单组分妥布霉素产生菌的选育

目的 提高妥布霉素产生菌的发酵单位。方法 以黑暗链霉菌UVS－51为出发菌株，采用UV和NTG为诱变剂，结合耐受妥布霉素，运用固体循环诱变筛选高产菌株。结果 获得两株高产稳定变株NS－81和NT－26，这两株菌的授瓶发酵效价分别比出发菌株提高53％和77％。经薄层层析检验，该两变株的产物仍为单一的氨甲酰妥布霉素。结论 出发菌株经UV和NTG诱变处理后结合耐自身代谢物的驯育，正变率显著提高。通过引入循环诱变筛选的思路，大大缩短的实验周期有交待 提高了诱变育种的工作效率，能在较短时间内选育到理想变株，因此，循环诱变筛选法是一种较好的选育方法。     

生物素与氨基酸对林可霉素生物合成的影响

在合成培养基中利用林可链霉菌发酵生产林可霉素。当向培养基中加入生物素和氨基酸时，林可霉素的产量受到很大影响。本研究中首先采用两水平因子设计法筛选出6个显著影响因子，即生物素、谷氨酸、缬氨酸、蛋氨酸、亮氨酸和酪氨酸；然后采用中心组合设计法．得到上述6因子的优化含量分别为30μg／L、100．5、83、29、117．5和58．5mg／L；最后在摇瓶中进行验证实验，优化条件下发酵液的生物效价为2116μg／ml；对照样品Ⅰ（培养基中无生物素和氨基酸）和Ⅱ（培养基中按原配方加入6个显著影响因子）的生物效价分别为893和1481μg／ml，与样品Ⅰ和Ⅱ比较，生物效价分别提高136．62％和42．88％。     

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。     

红霉素链霉菌遗传控制的研究 一 红霉素链霉菌无活性突变株回复突变株的筛选

利用亚硝基胍(NTG)对红霉素链霉菌(S.erythreus UV 80)活性菌株进行回复突变,获得了红霉素高产量变株。先以NTG(100μg/ml,1小时)处理母株,得无活性变株,再经诱变,得到回复突变株,其活性提高8％。进一步用NTG(1000μg/ml,1小时)诱变处理,得到了比原菌株红霉素产量高25％的变株。诱变剂的最适剂量为产生90～94％死亡率的剂量。同时还观察到红霉素链霉菌变株的培养特征和生产能力之间有相关性。     

金属离子对林可霉素生物合成的影响

采用优化设计方法研究金属离子在林可链霉菌(Streptomyces lincolnensis)林可霉素生物合成中的交互作用,并应用均匀设计法,以最终发酵液的生物效价为目标函数建立模型,通过软件Uniform Design Version 3.00分析得出优化配比:FeSO4.7H2O 0.45g/L,CuSO4.5H2O 1g/L,MnCl21g/L;按优化方案添加金属离子,使发酵液的最终生物效价较原配方提高20%,明显提高了林可霉素的发酵水平。     

铵离子对林可霉素发酵过程的动态影响

在林可链霉菌(Streptomyces lincolnensis)发酵生产林可霉素的过程中,经分析表明补入硫酸铵前,应待发酵液中的NH4 浓度消耗至1.0mmol/L以下,使迟效碳、氮源充分降解,可能促使林可霉素合成酶大量合成;20～24h,尽可能提高硫酸铵的平均补入速率(最高瞬时浓度＜19.0mmol/L),以缩短生物量的积累时间;24h后逐渐降低NH4 的平均补入速率,使主代谢流转入次级代谢;40h后将摄氧率和最低NHJ4 浓度分别控制在20～30mmol·(L·h)-1和3.0～4.0mmol/L,每24h生物效价的涨幅达1000μg/ml以上.     

响应面法优化林可霉素产量与组分

以林可霉素A(Lin A)产量提高以及林可霉素B组分(Lin B)含量降低为目标，在单因素实验基础上，利用响应面法对林可链霉菌18-8菌株的含硫前体物质(硫酸钠、甲硫氨酸、半胱氨酸)与戊糖磷酸途径(HMP)的关键前体(葡萄糖酸钙、葡萄糖酸钠)以及相关醇类、离子(肌醇、氯化钴)等物质进行组合优化。首先对7个因素进行Plackett-Burman实验，筛选得到3个显著性因子：葡萄糖酸钙、肌醇和氯化钴。再通过Box-Behnken设计3因素3水平实验，利用Design-Expert 8.5软件进行回归分析，得到最佳优化条件为：氯化钴7.97mg/L、葡萄糖酸钙6.0g/L、肌醇0.42g/L。摇瓶验证Lin A液相效价为5210mg/L，比优化前提高21%，Lin B含量为4.3%，比出发菌株的Lin B含量(6.0%)降低39.5%；在15L发酵罐中进行发酵实验，Lin A液相效价可达8980mg/L，比对照(6630mg/L)提高35%，Lin B含量在90h达到最低含量2.4%，相比对照Lin B含量(4.8%)降低50%，效果显著。     

透明颤菌血红蛋白基因的克隆及其在林可链霉菌中的表达

目的通过将透明颤菌血红蛋白基因vgb克隆到林可链霉菌染色体黑色素生物合成基因m elC位点,使m elC基因被破坏失活,vgb基因实现表达同时提高林可霉素产量。方法构建大肠杆菌-链霉菌重组质粒pYHT04,通过接合转移实验将重叠延伸PCR得到的具有红霉素抗性基因启动子的透明颤菌血红蛋白基因,以双交换的方式整合进林可链霉菌黑色素生物合成基因m elC中。采用不同装瓶量摇瓶发酵,HPLC检测发酵液中林可霉素含量。结果重组菌株颜色变浅,CO结合差光谱显示在420 nm处有明显吸收峰,随着装瓶量的增加重组菌株发酵液中林可霉素效价与对照菌株相比降低幅度较小。结论重组菌株m elC基因失活不能继续合成黑色素,vgb基因得到表达并表现出生物学功能,限氧条件下重组菌株发酵液中林可霉素效价高于对照菌株,有希望应用于工业发酵生产。     

红霉素高产菌株的选育

以红霉素链霉菌0－11－26为出发菌株，经LiCl和紫外线复合诱变处理，再用含有4种不同结构类似物的培养基定向筛选，获得4株突变株，其中M－03－59摇瓶效价6876u/ml，比起始菌株（5328u/ml）提高29.1%，在10t发酵罐上连续6批验证，平均发酵效价7016u/ml，比原生产水平（5652u/ml）提高24.1%，并且质检全部合格，对生产实际具有重要意义。     

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％)。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.