检测流感疫苗中宿主细胞DNA残留量的地高辛标记探针杂交法的建立

目的  建立地高辛标记DNA探针杂交法,检测基于Madin-Darby犬肾（Madin-Darby canine kidney,MDCK）细胞生产的流感疫苗中宿主DNA的残留量。方法  提取MDCK细胞基因组DNA,制备地高辛标记探针。对地高辛标记DNA探针杂交法进行特异性、灵敏度及稳定性验证, 并用此法检测3批MDCK细胞流感疫苗中的DNA残留量。结果 MDCK细胞基因组DNA的质量浓度为33 ng/μl,260 nm与280 nm波长处的吸光度比值为1.9。制备的探针与非同源细胞DNA无杂交,最低检测限度为10 pg。探针在－70 ℃放置9个月后,检测灵敏度仍可达到10 pg。经检测,MDCK细胞流感疫苗成品中的DNA残留量＜10 ng。结论  建立的地高辛标记探针杂交法特异性强、灵敏度高、稳定好,并且操作简便,可用于MDCK细胞流感疫苗中DNA残留量的检测。     

糖尿病对游离地高辛浓度的影响及其测定

目的比较在同一地高辛血药浓度水平下,单纯心力衰竭患者及心力衰竭伴糖尿病患者的游离地高辛浓度的差异,为临床制定合理的地高辛用药方案提供可靠的实验室数据。方法18例地高辛适应证患者按是否有糖尿病分为A、B两组。A组为单纯心力衰竭患者,B组为心力衰竭伴糖尿病患者,分别用荧光偏振免疫测定法(FPIA法)测定其地高辛血药浓度,用超滤-FPIA法测定其血清中游离地高辛浓度。采用成组t检验对两组数据进行统计分析,对两组间地高辛总浓度及游离地高辛进行比较。结果A组地高辛总浓度(1.29±0.29)ng/ml(0.81～1.63ng/ml);B组地高辛总浓度(1.45±0.42)ng/ml(0.88～1.98ng/ml)。A组游离地高辛浓度(0.63±0.11)ng/ml(0.42～0.74ng/ml);B组游离地高辛浓度为(0.87±0.19)ng/ml(0.59~1.07ng/ml)。结论在地高辛总浓度处于同一水平情况下,心力衰竭伴糖尿病患者的游离地高辛浓度与单纯心力衰竭患者的游离地高辛浓度具有统计学差异,前者浓度和游离百分率均明显高于后者(P<0.05)。     

抗生素影响地高辛的血液浓度

地高辛的治疗指数低,医院中应用地高辛的患者,中毒发生率为10～30%,其中老年、肾功能不全、慢性肺疾患和甲状腺机能低下患者更多见。放射免疫方法测定血清中地高辛的浓度可以精确地估计个体的剂量需要,多数房颤患者控制心率所需浓度在1.0和2.5nmol/l(0.8～2.0ng/l)之间。地高辛的剂型可以影响其血液浓度,许多药物可以与地高辛产生相互作用,灭吐灵和普鲁本辛能改变胃排空的速度而降低或增加地高辛的血液浓度,合并应用利尿剂常致血钾降低而使洋地黄中毒发生率增加。几种治疗心脏病的药物可使地高辛的血液浓度增加,如异博定(增加70～80%)、硝苯毗啶(增加45%)、乙胺碘呋酮(增加70%)。奎尼丁能使地高辛的平均血浓从1.1nmol/l 增加至2.0nmol/l(0.9ng/l 至1.6ng/l),据信其可能的原因之一是     

Sabin株脊髓灰质炎灭活疫苗宿主细胞DNA残留量检测方法研究

目的 建立Sabin株脊髓灰质炎灭活疫苗Vero细胞DNA残留量的检测方法.方法 提取Vero细胞基因组DNA,制备地高辛标记探针.比较样品不同处理方式的检测差异.对建立的残留DNA检测方法进行特异性、灵敏度和稳定性验证.结果 Vero细胞基因组DNA的质量浓度为295 μg/ml,260nm与280nm波长处的吸光度比值为1.88.探针灵敏度达到0.01 pg/μl.采用苯酚抽提方式处理DNA参考品,检测灵敏度为1 ng;而用碘化钠处理DNA参考品,灵敏度达到10 pg.特异性检测表明,探针与非同源DNA无杂交.灵敏度和稳定性检测结果显示,探针于-70℃保存9个月,灵敏度仍为10 pg.结论 建立了Sabin株脊髓灰质炎灭活疫苗Vero细胞DNA残留量的检测方法.采用碘化钠提取DNA,回收率高,操作简单,适用于Vero细胞的残留DNA检测.     

