洛法他汀产生菌的激光诱变育种研究

使用氦氖激光、Ｎｄ：ＹＡＧ倍频脉冲激光和铜蒸气激光,以没剂量对洛法他汀产生蓖：－１－１０９进行诱变照射,同时对激光修复作用进行探讨。结果表明,被照菌株产洛法能力有不同程度提高。氦氖激光照积压量高提高２３．５％,Ｎｄ：ＹＡＧ倍频脉冲激光照射最高提高２０．８％。铜蒸气激光照射６．３％。将菌株紫外线辐射后再以低剂量氦氖激光照射,获得一株高产优质新菌种Ｌ－２－１０１,产洛法能力提高３０％以上,遗传性能稳定     

睾酮转化菌的诱变育种

从雄甾二酮的转化产生菌偶发分枝杆菌MF2出发,经紫外诱变结合平板筛选,获得一株主要转化生产睾酮的突变株TS-N-121。该菌株可以胆固醇等单一或混合甾醇作为原料,经一步培养转化生产睾酮,其含量占总转化产物的90%以上,转化率大于80%。     

雄甾—1,4—二烯—3,17—二酮产生菌的诱变

用N′-甲基-N′-亚硝基-N~3-硝基胍(NTG)、溴化3,8-二氨基-5-乙基-6-苯基菲啶鎓(EB)和紫外线照射(UV)等分别对诺卡氏菌36野生型菌株进行诱变,发现NTG和UV的诱变效果比EB好,在NTG1000～2000μg/ml、UV2～3min条件下,经多次诱变处理,得到了一菌株诺卡氏菌22~#,它转化胆甾醇为雄甾-1,4-二烯-3,17-二酮的能力为原来36~#菌的4倍以上(底物浓度1％)。     

一株白僵菌对雄甾烯二酮转化产物的研究

利用白僵菌Beauveria bassiana HCCB00059对雄甾烯二酮（4AD）进行转化，对其主要产物进行分离纯化和结构鉴定，确认转化产生四个化合物，分别为：11-羟基-睾酮、6，11-羟基睾酮、6，11-羟基-雄甾烯二酮和11-羟基-18-氧杂D扩环雄甾烯二酮。     

利用高速逆流色谱从真菌HCCB00106转化液分离转化产物

采用高速逆流色谱法（HSCCC）对真菌HCCB00106的雄甾烯二酮（4AD）转化产物进行了分离研究，在选定体系中底物4AD、杂质及主要产物得到了较好的分离。经鉴定，主要产物为睾内酯（17α-氧代-D-的扩环-雄甾-1，4-二烯-3，17-二酮）。     

螺旋霉素产生菌的He-Ne激光诱变效果及其数量分析

目的利用He-Ne激光选育螺旋霉素产生菌生二素链霉菌（Streptomyces ambofaciens）的高产菌。方法利用功率密度为0．4mW／cm^2的He-Ne激光器，以不同的时间辐照螺旋霉素产生菌，筛选高产突变株。结果在激光照射8h的条件下，得到一株发酵效价比对照提高了38．6％的高产菌株，而且组分较出发菌株优异。结论通过对辐照剂量与致死率及正突变率问关系的数量分析，得到了一个基于自限模型和正态分布模型的用连续函数表达的诱变效果数学模型，根据模型优化的最优值，确定了He-Ne激光诱变的最佳作用时间为9h。     

半导体激光和紫外线对辅酶Q_(10)产生菌的复合诱变效应

目的选育辅酶Q10(CoQ10)高产菌株。方法采用紫外线、半导体激光及紫外线与半导体激光复合作用的方法,诱变产CoQ10红酵母菌SY-3,以提高CoQ10的产量。结果紫外线和半导体激光单独作用,诱变效果不佳;二者合用的效果却很好。用紫外照射120 s,再经半导体激光辐射8 min,得到一株叠氮钠和维生素K3双抗性突变株,产辅CoQ10的量达到157.7mg.L-1,比原始菌株提高了88.1%,并具有良好的遗传稳定性。结论所建方法简便可行。     

