木瓜凝乳蛋白酶动力学研究

以软骨蛋白多糖为底物,木瓜凝乳蛋白酶的最适反应温度为60°C(pH=7.0),最适pH值7.0(37℃).在pH值7.0、反应温度为37℃的条件下,木瓜凝乳蛋白酶的Km值为11.11 mg/ml,Vmax为0.03 mg/(min·ml).一定浓度下的NaCl和Ca2+对木瓜凝乳蛋白酶均有激活作用,并研究了其它金属离子对酶活性的影响.     

赤子爱胜蚓脱氧核糖核酸酶酶动力学研究

目的对赤子爱胜蚓中一组新型脱氧核糖核酸酶(EWDs)进行酶动力学研究。方法运用单相酶扩散法(SRED)、紫外分光光度法和林-贝氏(Lineweaver-Burk)作图法。结果EWDs的最适温度均为37℃。EWD1的最适pH为5.2,Km为1.58mg/ml,Vmax为5.36mg·ml-1·min-1;EWD2的最适pH为4.4,Km值是3.95mg/ml,Vmax值为2.87mg·ml-1·min-1;EWD3的最适pH为4.8,Km值是1.52mg/ml,Vmax值为4.89mg·ml-1·min-1。Mn2+和Ca2+是EWDs的抑制剂,Mg2+仅抑制EWD3的活性。结论蚯蚓组织中发现的此组脱氧核糖核酸酶具有特殊的酶学性质,区别于已知的脱氧核糖核酸酶类。     

石刁柏细胞的L-天冬酰胺酶发酵条件的研究

优化了石刁柏细胞L-天冬酰胺酶发酵条件并对其粗提进行了探索。其最佳产酶条件为:最适培养基为MS 蔗糖(35 g/L) 6-(苄胺基)嘌呤(0.01 mg/L) α-奈乙酸(3 mg/L)最适pH值为6.4、最适温度为27℃、最适转速为110 r/min、接种量为5.0×104个/ml。在此发酵条件下,L-天冬酰胺酶发酵液酶活力的峰值可达到22.37 U/ml、产酶稳定期持续7~8 d,利用吴氏提取工艺可以得到回收率为30.8%,纯化倍数为9.85,比活力为0.55 U/mg的L-天冬酰胺酶。     

聚乙二醇修饰牛胰核糖核酸酶

采用N-羟基珀酰亚胺活化酯法活化单甲氧基聚乙二醇,测定了聚乙二醇（PEG）的活化度为86.2%。以活化的PEG对牛胰核糖核酸酶进行化学修饰;分析了蛋白质被修饰程度。用毛细管电泳法给出了被修饰蛋白的修饰度与修饰蛋白分布的定量结果。比较了被修饰产物对大分子底物(酵母RNA)与小分子底物（2',3'-环磷酸胞嘧啶）的降解活力,其表观酶活力分别保留了52.8%和66.3%。结合毛细管电泳定量分析得到的修正酶活力略低于表观酶活力。     

亚甲基四氢叶酸还原酶的纯化及催化性质研究

对亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)的体外酶促反应性质进行研究。通过盐析、DEAE-Sepharose fast flow柱层析纯化猪肝中的MTHFR,采用荧光法分析测定该酶纯化结果和酶促动力学性质。结果表明,MTHFR的纯化倍数可达20倍以上;测得最适酶促反应条件为pH 6.8,37℃;NADPH浓度1.2 mmol/L;以5,10-亚甲基四氢叶酸为底物测得米氏常数km=70.3μmol/L。研究发现,别构抑制剂S-腺苷甲硫氨酸(AdoMet)可使MTHFR的体外活性降到原活性的60%以下。本文初步探讨了MTHFR的催化性质,对MTHFR催化活性的主要影响因素进行研究,为酶促法合成5-甲基四氢叶酸的进一步研究奠定了基础。     

透明质酸酶催化透明质酸水解的最适反应条件

目的确定透明质酸酶(HAase)催化透明质酸(HA)水解的最适条件。方法HAase不同条件下催化HA水解,反应结束后利用高效凝胶渗透色谱(HPGPC)法测量反应产物的相对分子质量及其分布系数。结果HAase催化HA水解受温度、pH值、酶浓度、底物浓度、反应时间等因素的影响。结论HAase催化HA水解的最适反应条件是底物浓度为10 g/L,酶浓度为150 000 U/L,pH为5.0,反应温度为50℃。     

