溶剂萃取法提取亮氨酸和异亮氨酸

研究了用二（２－乙基己基）磷酸（Ｐ２０４）作萃取剂,从毛发水解生产胱氨酸的二次母液中提取亮氨酸和异亮氨酸的方法和条件。研究表明,在常温条件下,水相初始ｐＨ为４．０时,用３０％的Ｐ２０４（体积分数）正庚烷溶液,萃取相比为１,可萃取出水相中近７０％的亮氨酸和异亮氨酸。     

合成5—氟尿嘧啶—N^1—甲酰基异亮氨酸的新方法

合成５－氟尿嘧啶－Ｎ~１－甲酰基异亮氨酸的新方法叶发青（咸宁医学院化教研室，咸宁４３７１００）５－氟尿嘧啶－Ｎ１－甲酰基异亮氨酸具有较高的抗肿瘤活性，并且毒副作用较低［１］。对此类药物的开发具有较广泛的应用前景。它也是合成含５－氟尿嘧啶的高分子抗癌药?..     

二（2—乙基己基）磷酸萃取L—异亮氨酸的研究

以二（２－乙基己基）磷酸（Ｄ２ＥＨＰＡ）－正辛烷及Ｄ２ＥＨＰＡ－正辛醇萃取Ｌ－异亮氨酸为对象,研究了Ｄ２ＥＨＰＡ浓度、Ｌ－异亮氨酸初始浓度以及ｐＨ值对萃取平衡分配系数的影响。结果表明,在实验研究涉及的ｐＨ值范围内,分配系数先随ｐＨ的增加而增大,在３．５〈ｐＨ〈５区域,ｐＨ值对分配系数的影响较小。分配系数还随Ｄ２ＥＨＰＡ浓度的增加而增大。正辛醇加入有机相,萃取分配系数增大。Ｄ２ＥＨＰＡ与Ｌ－异亮氨酸     

DL-异亮氨酸合成新工艺研究

以丁酮和海因为原料经Knoevenagel condensations缩合、催化加氢、碱水解合成了DL-异亮氨酸,收率大于71%。     

L-异亮氨酸生产菌发酵水平提高的研究

异亮氨酸在当今社会的应用越来越广泛,福建省麦丹生物集团有限公司福州研究中心对异亮氨酸的生产研究已有十多年的时间,在异亮氨酸生产菌株基础上进行研究,对于提高异亮氨酸的大罐生产水平具有一定的指导意义。     

原生质体诱变筛选阿维菌素高产菌株

阿维菌素是阿维链霉菌次级代谢产生的具有抗寄生虫活性的抗生素,菌种的改良是提高阿维菌素产量的关键。通过原生质体诱变,以L-异亮氨酸作为筛选模型来筛选阿维菌素高产菌株。首先制备阿维链霉菌原生质体,之后原生质体细胞在搅拌状态下进行紫外诱变,最后在含有0.5%L-异亮氨酸抗性平板上进行培养,得到了一株比出发菌株阿维菌素B1a效价提高35.4%的新菌株。     

用D_2EHPA-煤油溶剂萃取分离L-异亮氨酸

本文研究了从发酵液中用D2EHPA－煤油溶剂萃取分离L－异亮氨酸工艺．考察了萃取时pH值、萃取剂浓度和异亮氨酸浓度对于异亮氨酸和缬氨酸的萃取分配比的影响．在计算机上进行回归，得到以下分配模型：当1≤pH≤3．5时lgDIle＝0．09906pH2＋0．8625pH＋2．2696lgCHR－0．09188lg2CIle＋0．1766lgCIle－1．366lgDrmVal＝－0．0293pH2＋0．550pH＋1．2659lgCHR－0．2420lg2CIle＋0．4549lgCIle－1．696用此模型，应用分馏萃取理论，进行逐级计算，求取了萃取工艺的主要参数，并在φ20mm的多级离心萃取器上对上述结果进行了验证．结果表明：当CHR浓度为1．5mol／L，相比V：L：L’＝2：1：2，N’＝3，N＝6时，异亮氨酸的收率达到90％以上，纯度＞99％．实验取得了较为满意的结果．     