常压室温等离子体-紫外复合诱变选育新硫肽类抗生素166A高产菌株

摘要：目的 通过对新硫肽类抗生素166A产生菌小单孢菌TMD166进行诱变选育研究,以期获得产166A的高产菌株。方 法 使用多功能等离子体诱变系统(multifunctional plasma mutagenesis system, MPMS)对出发菌株TMD166的孢子进行等离子体-紫 外(MPMS-UV)复合诱变,单孢子悬液经照射处理、涂布培养后获得单菌落,并以牛津杯固体发酵高通量筛选方法对菌株进行初 筛,之后对高产菌株进行摇瓶复筛,利用突变株摇瓶发酵的化学效价筛选出正突变菌株。结果 对比MPMS-UV不同诱变剂量发 现,MPMS105s-UV60s复合诱变剂量获得的正突变率最高,达到41.43%,相应致死率为99.88%。最终筛选出一株166A高产突 变株TMD166-MU1,在培养温度为28℃、210 r/min的条件下,经过96~120 h培养后,166A产量相比出发菌株提高了7.47倍。结 论 采用MPMS-UV复合诱变方式,再结合牛津杯固体发酵高通量筛选方法和摇瓶复筛,可高效筛选获得166A高产菌株。     

通过获得链霉素抗性基因(str)突变株筛选梅岭霉素高产菌株

本文通过链霉素对梅岭霉素（meilingmycin）产生菌南昌链霉菌NS－41－80菌株孢子致死浓度的测定，采用诱变剂EMS四种不同诱变剂量对菌株的孢子进行诱变处理，诱变处理的孢子涂布在含链霉素致死浓度的高氏平板上，获得了大量的链霉素抗性基因（str）突变株。然后从链霉素抗性基因突变株进一步筛选到梅岭霉素高产菌株80－5．11－221，在摇瓶条件下，只产梅岭霉素不产南昌霉素，梅岭霉素活性效价达1500μg/ml，比出发菌株NS－41－80的摇瓶发酵效价855μg/ml提高了77．9％，该菌株连续传代六代进行摇瓶发酵，其F2代和F3代梅岭霉素发酵效价稳定，F4代至F6代随着传代数增加，其梅岭霉素发酵效价急速下降。通过EMS诱变剂量分别与抗药性突变率和链霉素抗性基因突变株产梅岭霉素产量的产势统计分析表明，菌株抗药性突变与产抗生素突变密切相关，产抗生素突变的EMS诱变剂量高于链霉素抗性基因突变诱变剂量。在0．03mol/L的EMS剂量作用下，菌株致死率为99．43％，而抗药性突变率为0．0440％，建立了梅岭霉素产生菌链霉素抗性基因突变筛选方法，为南昌链霉菌高产菌种选育研究作了有益的尝试，并有助于其它链霉菌属的抗生素产生菌育种研究。     

△4.9雌甾二烯—3,17双酮的合成

本文报道了合成RU-486的中间体Δ~(4,9)一雌二烯—3,17—双酮[Ⅰ]的制备。以1?—羟甲基—Δ~4—雄甾烯—3,17—双酮[Ⅷ]为起始原料,与Jones试剂反应得Δ~4—雄甾烯—3,17—双酮—10—羧酸[Ⅸ],收率为79～84%。[Ⅸ]在吡啶中用碘处理生成化合物[Ⅰ],收率为48～52%。化合物[Ⅰ]的结构经红外光谱和紫外光谱证实。     

Biohydroxylation of 7‐oxo‐DHEA,a natural metabolite of DHEA,resulting in formation of new metabolites of potential pharmaceutical interest

