基于网络药理学探究知柏地黄丸治疗乳腺癌内分泌耐药的作用机制

目的 运用网络药理学技术探究知柏地黄丸治疗乳腺癌内分泌耐药的活性成分、潜在靶点和作用机制。方法 通过TCMSP数据库、TCM-ID数据库和BATMAN-TCM数据库，筛选知柏地黄丸的有效活性成分和作用靶点，利用GeneCards数据库、OMIM数据库、TTD数据库和GEO数据库检索获得乳腺癌内分泌治疗耐药的相关靶基因，并与活性成分作用靶点取交集得到共同靶点；通过String 11.5数据库构建知柏地黄丸治疗乳腺癌内分泌耐药的蛋白质互作（PPI）网络，并借助Cytoscape 3.8.2软件的CytoNCA插件筛选核心靶点；通过Metascape基因功能注释分析工具对交集靶点进行基因本体（GO）和京都基因组百科全书（KEGG）通路富集分析，使用Cytoscape 3.8.2软件构建药物活性成分–靶点–通路相互作用网络，获取核心活性成分。结果 共筛选出知柏地黄丸活性成分80个，与疾病的交集靶点117个，主要涉及肿瘤蛋白p53（TP53）、蛋白激酶B1（Akt1）、肿瘤坏死因子（TNF）、白细胞介素-6（IL6）、雌激素受体1（ESR1）、丝裂原活化蛋白激酶1（MAPK1）等。KEGG通路富集分析显示磷脂酰肌醇3-激酶（PI3K）/Akt、MAPK、核因子-κB（NF-κB）、雌激素等信号通路可能是知柏地黄丸治疗乳腺癌内分泌耐药的关键信号通路，由药物活性成分–靶点–通路网络得出槲皮素、山柰酚、脱水淫羊藿素、β-谷甾醇、薯蓣皂苷元等是发挥作用的核心活性成分。结论 知柏地黄丸中的槲皮素、山柰酚、脱水淫羊藿素、β-谷甾醇、薯蓣皂苷元等核心活性成分，能够作用于TP53、Akt1、TNF、IL-6、ESR1、MAPK1等多个关键靶点，调节PI3K-Akt、MAPK、NF-κB、雌激素等信号通路，发挥治疗乳腺癌内分泌耐药的作用。     

基于网络药理学的丹参川芎嗪注射液入血成分治疗脑梗死机制分析

目的 应用网络药理学方法研究丹参川芎嗪注射液入血成分治疗脑梗死的作用机制。方法 通过文献挖掘确定丹参川芎嗪注射液入血成分,在TCMSP、Swiss Target Prediction数据库检索入血成分靶点,通过Gene Cards数据库搜集脑梗死靶点,将两者靶点筛重得到入血成分治疗脑梗死的作用靶点,借助Cytoscape软件构建入血成分-治疗脑梗死靶点网络;应用STRING平台构建入血成分靶点相互作用网络,并导入Cytoscape软件计算拓扑参数,筛选关键靶点;利用DAVID数据库进行KEGG通路富集分析。结果 丹参川芎嗪注射液中丹参素、丹酚酸B、川芎嗪等14种入血成分主要作用于NOS3、VEGFA、IL4、IL2、F2等关键靶点,调控PI3K-Akt信号通路、VEGF信号通路、TNF信号通路、雌激素信号通路和血小板激活等。结论 丹参川芎嗪注射液主要通过改善微循环、抗凝、保护血管内皮细胞等药理作用,发挥治疗脑梗死作用。     

