胚胎发育相关基因stk40的蛋白表达和生物信息学分析

目的 检测胚胎发育相关基因 stk40 的表达,并对其进行生物信息学分析.方法 取小鼠早期各发育阶段的胚胎样本,用Western blot方法检测stk40的蛋白表达.用生物信息学软件或数据库分析预测stk40基因及其编码蛋白的基因结构、染色体定位、蛋白质理化性质、二级结构、疏水性/亲水性及结构域.结果 证实stk40蛋白在小鼠8细胞发育阻滞胚胎中的表达显著低于发育正常的早期胚胎.生物信息学分析显示,stk40基因mRNA全长3877 bp,开放阅读框长1350 bp,编码449个氨基酸,相对分子质量50563.9;定位于染色体4D2.2区域,含13个外显子和12个内含子.蛋白结构域分析提示,stk40基因编码蛋白存在-STYKc结构域,可能与细胞有机体的发生有关.结论 stk40基因编码蛋白的成功检测及生物信息学分析为进一步研究该基因奠定了基础.     

巴戟天MoDXR基因及其启动子的克隆与分析

从巴戟天中克隆MEP途径中的1-脱氧-D-木酮糖5-磷酸还原异构酶基因MoDXR及其启动子序列,并进行生物信息学分析、启动子区顺式作用元件分析,以及进行原核表达分析。根据巴戟天转录组中DXR基因原始序列和NCBI-ORFfinder分析,设计特异性引物,并进行RT-PCR扩增和生物信息学分析;采用染色体步移克隆MoDXR基因的5′端启动子序列;通过亚细胞定位分析MoDXR基因在细胞中的位置;构建原核表达载体pET-28a-MoDXR,导入BL21(DE3)表达感受态细胞后,在IPTG诱导下表达。从巴戟天中克隆的MoDXR基因,其cDNA全长2 015 bp,预测的阅读框大小为1 425 bp,编码474个氨基酸,分子质量为51.27 kDa; BlastP序列比对分析表明MoDXR基因与其他植物的DXR基因具有高度同源性,如:咖啡树DXR (CaDXR)、萝芙木DXR (RvDXR);系统进化发育树分析显示,巴戟天MoDXR蛋白与小粒咖啡和栀子的DXR蛋白聚为一类,其亲缘关系最近;由亚细胞定位可知MoDXR基因所编码的蛋白定位于叶绿体上; MoDXR基因5′端启动子序列长度为1 493...     

钩藤STR基因及其启动子的克隆与分析

根据钩藤转录组中STR基因核心序列设计特异性引物,并进行RACE扩增,克隆到UrSTR基因(GeneBank:OL310251)的cDNA全长1 541 bp,编码345个氨基酸;采用染色体步移克隆到UrSTR基因启动子(GeneBank:OL310252)序列1 179 bp。系统进化发育树分析显示,钩藤UrSTR蛋白与同为茜草科的美丽帽柱木和短小蛇根草的STR蛋白聚为一类,其亲缘关系最近;亚细胞共定位实验表明UrSTR蛋白定位于液泡膜上;SDS-PAGE结果表明pET-28a-UrSTR重组蛋白成功表达且大小与预期相符;启动子顺式作用元件分析表明其含有光响应、胁迫响应和激素响应有关的多种调控元件;启动子活性分析UrSTR基因启动子具有转录活性。成功克隆了UrSTR基因及其启动子序列并对其进行生物信息学分析和启动子活性分析;后期需要优化UrSTR原核表达体系,为进一步纯化UrSTR蛋白研究其结构和功能奠定基础。     

TNMD基因在肥胖者脂肪组织中的表达及生物信息学分析

目的 验证差异表达基因-Tenomodulin(TNMD)基因在肥胖者脂肪组织中的表达及生物信息学特征.方法 采用实时荧光定量RT-PCR法验证肥胖与正常人(各8例)脂肪组织中TNMD基因的表达筹异,应用生物信息学方法初步分析该基因的核苷酸基本特征、蛋白质理化性质、疏水性/亲水性、跨膜结构域、亚细胞定位及结构域等.结果 TNMD基因差异低丰度表达于肥胖者脂肪组织中.该基因cDNA全长1360 bp,开放阅读框长954 bp,编码317个氨基酸,预测分子量37kDa,定位于染色体Xq21.33-q23区带,含7个外显子和6个内含子;其编码蛋白为一非分泌的跨膜蛋白.定位于线粒体的可能性较大,存在一BRICHOS结构域.结论 成功验证TNMD基因在肥胖与正常者脂肪组织中的差异表达并对其进行生物信息学分析,为进一步研究该基因提供依据.     