Metabolism of steroids in healthy and unhealthy human organs is the subject of extensive clinical and biomedical studies. For this kind of investigations, it is essential that the reference samples of new derivatives of natural, physiologically active steroids (especially those difficult to achieve in the chemical synthesis) become available. This study demonstrated for the first time transformation of 7‐oxo‐DHEA—a natural metabolite of DHEA, using Syncephalastrum racemosum cells. The single‐pulse fermentation of substrate produced two new hydroxy metabolites: 1β,3β‐dihydroxy‐androst‐5‐en‐7,17‐dione and 3β,12β‐dihydroxy‐androst‐5‐en‐7,17‐dione, along with the earlier reported 3β,9α‐dihydroxy‐androst‐5‐en‐7,17‐dione and 3β,17β‐dihydroxy‐androst‐5‐en‐7‐one. Simultaneously, the same metabolites, together with small quantities of 7α‐ and 7β‐hydroxy‐DHEA, as well as the products of their reduction at the C‐17 were obtained after transformation of DHEA under pulse‐feeding of the substrate. The observed reactions suggested that this micro‐organism contains enzymes exhibiting similar activity to those present in human cells. Thus, the resulting compounds can be considered as potential components of the eukaryotic, including human, metabolome.     

快速分析测定大豆甾醇侧链降解底物的方法

建立了快速分析测定大豆甾醇侧链降解过程中底物大豆甾醇和产物雄甾-4-烯-3，17-二酮(AD)及雄甾-1，4-二烯-3，17-二酮(ADD)的方法。采用薄板层析(TLC)，在板层为硅胶60GF254，流动相为石油醚-乙酸乙酯(6：4)的条件下，成功的分离了底物与产物，从而能快速简便的监测底物的消耗与产物的生成情况。另外根据底物与产物AD与ADD在紫外区的吸收情况，利用254nm与298nm双波长测定ADD与AD的生成量。上述两种方法便于工厂对生产情况的同步检测。     

Metabolic studies of 1‐testosterone in horses

1‐Testosterone (17β‐hydroxy‐5α‐androst‐1‐en‐3‐one), a synthetic anabolic steroid, has been described as one of the most effective muscle‐building supplements currently on the market. It has an anabolic potency of 200 as compared to 26 for testosterone. Apart from its abuse in human sports, it can also be a doping agent in racehorses. Metabolic studies on 1‐testosterone have only been reported for human in the early seventies, whereas little is known about its metabolic fate in horses. This paper describes the studies of in vitro and in vivo metabolism of 1‐testosterone in horses, with the aim of identifying the most appropriate target metabolites to be monitored for controlling the misuse or abuse of 1‐testosterone in racehorses. Six in vitro metabolites, namely 5α‐androst‐1‐ene‐3α,17β‐diol (T1a), 5α‐androstane‐3β,17β‐diol (T2), epiandrosterone (T3), 16,17‐dihydroxy‐5α‐androst‐1‐ene‐3‐one (T4 & T5), and 5α‐androst‐1‐ene‐3,17‐dione (T6), were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 800 mg of 1‐testosterone by stomach tubing. The results revealed that the parent drug and eight metabolites were detected in urine. Besides the four in vitro metabolites (T1a, T2, T3, and T5), four other urinary metabolites, namely 5α‐androst‐1‐ene‐3β,17α‐diol (T1b), 5α‐androst‐1‐ene‐3β,17β‐diol (T1c), 5α‐androstane‐3α,17α‐diol (T7) and 5α‐androstane‐3β,17α‐diol (T8) were identified. This study shows that the detection of 1‐testosterone administration is best achieved by monitoring the parent drug, which could be detected for up to 30 h post‐administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Factors affecting urinary excretion of testosterone metabolites conjugated with cysteine

The implementation of the athlete steroidal passport in doping control analysis aims to detect intra‐individual changes in the steroid profile related to the abuse of anabolic steroids. In this context, the study of intrinsic variations associated with each marker is of utmost importance. In the present work, the influence of several factors in the excretion of the recently reported testosterone metabolites conjugated with cysteine (Δ¹‐AED; 1,4‐androstadien‐3,17‐dione, Δ⁶‐AED; 4,6‐androstadien‐3,17‐dione, Δ⁶‐T; 4,6‐androstadien‐17β‐ol‐3‐one, and Δ¹⁵‐AD; 15‐androsten‐3,17‐dione) is evaluated for the first time. Degradation experiments at 37 °C proved that, although the cysteinyl moiety is released, the variation for urinary Δ¹‐AED/Δ⁶‐AED, Δ¹‐AED/Δ⁶‐T ratios is less than 30%. Moreover, freeze/thaw cycle testing resulted in RSDs values below 15% for all the analytes. Regarding infradian variability, moderate variations (below 40%) were observed. Additionally, notable alterations in the excretion of these compounds have been observed in the earliest stages of pregnancy. UGT2B17 polymorphism, responsible for the low T/E ratio found in some population, does not influence the excretion of cysteinyl compounds whereas the intake of exogenous substances (alcohol or 5α‐reductase inhibitors) dramatically affects their excretion. The urinary concentrations of Δ¹‐AED, Δ⁶‐AED, and Δ¹⁵‐AD decreased (<50 %) after the ethanol intake, whereas after the administration of dutasteride, an important increase was observed for the concentrations of Δ⁶‐AED, Δ⁶‐T and Δ¹⁵‐AD. Overall, the presented data describes the stability of the urinary cysteinyl steroids under the influence of many factors, proving their potential as suitable parameters to be included in the steroidal module of the athlete′s biological passport. Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolic studies of formestane in horses