生物法合成邻苯二醛——（Ⅰ）菌种筛选与野生型活细胞转化效果

以苯甲酸钠（Sodium Benzoate）为唯一碳源筛选到49株菌株，其中4株菌株表现出最佳的转化苯甲酸钠为邻苯二酚（Catechol）的能力。对B5进行了形态学和生理生化鉴定，初步确定其为假单胞菌。经紫外光谱和邻苯二酚特异性试验，证明该菌能转化苯甲酸钠合成邻苯二酚。该菌在6mg/mL的苯甲酸钠中培养24h，邻苯二酚的产量为1.6mg/mL。在培养基中加入甘油，利用静止细胞发酵培养16h，邻苯二酚的产量为2.1mg/mL，分子水平转化率达到46.11%。     

L-异亮氨酸对L-丙氨酸晶体生长速率的影响

结合实验和分子模拟研究了L-异亮氨酸对L-丙氨酸晶体主要晶面生长速率的影响。实验发现随着溶液中L-异亮氨酸浓度的增加,L-丙氨酸(120)面(轴面)的生长速率显著降低,而(011)面(端面)的生长速率增加,导致L-丙氨酸晶体的长径比增大。分子模拟的结果表明,L-异亮氨酸容易占据L-丙氨酸(120)晶面的台阶位从而阻碍(120)晶面的生长;但其不仅不容易吸附于(011)晶面的台阶位,反而会促进溶质分子在溶剂化界面的扩散,从而提高(011)晶面的生长速率。分子模拟的结果较好地解释了实验中L-异亮氨酸对L-丙氨酸不同晶面具有不同作用效果的现象。     

自动计量分布收集系统在L—异亮氨酸生产中的运用

以当今工控界流行的“集中监视，分散控制”观点为理论依据，采用ＰＣ机与可编程序控制器两级监控体系，解决了流速偿均匀的流体的累加体积难以精确实时计量的难题。介绍了该的原理。结构，功能以及在工业方面的应用。     

Discovery of a Novel Scaffold as an Indoleamine 2,3‐Dioxygenase 1 (IDO1) Inhibitor Based on the Pyrrolopiperazinone Alkaloid,Longamide B

Indoleamine 2,3‐dioxygenase 1 (IDO1) has emerged as a key target for cancer therapy, as IDO1 plays a critical role in the capacity of tumor cells to evade the immune system. The pyrrolopiperazinone alkaloid longamide B and its derivatives were identified as novel IDO1 inhibitors based on docking studies and small library synthesis. The thioamide derivative showed higher IDO1 inhibitory activity than longamide B, and displayed an activity similar to that of a representative IDO1 inhibitor, 1‐methyl‐tryptophan. These results suggest that the pyrrolopiperazinone scaffold of longamide B could be used in the development of IDO1 inhibitors.     

Indoleamine 2,3-Dioxygenase (IDO) Downregulates the Cell Surface Expression of the CD4 Molecule

Indoleamine 2,3-dioxygenase (IDO) has been implicated in preventing the fetus from undergoing maternal T cell-mediated immune responses, yet the mechanism underlying these kinds of IDO-mediated immune responses has not been fully elucidated. Since the CD4 molecule plays a central role in the onset and regulation of antigen-specific immune responses, and T cell is sensitive in the absence of tryptophan, we hypothesize that IDO may reduce cell surface CD4 expression. To test this hypothesis, an adenoviral vector-based construct IDO-EGFP was generated and the effect of IDO-EGFP on CD4 expression was determined on recombinant adenoviral infected C8166 and MT-2 cells, by flow cytometry and/or Western blot analysis. The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP. Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells. Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.     

Indoleamine 2,3 Dioxygenase 1—The Potential Link between the Innate Immunity and the Ischemia-Reperfusion-Induced Acute Kidney Injury?

