushrooms. Using three North American species, A. gallica, A. mellea and D. tabescens (syn., Armillaria tabescens), we documented a decline in luminescence of ten fold during the transition from mycelia to, immature mushrooms (i.e., pins) for the two Armillaria species. As pins matured, luminescence declined by an additional two or three orders of magnitude. Lower initial luminescence of D. tabescens mycelia declined to negligible levels during mushroom development. Further, light production was localized in the gills and lower stipe of A. mellea mushrooms. The decline in luminescence during mushroom formation was reversed by addition of hispidin to stipe or gills which significantly enhanced luminescence by one and three orders of magnitude, respectively. We conclude that the modulation of Armillaria and Desarmillaria luminescence is achieved by luciferin availability early in mushroom development. However, since the temporal regulation of bioluminescence differs between Armillaria species and other genera, we conclude that bioluminescence in Armillaria is under unique selective pressures.