Formestane (4‐hydroxyandrost‐4‐ene‐3,17‐dione) is an irreversible steroidal aromatase inhibitor with reported abuse in human sports. In 2011, our laboratory identified the presence of formestane in a horse urine sample from an overseas jurisdiction. This was the first reported case of formestane in a racehorse. The metabolism of formestane in humans has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the in vitro and in vivo metabolic studies of formestane in horses, with the objective of identifying the target metabolite with the longest detection time for controlling formestane abuse. In vitro metabolic studies of formestane were performed using homogenized horse liver. Seven in vitro metabolites, namely 4‐hydroxytestosterone (M1), 3β,4α‐dihydroxy‐5β‐androstan‐17‐one (M2a), 3β,4β‐dihydroxy‐5β‐androstan‐17‐one (M2b), 3β,4α‐dihydroxy‐5α‐androstan‐17‐one (M2c), androst‐4‐ene‐3α,4,17β‐triol (M3a), androst‐4‐ene‐3β,4,17β‐triol (M3b), and 5β‐androstane‐3β,4β,17β‐triol (M4) were identified. For the in vivo studies, two thoroughbred geldings were each administered with 800 mg of formestane (32 capsules of Formadex) by stomach tubing. The results revealed that the parent drug and seven metabolites were detected in post‐administration urine. The six in vitro metabolites (M1, M2a, M2b, M2c, M3a, and M3b) identified earlier were all detected in post‐administration urine samples. In addition, 3α,4α‐dihydroxy‐5α‐androstan‐17‐one (M2d), a stereoisomer of M2a/M2b/M2c, was also identified. This study has shown that the detection of formestane administration would be best achieved by monitoring 4‐hydroxytestosterone (M1) in the glucuronide‐conjugated fraction. M1 could be detected for up to 34 h post‐administration. In blood samples, the parent drug could be detected for up to 34 h post administration. Copyright © 2013 John Wiley & Sons, Ltd.     

雌酮的合成工艺

以雄甾-1，4-二烯-3，17-二酮为原料，经缩酮化、芳构化和水解三步反应合成雌酮，并对此工艺进行中试放大，总收率可达53.3％。     

调Q开关Nd：YAG染料700nm激光治疗雀斑的临床效果观察（附507例报告）

目的探讨调Q开关Nd：YAG染料700nm激光治疗雀斑临床效果。方法用调Q开关Nd：YAG染料激光治疗507例雀斑患者，治疗参数：波长700nm，能量密度为5．6J／cm。，光斑直径为1．5mm，固定脉宽7～10ns。结果507例患者痊愈率为85％，有效率为100％。所有患者治疗后均无疤痕。结论调Q开关Nd：YAG染料700nm激光治疗雀斑疗效好，安全性高。     

微生物来源白细胞介素1受体拮抗剂的研究Ⅱ．链霉菌660代谢产物的化学研究

从活性链霉菌660的发酵液中,经溶媒萃取,硅胶制备薄层层析及ODS等分离得到化合物4和化合物6,通过理化性质研究和UV、IR、MS、^1H-NMR、^13C-NMR、DEPT、^1H-^1H COSY、^1H-^13C COSY、HMBC等光谱分析确定化合物4为N－乙酰基酪胺（N-acetyltyramine),化合物6为3－丙基－7－甲基－六氢吡咯[1,2-a]并吡嗪－1,4－二酮（3－propyl-7-methyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione)。并对其活性进行研究。     

