高效液相色谱法测定虫草发酵菌丝粉中腺苷的含量

本文讨论了高效液相色谱法测定虫草发酵菌丝粉中腺苷含量的方法。实验表明:在ODSHypersil(5μm,125×4mm)色谱柱上,以0.06mol/L磷酸二氢钾-甲醇-四氢呋喃(150:10:1.5)为流动相,检测波长260nm,腺苷的检出限为50.25μg/mL,成功测定了不同批号虫草发酵菌丝粉中腺苷的含量,平均回收率为101%,分析方法灵敏、准确、重现性好。     

一株野生脉孢菌的鉴定及其纤维素乙醇发酵潜力研究

鉴定了一株野生型脉孢菌并研究了使用玉米芯为原料时的纤维素乙醇发酵潜力,对其形态特征和生理生化特性研究表明,该菌与脉孢菌属(Neurospora)菌株的特征一致;对其18S-28S rDNA的ITS区段进行序列分析,通过构建系统进化树和分析系统发育关系,确定该菌为脉孢菌属(Neurospora)的间型脉孢菌(Neurospora intermedia)。该脉孢菌能在以玉米芯粉和葡萄糖为碳源的液体培养基中快速生长,从接入孢子到形成有乙醇发酵能力的菌丝只需48h的时间。以10g·L-1的玉米芯配成培养基,分别进行脉胞菌、酿酒酵母直接乙醇发酵和脉孢菌与酿酒酵母共发酵试验。结果表明脉孢菌对玉米芯进行直接乙醇发酵时,乙醇产量为4.37g·L-1(产醇底物还包括培养菌丝时未用完的部分碳源),用酵母菌进行直接乙醇发酵时,乙醇产量仅为0.18g·L-1。而当培养脉孢菌菌丝后接入酿酒酵母同时糖化发酵时,得到的乙醇浓度提高到5.53g·L-1,因而,使用脉胞菌与酿酒酵母的同时糖化发酵更有利于玉米芯的纤维素乙醇发酵。     

番茄早疫病菌对7种杀菌剂的敏感性比较及其对苯醚甲环唑的敏感性基线建立

采用菌丝生长速率法和孢子萌发法测定并比较了7种杀菌剂对番茄早疫病菌的室内抗菌活性,结果表明.芳烃类杀菌剂百菌清抑制病菌孢子萌发的活性最高,同时对病菌菌丝生长也有较高活性;苯并咪唑类杀菌剂多菌灵对菌丝生长和孢子萌发的活性均较低;二甲酰亚胺类杀菌剂腐霉利、异菌脲对番茄早疫病菌的菌丝生长和孢子萌发两个阶段均有较高活性;三唑类杀菌剂苯醚甲环唑虽然对孢子萌发的抑制活性较低,但其对菌丝生长的活性最高;甲氧基内烯酸酯类杀菌剂醚菌酯和嘧菌酯在离体条件下对菌丝生长的抑制活性较低,但对孢子萌发有较高活性.采用菌丝生长速率法测定了从湖北和山东采集的60株番茄早疫病菌对苯醚甲环唑的敏感性,结果表明这些菌株对苯醚甲环唑均敏感,未出现对苯醚甲环唑敏感性明显下降的抗药菌株,因此,这些菌株的平均EC5o值为(0.3050±0.1361)mg/L,可以作为番茄早疫病菌对苯醚甲环唑的敏感性基线.     

香蕉弯孢霉叶斑病菌的药物敏感性测定

选用5种杀菌剂对香蕉弯孢霉叶斑病菌菌丝生长进行了药物敏感性测定.药物敏感性试验结果表明,6%戊唑醇ME、25%丙环唑EC、25%咪鲜胺EW、25%戊唑醇EC和25%苯醚甲环唑EC对香蕉弯孢霉叶斑病菌菌丝生长均有较好的抑制效果,其中25%咪鲜胺EW和25%苯醚甲环唑EC的抑菌效果最好.     

