一种新型聚乙二醇化重组人生长激素的制备及性质考察

目的制备一种新型聚乙二醇化重组人生长激素(PEG-(rhGH)2),并对其理化性质及药理作用进行研究。方法以分子质量为2×104u的聚乙二醇二丙醛(PEG-(ALD)2)对重组人生长激素(rhGH)进行修饰,采用Sephacryl S-200凝胶过滤色谱、Q Sepharose FF离子交换色谱对修饰产物进行分离纯化,SDS-PAGE鉴定各组分,通过SEC-HPLC、IEF、MALDI-TOF-MS等方法对目标产物进行分析,并采用去脑垂体大鼠模型研究其体内生物活性与长效作用。结果获得了一种新型聚乙二醇化重组人生长激素(PEG-(rhGH)2)。其纯度质量分数达到97%以上,等电点为5.27,分子质量为6.6×104u。与未修饰的rhGH相比,PEG-(rhGH)2具有高效、长效的促进机体生长的药理作用。结论新型聚乙二醇化重组人生长激素PEG-(rhGH)2是一种具有开发前景的长效蛋白质药物,值得进一步深入研究。     

[D-Ala~2,D-Ala~5]-脑啡肽及其聚乙二醇修饰对热板致痛小鼠的镇痛作用

<正>我们曾经报告,相对分子质量2 ku、5 ku直链甲氧基聚乙二醇(mP EG)修饰的脑啡肽(Tyr-Gly-Gly-Phe-Met,ENK)半衰期延长、清除率下降、镇痛药效增强[1]。在此基础上,本文对D-Ala替换ENK第2、5位氨基酸([D-Ala~2,D-Ala~5]-ENK,DADAE)、及其5 ku分子质量mP EG修饰双重结构改造的合成脑啡肽类似物进行了镇痛药效分析。     

新型定点修饰的聚乙二醇化重组人生长激素修饰位点研究

目的:建立新型定点修饰的聚乙二醇化重组人生长激素(PEG-rhGH)修饰位点的研究方法,为该类新型修饰技术产物的质量评价提供依据。方法:采用MALDI-TOF质谱分析修饰产物中聚乙二醇(PEG)的修饰个数及修饰肽段的质量数;采用LC-Q-TOF质谱分析修饰位点,分析色谱柱为UPLC色谱柱(Protein ACQUITY BEH C₄ Column, 150 mm×2.1 mm, 1.7μm, 300?),以0.1%甲酸水溶液-0.1%甲酸乙腈溶液为流动相,进行梯度洗脱,柱温为40℃。质谱数据采集条件为MS~E模式,一级质谱能量4 eV,二级碎裂电压30～55 eV。结果:修饰蛋白中PEG的平均相对分子质量为30 000,并且每个PEG-rhGH分子中仅存在1个PEG修饰;原型蛋白重组人生长激素中第134位精氨酸被替换为赖氨酸,且该赖氨酸的ε-氨基与连接子HOOC-O-CH₂-CH₂-N₃中的羧基端共价结合,连接子另一端的叠氮基团与活化后的PEG偶联后生成修饰产物。结论:联合运用多种质谱技术,通过比对...     

单甲氧基聚乙二醇5000修饰胶原酶的初步研究

为解决胶原酶免疫原性，采用经氯甲酸对硝基苯酯活化的单甲氧基乙二醇来修饰胶原酶。每个酶分子接上４￣５个单甲氧基聚乙二醇分子，可保持原有酶活力的４５％以上。     

聚乙二醇化新型干扰素

干扰素临床上用于治疗慢性病毒性肝炎、各种肿瘤已有近 2 0年的历史 ,其疗效和安全性得到了广泛的认同。由于干扰素是基因重组蛋白类药物 ,多采用注射剂型 ,且其半衰期很短、难以充分发挥疗效 ,长期治疗、频繁给药给病人也带来很大的不便和痛苦。如何延长重组蛋白在体内的存留时间 ,是基因药物二次开发的一个热点。聚乙二醇 (ｐｏｌｙｅｔｈｙｌｅｎｅｇｌｙｃｏｌ,简称ＰＥＧ)是一种无毒的线型分子 ,因其结构特点而具有水溶性、免疫学惰性和分子大小可改变性。聚乙二醇经活化后用于修饰蛋白 ,在蛋白分子外形成“一保护层” ,以保护蛋白被降…     

聚乙二醇修饰重组人干扰素α-2b修饰产物的初步分析研究

目的 对聚乙二醇(PEG)修饰重组人干扰素α-2b(rhIFNa-2b)的修饰产物进行了初步分析研究。方法以单甲氧基聚乙二醇甲酸琥珀酰亚胺酯对rhIFNα-2b进行修饰,修饰产物以Superdex 75 Highload制备型凝胶色谱柱进行分离、纯化,SDS-PAGE鉴定各组分,采用Mariner电喷雾飞行时间质谱对单修饰IFNα-2b(Inono-PEG-IFNα-2b)进行分析。结果 分别获得了纯化的mono-PEG-IFNα-2b和多修饰IFNα-2b(poly-PEG-IFNα-2b)。ESI-TOF质谱也证明mono-PEG-IFNα-2b分子量比rhIFNα-2b多5 000左右;紫外光谱分析表明,PEG修饰产物的结构没有发生改变。结论获得了纯化的PEG修饰产物。     

聚乙二醇修饰水蛭素的分离纯化与活性分析

用活化的聚乙二醇(PEG)对水蛭素进行化学修饰，质谱分析表明，修饰产物是修饰度不同的分子质量相差5000u的水蛭素的混合物。用离子交换层析和凝胶过滤层析对反应产物进行分离纯化，离子交换层析难以达到完全分离；而采用凝胶过滤层析方法可较好地分离修饰度不同的各组份。生物学活性分析表明，不同的PEG修饰度对水蛭素的活性具有显著影响，连接1～2个PEG的水蛭素保持了原有活性，而连接3个PEG的水蛭素活性显著下降。     

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。     

聚乙二醇干扰素

聚乙二醇(PEG)具有溶解性良好、毒性很小和无抗原性的特性.PEG修饰蛋白质使分子量增加,在体内半衰期延长,可以减少用药次数,提高治疗效果.本文对PEG的结构、性状、对干扰素修饰的方法、修饰后的药代动力学和临床效果进行综述.PEG修饰重组人干扰素α2a和PEG修饰重组人干扰素α2b在体内药代动力学均有明显改善,半衰期明显延长.两种PEG干扰素均已进行临床试验.每周注射1次治疗丙型肝炎,效果明显较普通干扰素为好.其安全性与普通干扰素基本相似.     

聚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。     

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.     

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.