Ischemia-reperfusion injury (IRI) is of the most common causes of acute kidney injury (AKI); nevertheless, the mechanisms responsible for both early kidney injury and the reparative phase are not fully recognised. The inflammatory response following ischemia is characterised by the crosstalk between cells belonging to the innate immune system—dendritic cells (DCs), macrophages, neutrophils, natural killer (NK) cells, and renal tubular epithelial cells (RTECs). A tough inflammatory response can damage the renal tissue; it may also have a protective effect leading to the repair after IRI. Indoleamine 2,3 dioxygenase 1 (IDO1), the principal enzyme of the kynurenine pathway (KP), has a broad spectrum of immunological activity from stimulation to immunosuppressive activity in inflamed areas. IDO1 expression occurs in cells of the innate immunity and RTECs during IRI, resulting in local tryptophan (TRP) depletion and generation of kynurenines, and both of these mechanisms contribute to the immunosuppressive effect. Nonetheless, it is unknown if the above mechanism can play a harmful or preventive role in IRI-induced AKI. Despite the scarcity of literature in this field, the current review attempts to present a possible role of IDO1 activation in the regulation of the innate immune system in IRI-induced AKI.     

New 4‐Amino‐1,2,3‐Triazole Inhibitors of Indoleamine 2,3‐Dioxygenase Form a Long‐Lived Complex with the Enzyme and Display Exquisite Cellular Potency

Indoleamine‐2,3 dioxygenase 1 (IDO1) has emerged as a central regulator of immune responses in both normal and disease biology. Due to its established role in promoting tumour immune escape, IDO1 has become an attractive target for cancer treatment. A novel series of highly cell potent IDO1 inhibitors based on a 4‐amino‐1,2,3‐triazole core have been identified. Comprehensive kinetic, biochemical and structural studies demonstrate that compounds from this series have a noncompetitive kinetic mechanism of action with respect to the tryptophan substrate. In co‐complex crystal structures, the compounds bind in the tryptophan pocket and make a direct ligand interaction with the haem iron of the porphyrin cofactor. It is proposed that these data can be rationalised by an ordered‐binding mechanism, in which the inhibitor binds an apo form of the enzyme that is not competent to bind tryptophan. These inhibitors also form a very tight, long‐lived complex with the enzyme, which partially explains their exquisite cellular potency. This novel series represents an attractive starting point for the future development of potent IDO1‐targeted drugs.     

Improving the Potency of Cancer Immunotherapy by Dual Targeting of IDO1 and DNA

Herein we report the first exploration of a dual‐targeting drug design strategy to improve the efficacy of small‐molecule cancer immunotherapy. New hybrids of indoleamine 2,3‐dioxygenase 1 (IDO1) inhibitors and DNA alkylating nitrogen mustards that respectively target IDO1 and DNA were rationally designed. As the first‐in‐class examples of such molecules, they were found to exhibit significantly enhanced anticancer activity in vitro and in vivo with low toxicity. This proof‐of‐concept study has established a critical step toward the development of a novel and effective immunotherapy for the treatment of cancers.     

生物催化不对称还原法制备(R)-和(S)-2-羟基-4-苯基丁酸乙酯

用全细胞催化法合成2-羟基-4-苯基丁酸乙酯(EHPB)的两种对映异构体。筛选得到两株高立体选择性菌株,能催化前手性酮还原分别生产相应的手性醇。考察了这两株菌株的反应特性,得到了合适的反应条件:菌株短小芽孢杆菌(Bacillus pumilusPhe-C3),反应24 h,底物浓度25 mmol/L,体系pH7.0,温度30℃,R-EHPB的产率达74.5%,对映体过量值(e.e.)达97%;菌株肺炎克雷伯菌(Klebsiella pneumomiaePhe-E4),反应20 h,底物浓度15 mmol/L,体系pH7.0,温度30℃,S-EHPB的产率达71.7%,e.e.达95%。     