蜜环菌菌索抗氧化作用初步研究

以芦丁为对照,从活性氧自由基清除效果及对DPPH自由基清除能力等方面对云南昭通蜜环菌乙醇提取物的抗氧化活性进行实验研究。结果表明:云南昭通蜜环菌乙醇提取物对光照核黄素自氧化产生的超氧阴离子、Fenton反应产生的羟自由基和DPPH自由基均有一定的清除作用,对DPPH·的清除率最高,而对超氧阴离子的清除能力较低。     

青霉素发酵过程接种量的研究

传统青霉素发酵一般采用10%～15%的接种量.通过对比研究移种或倒种不同接种量引起的菌丝浓度的变化,以及带来菌龄的分布,寻求合适的接种量.适当提高接种量(30%～35%)以后,可以大大缩短迟滞期和菌丝生长期,从而提高了发酵生产水平和生产效率.     

中药蝉花菌株筛选及发酵条件优化研究

目的:从蝉花中分离真菌,并采用液体培养考察菌丝收率。方法:在单因素实验基础上,利用正交实验设计,对蝉花菌液态深层发酵条件如摇床转速、接种量、p H等进行了优化。结果:最优的发酵工艺为初始p H 7.0、摇床转速160 r/min、发酵温度25℃。结论:优化发酵工艺条件下蝉花菌发酵生物量为6.57 g/L。     

摇床转速对VB12发酵的影响

通过设置不同摇床转速来调节通气量,以VB12脱氮假单孢菌发酵培养过程中的pH值、OD值、糖耗速率、菌体形态、发酵单位为指标,研究通气量对VB12脱氮假单孢菌生长的影响。结果表明：①通气量不仅影响菌丝生长而且影响菌体代谢产物VB12的合成。②可将脱氮假单孢菌发酵培养过程分为前中后3个阶段,对摇床转速进行调控,改变通气量,提高VB12发酵单位。     

青霉素发酵过程菌丝成球的工程特性研究

研究了产黄青霉菌８７６菌株发酵过程中菌丝成球现象。菌丝成球后，由于在菌球内外形成一个氧浓度分布梯度，菌丝临界氧由２５％提高至４０％，从而对反应器供氧提出了更高的要求。一旦反应器供氧不足，菌体碳、氮源代谢能力减弱，不利于青霉素合成。这种代谢与工程因素的交互影响，为发酵过程优化控制提出了一个值得探讨的，既有理论学术价值又有实际意义的研究课题。     

吡唑醚菌酯与苯醚甲环唑混剂对花生褐斑病的防治

[目的]明确吡唑醚菌酯和苯醚甲环唑混合对花生褐斑病毒力增效作用。[方法]室内联合毒力测定和田间药效试验。[结果]筛选得到吡唑醚菌酯与苯醚甲环唑的增效型混剂。[结论]吡唑醚菌酯与苯醚甲环唑以1:1混配对抑制菌丝生长增效最为明显,共毒系数为138.86。田间药效试验中,20%吡唑醚菌酯·苯醚甲环唑悬浮剂对花生叶斑病的防治效果达到84.50%,显著优于2个单剂的防效。     

北虫草废料酶解液中腺苷和虫草素含量的HPLC分析

测定人工北虫草及废料酶解液中腺苷和虫草素含量,判断酶解过程对腺苷和虫草素的影响。以kromasil 5u C18色谱柱,甲醇-水为流动相,采用梯度洗脱,流速0.8 mL/min,检测波长260 nm,柱温30℃。人工北虫草废料酶解液中腺苷和腺苷的含量为11.24 mg/L和70.40 mg/L。废料酶解液中含有一定量的腺苷和虫草素,具有利用价值。     

Base pairing at the abasic site in DNA duplexes and its application in adenosine aptasensors

The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (K(d) =14.1 μM for adenosine and 41.8 μM for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensors-a limit of detection of 0.7 μM was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.     

