金色链霉菌原生质体电融合

为了提高去甲基金霉素的发酵效价,将金霉素链霉菌产生去甲基金霉素的变株的原生质体与金霉素高产变株热灭活的原生质体,用非交流电场法和交流电场法进行了电融合。在非交流电场法实验中。先用25％PEG将原生质体聚集,再以BAEKON 2000和SDⅡ型二种基因转移仪,采用几种不同的实验参数(脉冲强度、宽度和个数)进行融合,皆未能提高融合频率。交流电场法融合实验用SD3000细胞融合仪,当原生质体在交流电场(频率0.5MHz,强度800v/cm,作用时间60s)中形成串珠状后,施加直流脉冲(脉冲强度7kv/cm,宽度20μs,间隔0.5s,个数3),融合频率为2×10~(-4)左右,比50％PEG诱导的融合频率(2.5×10~(-5)高约一个数量级。对225株融合子进行了初筛发酵和效价测定。结果表明,其中效价高菌株所占百分数高于对照组(去甲基金霉素产生菌不同营养缺陷型的融合子),而且85％以上的融合子只产生去甲基组分。尽管融合子的发酵效价皆不稳定,但经过几次自然分离后仍获得了产量比出发菌株提高一倍左右并只产生去甲基组分较为稳定的融合子菌株。     

地中海拟无枝酸菌原生质体电融合及其在提高利福霉素SV发酵效价中的应用

对产利福霉素SV的地中海拟无枝酸菌 (Amycolatopsismediterranei)原生质体进行热灭活和紫外灭活标记 ,其热灭活条件为 5 2℃ ,1.5h ,紫外灭活条件为紫外线照射 ( 15W ,2 5cm ,2 70nm) 6 0min。将双灭活标记的原生质体用CRY 3型细胞融合仪进行电融合 ,得出 :成串电流频率 1.5MHz ,电压 5 8V ,成串时间 2 0s ,融合脉冲幅宽 80 μS ,融合电压 5 0 0V ,融合槽间距 0 .5mm时 ,融合率最高为 9.3× 10 -4 。对筛选的融合子产量试验表明 ,5 0 %以上的融合子产量高于出发菌株 ,有明显的正变作用 ,将融合子在利福霉素代谢类似物平板上分离纯化 ,获得了化学效价提高 30 %以上的产量稳定菌株。     

抗生素链霉菌与弗氏链霉菌原生质体的种间电融合

许多链霉菌能够合成抗生素、酶及抗肿瘤剂等各种重要产物。最近,原生质体融合已经成为菌株改良的有效技术。对于链霉菌原生质体融合,几乎专门使用聚乙二醇(PEG)作促融剂。然而,同新发展的电融合法比较,该法存在许多缺点。因此,电融合法正成为酵母、植物及动物细胞融合的常规方法。虽然电融合法正在不同范围内推广使用,但对于像链霉菌这么小的原生质体,至今尚未加以应用。本文论述由抗生素链霉菌a20和弗氏链霉菌f2所制备的原生质体的电融合。利用由显微镜载玻片和铝箔构成的微型融合室来监测链霉菌原生质体的融合过程。     

基因组重组技术选育埃博霉素B高产菌株

目的 用基因重组技术选育埃博霉素高产菌株.方法 本研究首先从不同生境中分离筛选到4株能产生埃博霉素B的纤维堆囊菌,以这些野生菌株作为Genome shuffling递归原生质体融合出发菌株.结果 通过五轮基因组重组成功选育到了3株遗传稳定的高产埃博霉素B重组菌株,其中一株重组菌株So F5-09的埃博霉素B产量达到了15.8mg/L,比原始出发菌株So 07-9埃博霉素B产量提高了35.1倍.结论 本研究证明使用野生菌株作为Genome shuffling递归原生质体融合出发菌株也能有效提高目标代谢产物的产量.     

他克莫司高产菌株选育及发酵配方优化

目的 选育他克莫司高产菌株,优化培养基配方,提高发酵产量。方法 采用原生质体融合技术对2株筑波链霉菌进行基因组重排,并经响应面设计对发酵培养基配方进行优化。结果 以紫外灭活作为遗传标记经过4轮基因组重排育种后,摇瓶筛选获得3株高产菌株,其中菌株FK22-71菌丝球松散、椭圆状,发酵浸泡液颗粒状。该菌株稳定高产,采用响应面设计优化后的配方在50 L罐发酵产量平均达1684 mg/L。结论 基因组重排技术应用于他克莫司高产菌株选育,可大幅提高发酵产量,为其工业化发酵生产奠定了基础。     