基于网络药理学和实验验证探究白藜芦醇治疗糖尿病视网膜病变的作用机制

目的 采用网络药理学的方法探讨白藜芦醇在糖尿病视网膜病变治疗中的潜在机制,为白藜芦醇防治糖尿病视网膜病变提供理论依据。方法 使用CTD、DGIdb、Drugbank、Swiss Target Prediction、TCMSP数据库获得白藜芦醇的作用靶点。从GeneCard、DisGeNET、OMIM、DrugBank数据库获得糖尿病视网膜病变疾病相关靶点。使用Venn图取交集即为白藜芦醇抗糖尿病视网膜病变作用靶点。将白藜芦醇抗糖尿病视网膜病变靶点蛋白上传至String数据库,将所得数据导入Cytoscape 3.7.1,构建白藜芦醇抗糖尿病视网膜病变靶点蛋白的蛋白互作（PPI）网络,并筛选出核心作用靶点。使用DAVID数据库对白藜芦醇抗糖尿病视网膜病变靶点基因进行基因本体（GO）和京都基因与基因组百科全书（KEGG）通路富集分析。对核心作用靶点与白藜芦醇结合力进行分子对接验证。利用细胞活力实验观察白藜芦醇对高糖作用下的人视网膜血管内皮细胞（HRCECs）细胞增殖的影响;检测不同浓度（40、80、160 μmol/L）白藜芦醇对高糖作用下HRCECs细胞STAT3、VEGFA、TNF mRNA表达的影响。结果 共获得白藜芦醇抗糖尿病视网膜病变作用靶点130个,并筛选出核心靶基因10个。KEGG途径分析富集的信号通路包括TNF信号通路、HIF-1信号通路、FoxO信号通路等。分子对接显示,核心靶基因AKT1、IL-6、TNF、VEGFA、IL-1B、MAPK3、EGFR、JUN、STAT3及CASP3与白藜芦醇亲和力较好,尤其与STAT3、VEGFA和TNF具有强烈的结合活性。细胞实验显示,与模型组相比,白藜芦醇能明显抑制高糖作用下HRCECs的细胞增殖能力（P＜0.05、0.01）。同时能明显降低高糖作用下HRCECs细胞STAT3、VEGFA、TNF mRNA表达（P＜0.05、0.01）。结论 白藜芦醇治疗糖尿病视网膜病变具有多靶点、多途径的特点。白藜芦醇可能通过作用于VEGFA、AKT1、CASP3、IL-6、STAT3、EGFR、TNF、和MAPK3等核心靶点基因,影响TNF信号通路和HIF信号通路等发挥对糖尿病视网膜病变的治疗作用。     

猪肺磷脂注射液联合高频振荡通气治疗新生儿呼吸衰竭的临床研究

目的 基于网络药理学方法探讨麻杏石甘汤的主要活性成分及治疗支气管哮喘的作用机制。方法 在中药系统药理学分析平台（TCMSP）数据库中检索麻杏石甘汤的化学成分及靶点,采用Cytoscape 3.7.2软件构建活性成分-预测靶点网络图。在Genecards数据库中获取支气管哮喘疾病靶点,与药物靶点映射获得麻杏石甘汤作用于支气管哮喘的预测靶点。通过STRING数据库构建靶点相互作用网络,导入Cytoscape 3.7.2中筛选麻杏石甘汤的核心靶点。利用DAVID数据库及Cytoscape3.7.2对麻杏石甘汤的核心靶点进行KEGG信号通路和GO生物过程富集分析,并构建麻杏石甘汤活性成分-核心靶点-信号通路网络图。结果 预测结果显示,麻杏石甘汤中136个主成分可靶向98个哮喘相关靶点,关键有效成分包括麻黄碱、槲皮素、木犀草素、山柰酚等,核心靶点包括IL-6、MAPK3、TNF、TP53、VEGFA、JUN、EGFR、EGF、NOS3、CAT,涉及HIF-1、PI3K-Akt、MAPK、雌激素等71个信号通路和RNA聚合酶II启动子转录、细胞凋亡过程、ERK1和ERK2级联的正调控、上皮细胞增殖、平滑肌细胞增殖等20个生物过程。结论 初步揭示了麻杏石甘汤的药效物质基础及治疗支气管哮喘的机制,为麻杏石甘汤治疗支气管哮喘的研究提供参考。     