用柱切换HPLC法测定大鼠、狗及人血浆中的氢氯噻嗪

已有的 HPLC 测定生物液体中氢氯噻嗪(Ⅰ)浓度的方法大多需繁琐的样品处理步骤,采用本文方法,血浆样品可直接注入色谱系统作全自动分析。溶液的制备分别配制1.0mg/ml 的(Ⅰ)和内标氢氟噻嗪(Ⅱ)的甲醇溶液作为贮备液,于-20℃保存。将(Ⅰ)的贮备液用10mmol/L 的磷酸盐缓冲液(pH7.2)稀释至100μg/ml,并以空白血浆(先离心去微粒)进一步稀释制得血浆标准液。以相同缓冲液稀释内标液至1500ng/ml。血浆样品室温解冻后离心(3000rpm,10min)去微粒,将150μl 血浆标准液或样品液与150μl 内标液混合,采用自动进样器将100～200μl 混合液注入 HPLC 仪。色谱系统包括提取柱(两个串联的Guard-Pak C_(18)柱,柱长0.4cm)和串联Guard-Pak C_(18)保护柱的分析柱(HypersilODS,5μm,4.6×250mm),分别联于六孔     

疾病对老年人游离地高辛浓度的影响及意义

目的:比较在同一地高辛血药浓度水平下,单纯老年心衰患者及心衰伴糖尿病患者的游离地高辛浓度的差异,为临床制订合理的地高辛用药方案提供可靠的实验室数据.方法:18例地高辛适应症患者按是否伴有糖尿病分为两组,A组为单纯心衰患者,B组为心衰伴糖尿病患者.分别用FPIA方法测定患者地高辛血药浓度,用超滤-FPIA法测定血清中游离地高辛浓度.结果:地高辛总浓度和游离浓度,A组分别为(1.286±0.294)ng/mL,(0.626±0.106)ng/mL,B组分别为(1.45±0.417)ng/mL,(0.870±0.190)ng/mL.结论:在地高辛总浓度处于同一水平的情况下,心衰伴糖尿病患者与单纯心衰患者的游离地高辛浓度具有统计学差异,前者浓度和游离百分率均明显高于后者(P＜0.05).     

标记死亡受体5、诱捕受体2地高辛探针

目的标记死亡受体5(death receptor-5,DR5/TRAILR2)和诱捕受体2(death decoyreceptor 2,DcR2/TRAILR4)地高辛探针。方法查阅核酸序列数据库上DR5和DcR2的基因全序列,计算机辅助设计引物,RT-PCR扩增目的条带,用地高辛(Digoxin,DIG)进行标记,制作DR5和DcR2地高辛探针。结果地高辛标记的DR5和DcR2电泳能力下降,标记成功。结论死亡受体5,诱捕受体2地高辛探针的标记成功为进一步研究其与喉鳞状细胞癌的关系奠定了一定的基础。     

特异性单克隆抗体对地高辛抑制家兔红细胞膜Na~+,K~+-ATP酶活性的拮抗作用

本文研究结果表明抗地高辛特异性MAb可有效地拮抗地高辛引起的RRBC膜Na~+,K~+-ATP酶活性抑制。1.8×10~4倍稀释的FD8 MAb腹水,在试管内可使4ng/ml浓度地高辛引起的酶活性抑制恢复达50％。给地高辛中毒兔iv FD8 MAb 腹水后,被抑制的RRBC膜Na~+,K~+-ATP酶活性迅速恢复,酶抑制造成的RRBC内Na~+,K~+浓度异常也随之得到恢复。本研究从受体水平证实了特异性MAb解除地高辛毒性的作用机理     

神经生长导向因子Slit mRNA在人胚胎脊髓的原位杂交定位

目的 检测神经生长导向因子Slit mRNA在人胚胎脊髓中的定位表达。方法 地高辛标记的cRNA探针原位杂交技术。结果 Slit mRNA在脊髓中央管、前角和后角处表达。结论 神经生长导向因子Slit在人胚胎脊髓发育期轴突投射和神经通路形成中可能起重要作用。     

限制性片段长度多态性分析酶切条件的优化

目的：对结核分枝杆菌RFLP—DNA分型技术的各个环节进行优化，以提高DNA指纹的清晰度和稳定性。方法：提取结核分枝杆菌DNA，经PvuII酶切后进行琼脂糖凝胶电泳、southern转印，并用经地高辛随机引物标记试剂盒标记IS6110的245bp探针进行杂交。结果：用于结核分枝杆菌DNARFLP分析的最佳DNA浓度是2μg，内切酶浓度为2u／μgDNA，酶切反应时间为4h。结论：RFLP的酶切条件经过合理优化，可以在消耗最低的情况下取得最佳的实验效果。     

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.     

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.