通过两种糖多孢菌属菌株原生质体融合提高多杀菌素产量研究

多杀菌素是由放线菌刺糖多孢菌产生的一种新型绿色环保杀虫剂，兼具化学农药的高效性与生物农药的安全性。本研究通过将红色糖多孢菌和刺糖多孢菌两种菌株做原生质体融合探究来得到高产多杀菌素的菌株。首先构建了不产红霉素的红色糖多孢菌工程菌株，然后将其与刺糖多孢菌做原生质体融合，经加有抗生素平板培养基及发酵液HPLC分析筛选，得到了产多杀菌素的4株融合菌株。基于4株融合菌株多杀菌素产量都不及起始菌株高，接着采用不加抗生素直接筛选的方法，成功筛选到若干高产多杀菌素的融合菌株，其中菌株B-2多杀菌素产量提高最为明显，较原始菌株增加了331%，并用质谱分析做了进一步的验证。融合菌株B-2后续传代培养多杀菌素产量稳定，表明遗传稳定性较好。本文对红色糖多孢菌和刺糖多孢菌原生质体融合进行了探究并且成功提高了多杀菌素的产量，这为具有优良发酵特性的红色糖多孢菌和刺糖多孢菌融合菌株的构建提供了重要基础。     

基因组重排选育麦迪霉素高产菌株

目的以生米卡链霉菌(Streptomyces mycarofaciens)突变株为研究对象,选育麦迪霉素高产菌株。探索并验证基因组重排技术在菌种选育中的重要作用。方法通过2轮多亲株灭活原生质体融合技术实现基因组重排。将来自不同育种方法的5株麦迪霉素高产菌株B64-5、01-GM-1、H-101、H-106和H-108作为第一轮亲本,在质量浓度为3 g.L-1溶菌酶3、2℃水浴60 min条件下制备原生质体,分别采用紫外线照射148 s和52℃加热60 min灭活原生质体,然后采用质量分数35%PEG 4000、37℃保温2 min诱导原生质体融合。融合子经过初筛和复筛,获得产量进一步提高的重组子作为亲本进行第二轮的多亲株灭活原生质体融合实验。用生物学方法测定麦迪霉素效价,用HPLC方法测定麦迪霉素A1含量。结果与结论经过2轮基因组重排实验成功选育出3株高产且遗传稳定的麦迪霉素生产菌株。其中1株菌株GSZ2-32发酵前补加前体X-1后摇瓶产量比原始出发菌株Streptomyces mycarofaciens var.464提高1.1倍,麦迪霉素A1(MDMA1)质量分数含量保持在80%以上。     

非对称灭活原生质体融合法选育头孢菌素C高产菌株

目的选育高产头孢菌素C产生菌。方法分别以紫外线和加热灭活头孢菌素C产生菌Cephalosporium acremonium 18-U5和Cephalosporium acremonium 18-M6原生质体,并将两种灭活的原生质体经PEG4000融合,从融合株中筛选头孢菌素C高产菌株。结果获得高产头孢菌素C的顶头孢霉融合株Cephalosporium acremonium F20,发酵单位达4 655 mg.L-1。与双亲菌株相比分别提高了42.4%和34.9%。结论非对称灭活原生质体融合法选育头孢菌素C高产菌株的方法值得推广。     

原生质体诱变及高通量筛选选育链霉素高产菌株

目的 通过诱变和筛选方法的研究，提高灰色链霉菌(Streptomyces griseus)生产链霉素的水平。方法 优化灰色链霉 菌的原生质体的生成和再生条件，并对得到的原生质体进行紫外诱变，然后利用微孔板高通量方式对获得菌株进行筛选。结果 经过诱变选育获得一株菌NP-11703，其链霉素产量在100L罐上比出发菌株提高了21.8%。结论 用紫外诱变原生质体，可以有 效提高灰色链霉菌产链霉素的能力。结合高通量筛选模型的应用，可大大加快高产菌株的筛选效率。     

双亲灭活原生质体融合法选育阿维菌素高产菌株

目的选育高产阿维菌素产生菌。方法分别以紫外线和加热灭活阿维菌素产生菌Strepto-myces avermitilis620和Streptomyces avermitilis632原生质体,并将2种灭活的原生质体用PEG4000融合,从融合株中筛选阿维菌素高产菌种。结果获得高产阿维菌素融合株StreptomycesavermitilisF32,总发酵单位达3 904 mg.L-1,其中B1a组分产量较高,达1 016 mg.L-1,分别较出发菌株S.avermitilis 620、S.avermitilis 632提高117.1%和103.6%。结论双亲灭活原生质体融合法选育阿维菌素高产菌株是值得推广的一种选育方法。     

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.     

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.     

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.