基于网络药理学和实验验证探讨石榴皮对非酒精性脂肪性肝炎的作用机制

目的 基于网络药理学和细胞实验验证的方法探讨石榴皮改善非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)作用机制。方法 利用中药系统药理学数据库(TCMSP)进行条件检索,获取石榴皮的活性成分及其对应的作用靶点。利用人类基因数据库(GeneCards)等5个数据库获取NASH相关的靶点。将获得的石榴皮和NASH靶点进行筛选,通过韦恩图得到共同靶点。使用蛋白相互作用数据库(STRING)构建蛋白质-蛋白质相互作用网络,并用Cytoscape 3.7.1建立“石榴皮-成分-靶点-NASH”网络。利用Metascape软件进行基因本体(gene ontology,GO)富集分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析。最后借助人肝癌细胞HepG2观察石榴皮中主要活性成分对NASH的影响。结果 石榴皮活性成分有7个,获得靶点基因191个,NASH靶点1 818个,两者交集靶点98个,拓扑学分析显示,石榴皮治疗NASH的核心成分为槲皮素、山奈酚和木犀草素,核心靶点为蛋白激酶B (protein kinase B,Akt1)、白细胞介素-1β(interleukin 1β,IL-1β)、白细胞介素-6(interleukin 6,IL-6)、肿瘤坏死因子(tumor necrosis factor,TNF)等。KEGG通路分析结果显示石榴皮治疗NASH主要涉及磷脂酰肌醇-3-激酶(phosphatidylinositol 3 kinase,PI3K)/Akt、核因子κB (nuclear factor kappa-B,NF-κB)等信号通路。体外细胞试验结果显示,与对照组相比,模型组磷酸化蛋白激酶B (phosphorylated-Akt^Thr308,p-Akt^Thr308)、IL-6等蛋白表达水平升高(P<0.05);与模型组相比,石榴皮活性成分可显著降低p-Akt^Thr308、IL-6等蛋白的表达水平(P<0.05),也能够降低IL-6、TNF-α基因的mRNA表达水平(P<0.05)。结论 石榴皮可通过多成分、多靶点、多通路发挥抗NASH的作用,其机制可能与石榴皮中活性成分槲皮素、山奈酚和木犀草素影响Akt1等核心靶点及调控PI3K/Akt、NF-κB等信号通路,进而抑制相关炎症因子表达有关。     

基于网络药理学与分子对接的加减香砂六君子汤治疗新型冠状病毒肺炎恢复期肺脾气虚证的机制研究

目的 探讨加减香砂六君子汤治疗新型冠状病毒肺炎（COVID-19）恢复期肺脾气虚证的作用机制。方法 利用TCMSP数据库筛选中药活性成分和靶点，利用Gendcars、OMIM数据库筛选疾病靶点，STRING平台对靶点进行PPI网络构建，并进行GO和KEGG分析，利用Cytoscape 3.7.2软件构建“药物-活性成分-靶点-疾病”网络图，然后通过拓扑学参数分析获得加减香砂六君子汤主要活性成分和靶点，最后将主要活性成分与靶点进行分子对接验证。结果 得到槲皮素、木犀草素、山柰酚、柚皮素等115个活性成分，PTGS2、NOS2、PPARG、PTGS1、MAPK14等48个靶点，主要涉及IL-17、TNF、T细胞受体、MAPK、VEGF等155条通路。分子对接显示，槲皮素、木犀草素、山柰酚、柚皮素与3CL水解酶、ACE2、PTGS2具有较好的结合活性，与第七版推荐抗病毒药物结合能相近。结论 加减香砂六君子汤活性成分可通过与3CL水解酶、ACE2、PTGS2结合，调控IL-17、TNF、T细胞受体、MAPK、VEGF等信号通路来抑制炎症反应，调节机体免疫，减轻肺损伤，促进细胞生长分化和肺血管生成，从而改善患者症状，促进恢复期患者康复，提高机体免疫力，降低疾病复发或再感染的风险。     