小反刍兽疫病毒F蛋白的原核表达及其多克隆抗体的制备

目的原核表达小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)F蛋白,并制备多克隆抗体。方法根据GenBank中登录的PPRV Nigeria 75/1株F基因全长序列,采用DNAStar软件设计去除F基因信号肽和跨膜区的引物,进行RT-PCR扩增,并克隆至原核表达载体pET-32a(+)中,转化E.coli BL21(DE3),经IPTG诱导表达。表达的重组蛋白纯化后,进行SDS-PAGE和Western blot分析,免疫BALB/c小鼠,制备多克隆抗体,并初步应用于PPRV的检测。结果重组原核表达质粒pET-32a(+)-PPRV-F经双酶切和测序证实构建正确;表达的重组F蛋白相对分子质量约59 000,诱导6 h表达量最高,且主要以包涵体形式表达;纯化的重组蛋白纯度可达95%以上;免疫小鼠制备的多抗能与纯化的重组蛋白发生特异性反应,抗体效价高于1∶64 000;间接免疫荧光试验显示,制备的多抗能够识别PPRV Nigeria 75/1株全病毒抗原。结论原核表达了PPRVF蛋白,并制备了高效价的多克隆抗体,为其进一步研究及小反刍兽疫临床快速检测方法的建立奠定了基础。     

重组大肠杆菌全细胞催化合成S-苷甲硫氨酸

从大肠杆菌(E.ColiK12)基因组DNA中克隆出甲硫氨酸腺苷转移酶基因,构建了能高效表达甲硫氨酸腺苷转移酶的重组大肠杆菌E.ColiJM109(pBR322-MAT)。将重组大肠杆菌细胞用于催化合成S-腺苷甲硫氨酸(SAM)的研究中。实验发现,用φ(甲苯)=2%的水溶液对重组细胞进行通透化处理后,能大幅提高SAM的产率。探讨了底物浓度、温度、pH、反应时间以及菌体密度对反应转化率的影响。最佳反应条件为:底物浓度(ATP)30 mmol/L,反应温度35℃,pH=7.0,反应时间8 h,细胞密度80 g湿细胞/L反应液。在此条件下,底物ATP的转化率超过95%。     

Spiro-Oxindole Skeleton Compounds Are Efficient Inhibitors for Indoleamine 2,3-Dioxygenase 1: An Attractive Target for Tumor Immunotherapy

Indoleamine 2,3-dioxygenase 1 (IDO1) is an attractive heme enzyme for its significant function in cancer immunotherapy. Potent IDO1 inhibitors have been discovered for decades, whereas no clinical drugs are used for cancer treatment up to now. With the goal of developing medically valuable IDO inhibitors, we performed a systematic study of SAR405838 analogs with a spiro-oxindole skeleton in this study. Based on the expression and purification of human IDO1, the inhibitory activity of spiro-oxindole skeleton compounds to IDO1 was evaluated by IC₅₀ and K_i values. The results demonstrated that inhibitor 3 exhibited the highest IDO1 inhibitory activity with IC₅₀ at 7.9 μM among all inhibitors, which is ~six-fold of the positive control (4−PI). Moreover, inhibitor 3 was found to have the most effective inhibition of IDO1 in MCF-7 cancer cells without toxic effects. Molecular docking analysis revealed that the hydrophobic interaction stabilized the binding of inhibitor 3 to the IDO1 active site and made an explanation for the uncompetitive mode of inhibitors. Therefore, this study provides valuable insights into the screen of more potent IDO1 inhibitors for cancer immunotherapy.     

肾综合征出血热灭活疫苗毒株L99株的全基因序列分析

目的 了解我国肾综合征出血热疫苗生产毒株Ｌ９９株的分子特征及抗原性的分子基础。方法 设计出不同的引物，用ＰＣＲ方法提取细胞总ＲＮＡ，逆转录扩增产物纯化后克隆Ｔ载体，纯化后测序，序列用ＤＮＡＳ ＴＡＲ软件拼接，并对其序列进行分析。结果Ｌ９９株全基因组由Ｌ片段６５３３个，Ｍ３６５２个，Ｓ１７６４个核苷酸组成， 分别编码２１５１、１１３３、４２９个氨基酸，Ｌ９９与同型病毒间同源率高达９５．５％－９９．４％。结论 为研究疫苗毒株的生物 学特性、致病机理等提供了分子基础。