On the adsorption and kinetics of phase transients of adenosine at the different carbon electrodes modified with a mercury layer

Optical roughness of a pyrolytic graphite electrode with basal (PGEb) and edge orientation (PGEe), a glassy carbon electrode (GCE), and the same electrodes modified with mercury (MFE) was studied by an optical diffractive element (DOE) based sensor. Electrochemical characterisation of these electrodes used was performed by cyclic voltammetry (CV) and capacitance measurements (C-E curves and electrochemical impedance spectroscopy, EIS). The kinetics of phase transients of adenosine adsorbed on PGEb, and GCE modified with mercury layer of different thicknesses (thickness was changed from 0.02 to 2 μm) was studied by chronoamperometry (j-t curves) and capacitance measurements (C-E curves). In acidic (pH 5) solution adenosine forms two different two-dimensional (2D) physisorbed condensed layers on the MFE. The first of these (region I) is located at more positive potential; the centre of this adlayer is situated around −0.4 V. The second 2D physisorbed film (region III) is formed at more negative potentials; the centre of the region III is around −1.3 V. The 2D condensed films (adlayer I and III) of adenosine still exist on the PGEb substrate modified by 0.02 μm thick mercury layer. Adlayers III and I of adenosine exist on the GCE modified with mercury layer down to 0.02 and 0.2 μm, respectively. The kinetics of phase transients of the adenosine films taking place by a potential jump from dilute adsorption region (state II and IV) to the 2D physisorbed film (region I and III) at the PGEb and GCE substrates modified with mercury (Hg-PGEb and Hg-GCE) were studied, respectively. During both of the phase transients of IV→III and II→III of adenosine a polynucleation and growth process on both the Hg-PGEb and Hg-GCE was detected. The phase transients of II→I are characterised by an exponential decay of the current without the current maximum (the adsorption process took place only). It was observed that the phase transients of IV→III are the fastest at the HMDE and gradually slow down on 2 μm Hg-GCE and 2 μm Hg-PGEb, respectively.     

腺苷的合成与应用

腺苷是一种重要的医药原料 ,在医药工业中有很多重要的用途。本文介绍了腺苷生产的两种主要方法 ,即生物合成法 (发酵法或RNA降解法 )和化学合成法。其中采用糖质原料直接发酵生产腺苷的成本最低 ,但在国内此方法尚未完全成熟 ,国内目前主要仍以化学合成法为主。此外 ,还简要介绍了腺苷在医药工业方面的应用和市场发展前景     

纸层析-分光光度法测定腺苷的含量

通过对点样量、层析溶剂及洗脱剂、最佳吸收波长、最佳洗脱时间的确定,建立了以纸层析分离洗脱法定量测定腺苷的方法。结果表明,此法定量测定腺苷简单易行,可作为腺苷分析测定的常规手段。     

Generation of a Retargeted Oncolytic Herpes Virus Encoding Adenosine Deaminase for Tumor Adenosine Clearance

Background: Oncolytic viruses are immunotherapeutic agents that can be engineered to encode payloads of interest within the tumor microenvironment to enhance therapeutic efficacy. Their therapeutic potential could be limited by many avenues for immune evasion exerted by the tumor. One such is mediated by adenosine, which induces pleiotropic immunosuppression by inhibiting antitumor immune populations as well as activating tolerogenic stimuli. Adenosine is produced starting from the highly immunostimulatory ATP, which is progressively hydrolyzed to ADP and adenosine by CD39 and CD73. Cancer cells express high levels of CD39 and CD73 ectoenzymes, thus converting immunostimulatory purinergic signal of ATP into an immunosuppressive signal. For this reason, CD39, CD73 and adenosine receptors are currently investigated in clinical trials as targets for metabolic cancer immunotherapy. This is of particular relevance in the context of oncovirotherapy, as immunogenic cell death induced by oncolytic viruses causes the secretion of a high amount of ATP which is available to be quickly converted into adenosine. Methods: Here, we took advantage of adenosine deaminase enzyme that naturally converts adenosine into the corresponding inosine derivative, devoid of immunoregulatory function. We encoded ADA into an oncolytic targeted herpes virus redirected to human HER2. An engineered ADA with an ectopic signal peptide was also generated to improve enzyme secretion (ADA-SP). Results: Insertion of the expression cassette was not detrimental for viral yield and cancer cell cytotoxicity. The THV_ADA and THV_ADA-SP successfully mediated the secretion of functional ADA enzyme. In in vitro model of human monocytes THP1, this ability of THV_ADA and THV_ADA-SP resulted in the retrieval of eADO-exposed monocytes replication rate, suggesting the proficiency of the viruses in rescuing the immune function. Conclusions: Encoding ADA into oncolytic viruses revealed promising properties for preclinical exploitation.     