Analysis of the nutritional supplement 1AD, its metabolites, and related endogenous hormones in biological matrices using liquid chromatography-tandem mass spectrometry

1,5alpha-Androsten-3beta,17beta-diol and/or 1,5alpha-androsten-3,17-dione (1AD) is an over-the-counter pro-hormone nutritional supplement designed to enhance strength and performance in athletes. 1AD purportedly mimics the pharmacological activity of testosterone through activation of the pro-hormones to their active form 1,5alpha-androsten-17beta-ol-3-one or Delta(1)-testosterone. This testosterone analogue ostensibly possesses strong androgenic potency without the adverse effects associated with aromatization of testosterone to estrogens. We have developed a highly sensitive and selective liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of 1AD, its analogues, and several structurally related endogenous hormones in plasma and urine. The limits of quantitation for the analytes ranged from 0.25 to 0.5 ng/mL. The accuracy of the assay was 92-113% with a precision of 2-12% relative standard deviation (RSD) for all analytes at 1.0, 5.0, and 15.0 ng/mL, respectively. The interassay precision was 6-16% RSD, and the accuracy was 90-105%. We have used this assay to determine the unconjugated and total (conjugated and unconjugated) concentrations of 1AD, its analogues, androstenediol, androstenedione, testosterone, dihydrotestosterone, and estradiol, in plasma and urine, as well as to investigate the metabolic fate of the three 1AD analogues (diol, dione, and active forms) when incubated with rat liver microsomes or rat testicular homogenates. Concentrations of both unconjugated and total testosterone in plasma were approximately 1.5 ng/mL and ranged from undetectable to 4.1 ng/mL in urine. 1AD and its analogues were not detected in plasma or urine. In vitro metabolism experiments using rat liver microsomes and testicular homogenates provided evidence for the interconversion of the three 1AD analogues, biosynthesis, and decomposition of several endogenous hormones, as well as evidence for 1AD analogue-induced changes in the typical profiles of testosterone and androstenedione in testicular tissue.     

7‐keto‐DHEAmetabolism in humans. Pitfalls in interpreting the analytical results in the antidoping field

7‐keto‐DHEA (3β‐hydroxy‐androst‐5‐ene‐7,17‐dione) is included in section S1 of the World Antidoping Agency (WADA) List of Prohibited Substances. The detection of its misuse in sports needs special attention, since it is naturally present in urine samples. The main goal of this study is to investigate the in vivo metabolism of 7‐keto‐DHEA after a single administration to healthy volunteers and to better describe the relationship between arimistane (androst‐5‐ene‐7,17‐dione) and 7‐keto‐DHEA after the application of the common routine procedures to detect anabolic steroids in WADA accredited antidoping laboratories. Free, glucuro‐, and sulpho‐conjugated steroids extracted from urine samples obtained before and after the administration of 7‐keto‐DHEA were analyzed by different gas chromatographic (GC)–mass spectrometric (MS) techniques. Gas chromatography coupled to tandem MS to study the effect on the endogenous steroid profile, coupled to isotope ratio mass spectrometry (IRMS) to investigate the potential formation of androgens derived from DHEA and coupled to high resolution accurate mass spectrometry (HRMS) to investigate new diagnostic metabolites. The analysis by IRMS confirmed that there is no formation of DHEA from 7‐keto‐DHEA. Ten proposed metabolites, not previously reported, were described. These include reduced and hydroxylated structures that are not considered part of the steroid profile in antidoping analyses. They showed considerable responses in all fractions analyzed. Some deoxidation reactions (including arimistane formation) were found and most probably can be linked to the sample preparation or instrumental analysis. This is important when interpreting the results after the application of procedures to detect steroids in urine currently used in antidoping laboratories. 7‐keto‐DHEA metabolism in humans for antidoping purposes was studied and unexpected results were found. This could lead to a misinterpretation of the data, depending on the procedure applied and the analytical instrumentation used.