重组L-门冬酰胺酶的聚乙二醇化学修饰及修饰后酶的稳定性研究

目的单甲氧基聚乙二醇 (mPEG)化学修饰大肠杆菌重组L 门冬酰胺酶 (L ASP) ,考察经过修饰的酶的稳定性。方法N 羟基琥珀酰亚胺 (NHS)活化酯法活化mPEG ,生成的单甲氧基聚乙二醇琥珀酰琥珀酸亚胺酯 (SS mPEG)按不同摩尔比例与L ASP偶联 ,确定适合的反应时间和反应pH值。通过聚乙二醇化学修饰后的酶 (L ASP PEG) ,酶活力和纯度通过奈氏法和丙烯酰胺凝胶电泳 (SDS PAGE)检测 ,高效液相色谱检测L ASP PEG相对分子质量并考察了L ASP PEG体外稳定性等。结果SDS PAGE显示mPEG已经偶联到L ASP分子上 ,以两者摩尔比 1 0∶1为最佳 ,反应pH条件为 8.5 ,获得的L ASP PEG平均比活单位为 6 4 .8IU/mg ,相对分子质量为 30 1 80 0 ,体外稳定性高于L ASP。结论此实验确定了mPEG化学修饰L ASP最佳反应条件为 2 5℃反应 30min ,两者投料摩尔比为 1 0∶1 ,获得的L ASP PEG比L ASP稳定性高     

右旋糖苷对大肠杆菌L—天门冬酰胺酶Ⅱ的化学修饰

用高碘酸氧化法活化的右旋糖苷对大肠杆菌Ｌ－天门冬酰胺酶进行化学修饰，修饰反应的酶活力回收率为４６．８％，修饰酶冻干品比活为１１８．４ＩＵ／ｍｇ蛋白质，酶经修饰后，对底物的亲和力未发生变化，但抗胰蛋白酶水解能力明显提高，抗显性显著减弱。     

海藻酸钠法固定化谷氨酸脱羧酶的研究

目的研究海藻酸钠法固定化谷氨酸脱羧酶的最适条件。方法以海藻酸钠为载体,分别研究了海藻酸钠浓度、CaCl2浓度、固定化时间和固定化酶的最适pH、最适温度、最适底物浓度及反复利用的稳定性。结果最适固定化条件为3%海藻酸钠、0.1 mol/L CaCl2、固定化时间6 h。固定化酶的最适pH为5.0、最适温度为42℃、最适底物浓度1%,反应进行4次后一半的酶活损失,底物转化率在90%以上。结论海藻酸钠法固定化谷氨酸脱羧酶可以很好的转化谷氨酸钠生成γ-氨基丁酸,具有实用的潜力。     

牛肝β-D-葡萄糖醛酸苷酶的分离纯化和性质研究

采用自溶、35 %硫酸铵分部沉淀、CM 纤维素柱层析、羟基磷灰石柱层析由动物肝脏中分离纯化β D 葡 萄糖醛酸苷酶 ,获得比活 2 6 70 0U/mg的纯酶 ,活力回收率为 4 2 .8% ,纯化的酶经SDS PAGE呈单一条带。该酶以 β D 酚酞葡萄糖醛酸苷为底物的Km值为 2 .0× 10 -4mol/L ,以对硝基苯酚葡萄糖醛酸苷为底物的Km值为 2 .5× 10 -4mol/L。在 pH值 5～ 6的范围内 ,该酶的活力较高 ,最适 pH值为 5 .5 ,在pH值 5 .5时稳定性最好。在 30～ 5 0℃范围内酶活力均较稳定     

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.     

Pharmacological treatment of COVID-19: an opinion paper

The precocity and efficacy of the vaccines developed so far against COVID-19 has been the most significant and saving advance against the pandemic. The development of vaccines has not prevented, during the whole period of the pandemic, the constant search for therapeutic medicines, both among existing drugs with different indications and in the development of new drugs. The Scientific Committee of the COVID-19 of the Illustrious College of Physicians of Madrid wanted to offer an early, simplified and critical approach to these new drugs, to new developments in immunotherapy and to what has been learned from the immune response modulators already known and which have proven effective against the virus, in order to help understand the current situation.     

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.