Comparative analysis of putative agonist-binding modes in the human A1 adenosine receptor

A recent study reported a model of the human A(1) adenosine receptor and its agonist binding site, proposing two putative binding modes in the same binding site for the natural agonist, adenosine. The present work investigates the flexibility of this binding site by exhaustive exploration with the natural agonist and with three other adenosine derivatives: N6-cyclopentyladenosine (CPA), 2-chloro-N6-cyclopentyladenosine (CCPA), and 5'-N-ethylcarboxamidoadenosine (NECA). Our aim was to find a common binding mode for agonists that would explain the role in the binding process of the different substitutions allowed at the 2, N6, and 5' positions of adenosine. This problem was addressed through docking simulations, molecular dynamics studies, and estimations of the ligand-binding free energy with both the AUTODOCK scoring function and the linear interaction energy (LIE) approach. The results point to a single receptor-binding position that explains the effects of the different chemical modifications on the adenosine derivatives considered here.     

多元醇PE——AMP二元体系相图

多元醇在固-固相变时能够可逆地吸收和释放大量的热而被用于贮热材料。为了选择适宜的贮热材料,对季戊四醇(PE)和2-氨基-2-甲基-1,3-丙二醇(AMP) 两种多元醇利用差热分析 (DTA)、X射线衍射和变温红外光谱3种技术构筑了二元体系相图。由所得PE-AMP二元相图可知,在一定的组成范围内相图中存在3个相平衡温度：低转析温度86℃、低共析温度143℃和转熔温度176℃。X射线衍射确定了二元体系不同温度下的相态。变温红外光谱测得多元醇—OH特征吸收峰位移发生突跃的温度区间恰好与体系各自DSC测得的相转变温度相吻合,多元醇的晶体结构、分子的大小是影响二元体系不同相态间互溶度的重要因素。     

反相高效液相色谱测定中华苦荬菜中的木犀草素

目的：建立中华苦荬菜中木犀草素含量的测定方法。方法盐析法消除植物多酚的干扰；反相高效液相色谱法测定：C18为固定相；甲醇-水-醋酸(52：47．6：0．4v／v；pH=4．0)为流动相；检测波长254nm；柱温25℃。结果：木犀草素与其它物质得到了基线分离。结论：该方法为天然植物中木犀草素的开发提供了简便、灵敏、准确的测定方法。     

Highlights on the development of A(2A) adenosine receptor agonists and antagonists

Although significant progress has been made in the past few decades demonstrating that adenosine modulates a variety of physiological and pathophysiological processes through the interaction with four subtypes of a family of cell-surface G-protein-coupled receptors, clinical evaluation of some adenosine receptor ligands has been discontinued. Major problems include side effects due to the wide distribution of adenosine receptors, low brain penetration (which is important for the targeting of CNS diseases), short half-life of compounds, or a lack of effects, in some cases perhaps due to receptor desensitization or to low receptor density in the targeted tissue. Currently, three A(2A) adenosine receptor agonists have begun phase III studies. Two of them are therapeutically evaluated as pharmacologic stress agents and the third proved to be effective in the treatment of acute spinal cord injury (SCI), while avoiding the adverse effects of steroid agents. On the other hand, the great interest in the field of A(2A) adenosine receptor antagonists is related to their application in neurodegenerative disorders, in particular, Parkinson's disease, and some of them are currently in various stages of evaluation. This review presents an update of medicinal chemistry and molecular recognition of A(2A) adenosine receptor agonists and antagonists, and stresses the strong need for more selective ligands at the A(2A) human subtype.