一条脑胶质瘤相关的全长基因的生物信息学分析

目的报道一条脑胶质瘤相关的全长新基因的生物信息学分析及其研究的结果。方法对507E08克隆子进行了测序,生物信息学分析和Northern blot分析。结果507E08基因为全长基因,长度为2002bp,共编码203个氨基酸,命名为人核糖体蛋白L14．22。经Northern blot证实507E08基因在人正常脑组织中低表达,而在人脑胶质瘤中高表达。结论生物信息学是一种有效分析未知基因的工具。507E08基因可能是与人脑胶质瘤形成有关的一条全长新基因。     

豚鼠钙调蛋白1基因的克隆及序列分析

目的克隆豚鼠钙调蛋白1(CaM1)基因,对所克隆基因进行生物信息学分析。方法从豚鼠心室肌组织提取总RNA,应用RT-PCR方法扩增CaM1基因编码区447bp的cDNA片段,插入克隆载体构建重组质粒后基因测序及序列分析。结果克隆出的基因片段编码CaM1全部149个氨基酸,核苷酸序列与多种哺乳动物相应基因的同源性大于90%。获得了该序列的限制性内切酶酶切位点等信息。结论成功获得了豚鼠CaM1基因编码区序列,为进一步重组表达CaM及其功能研究奠定基础。     

华支睾吸虫硫氧还蛋白跨膜蛋白基因的生物信息学分析

目的对华支睾吸虫硫氧还蛋白跨膜蛋白(Cs TMX)基因进行生物信息学分析。方法利用生物信息学方法(Inter Pro Scan、Signal P、TMHMM和SWISS-MODEL等相关软件)对Cs TMX基因及相应氨基酸序列的同源性、理化性质、保守结构域、信号肽、亲水性/疏水性、三级结构进行预测分析。结果 Cs TMX基因的开放阅读框(ORF)包含414 bp,编码137个氨基酸,理论分子质量为16.04×103,等电点为5.16。TMHMM和Signal P3.0分析结果显示Cs TMX含信号肽,其在第19~20位氨基酸之间有1个切割位点;Inter Pro Scan和SWISS-MODEL分析结果显示,TMX特征性结构域位于第21~27位氨基酸之间,并且具有高度保守的CPAC基因序列。结论利用生物信息学知识和各种分析软件对Cs TMX所编码的c DNA序列及理论蛋白质的理化性质、结构和功能域等进行预测和分析,能够为开展Cs TMX的表达及其功能研究提供理论依据。     

AY887902全长扩增及其生物信息学研究

目的①对表达序列标签(EST)片段BQ135230进行全长扩增并应用生物信息学推测其开放阅读框(ORF)、染色体定位和功能。方法 ①从新鲜的膀胱肿瘤组织中提取RNA,并用快速cDNA末端扩增技术(RACE)方法获得BQ135230的5′端和3′端的DNA序列,然后应用GeneTool软件合成RACE测序结果。②根据NCBI提供的ORF Finder程序、电子-聚合酶链反应(e-PCR)程序、Blastn、Blastp,同时应用ExPASY ProtParam Tool、JUFO软件并结合Primer软件对AY887902全长及其相应蛋白AAX14401进行分析。Sequin软件编辑前述结果,上报NCBI GenBank,获取基因库获得序列号(GenBank accession number)。结果通过RACE得到了BQ135230的5′和3′端,GeneTool合成一个774 bp的全长序列。该序列在GenBank获得序列号(accession number):AY887902,初步分析含有完整的ORF和poly A尾。ORF翻译的多肽序列获得蛋白ID号AAX14401;染色体定位11q13,与GSTP1基因位置完全一致。AY887902的193¹95位编码赖氨酸的碱基AAA突变为终止子TGA,从而使有210个氨基酸的GSTP1突变为只有52个氨基酸的多肽,该假定蛋白不是跨膜蛋白和信号肽。结论①获得BQ135230的全长序列AY887902,推测该序列是GSTP1基因的突变体。②AAX14401在膀胱肿瘤的表达可能是膀胱癌发生发展的新的机制,为膀胱癌Ⅱ类癌提供了分子学标志,同时它有可能成为新的治疗靶点。     

美洲茶藨子查耳酮合成酶基因电子克隆和生物信息学分析

摘 要 目的：对美洲茶藨子查耳酮合成酶基因进行电子克隆及生物信息学分析。方法: 以黑茶藨子查耳酮合成酶基因序列和美洲茶藨子表达序列标签为基本材料（探针）,运用CAP3在线软件进行序列拼接,并对其进行了生物信息学分析。结果: 美洲茶藨子查耳酮合成酶基因全长1 423 bp,包含1 173 bp的开放阅读框,编码390个氨基酸,该蛋白是定位于细胞质的亲水性蛋白,存在三处跨膜区,二级结构主要由α-螺旋构成。结论: 研究结果有利于美洲茶藨子查耳酮合成酶基因的分子作用机制等方面的研究。     

薏苡delta-12脂肪酸去饱和酶基因FAD2的克隆和表达分析

本文克隆了薏苡delta-12脂肪酸去饱和酶基因FAD2 (fatty acid desaturase),并对其分子结构特征和功能进行了研究.结果 表明,薏苡FAD2基因的cDNA全长序列为936 bp,编码311个氨基酸残基.生物信息学预测结果显示,该基因编码蛋白为碱性亲水不稳定蛋白,分子质量34.87 kDa,含有...     

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.     

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.