基于网络药理学研究柴胡达胸合剂治疗新型冠状病毒肺炎的作用机制

目的 基于网络药理学方法探讨柴胡达胸合剂治疗新型冠状病毒肺炎（COVID-19）的潜在药理作用机制。方法 利用中药系统药理学数据库和分析平台（TCMSP）筛选柴胡达胸合剂活性成分和对应的作用靶点,并通过Uniprot数据库标准化靶点名称;在GeneCards和OMIM数据库检索冠状病毒相关基因,并与柴胡达胸合剂作用靶点取交集,筛选出治疗作用靶点;利用Cytoscape 3.7.2软件,构建和分析“药材-活性成分-靶点”网络图;通过String平台分析靶点蛋白相互作用,并使用R软件的相关包进行GO基因注释和KEGG信号通路分析。结果 筛选出165个有效成分和51个作用靶点,进一步分析发现主要活性成分为β-谷甾醇和11个黄酮类化合物,核心作用靶点为CASP3、MAPK3、IL-6、MAPK8、IL-10、CXCL8、MAPK1、IL-1B等。GO基因注释得到GO条目共1 722个（P<0.05）,其中生物学过程条目1 612个,细胞组成条目30个,分子功能条目80个。KEGG信号通路筛选出信号通路156条（P<0.05）,富集基因较多的信号通路为糖尿病并发症中的AGE-RAGE信号通路、甲型流感、IL-17信号通路、TNF信号通路和乙型肝炎。结论 该研究初步揭示了柴胡达胸合剂多成分、多靶点、多通路对COVID-19发挥治疗作用的特点,为进一步阐明柴胡达胸合剂治疗COVID-19的药理作用机制提供理论依据。     

黄芪-川芎药对治疗脑卒中的网络药理学研究

目的 探讨黄芪-川芎药对治疗脑卒中的药理机制。方法 通过多个数据库并结合文献调研检索黄芪、川芎的有效化学成分和相关靶点;采用Cytoscape软件绘制成分靶点可视化网络,并进行拓扑分析;以CTD在线分析平台挖掘脑卒中的相关靶点,借助STRING数据库构建化合物靶点蛋白互作网络、脑卒中靶点蛋白互作网络,应用Cytoscape软件进行网络合并获取核心网络,并对核心靶点基因进行KEGG通路富集分析。结果 共筛选出黄芪、川芎中49个有效化合物,靶点蛋白272个,核心靶点蛋白39个,KEGG富集通路10条。作用通路涉及肿瘤坏死因子（TNF）信号通路、白介素17（IL-17）信号通路、松弛素信号通路、核因子（NF）-κB信号通路、低氧诱导因子-1（HIF-1）信号通路、C型凝集素受体信号通路、Toll样信号通路、核苷酸结合寡聚化结构域（NOD）样信号通路、血管内皮生长因子（VEGF）信号通路、p53信号通路。结论 黄芪-川芎药对中槲皮素、山柰酚等化合物可能通过肿瘤坏死因子（TNF）信号通路、白介素17（IL-17）信号通路等多条通路发挥治疗脑卒中的作用。     

基于网络药理学和分子对接探究布渣叶改善消化不良的作用机制

目的 基于网络药理学和分子对接技术探讨布渣叶Microcos paniculata Linn.对消化不良的潜在物质基础和作用机制。方法 通过检索TCMSP数据库、文献和SwissTargetPredicted数据库收集成分和靶点信息,利用GeneCards和OMIM数据库获得疾病靶点,利用软件R 4.2.1中Venndigram包构建韦恩图,获得布渣叶和疾病的共同靶点;将得到的共同靶点通过软件R 4.2.1做基因本体(GO)和京都基因和基因组百科全书(KEGG)分析,通过STRING网站和Cytoscape 3.9.2做PPI分析得到核心靶点;利用Cytoscape 3.9.2构建“成分-靶点”网络图获得核心成分,将获得的核心成分和核心靶点利用Mastro进行分子对接。结果 筛出154个靶点,核心靶点主要有蛋白激酶B1(Akt1)、细胞肿瘤抗原(TP53)、血管内皮生长因子(VEGFA)、表皮生长因子受体(EGFR)、白细胞介素-6(IL-6)、非受体酪氨酸激酶(SRC)、肿瘤坏死因子(TNF),布渣叶改善消化不良可能通过正向调节酶活性、伤口愈合、丝裂原活化蛋白激酶(MAPK)级联调控等生物过程,与磷脂酰激醇3-激酶(PI3K)-Akt信号通路、脂质代谢与动脉粥样硬化通路、流体剪切应力和动脉粥样硬化有关。分子对接结果表明,核心靶点与相对应的核心成分有比较稳定的对接,其中紫云英苷与SRC有强烈的对接活性。结论 布渣叶中多种活性成分通过多途径、多靶点来调节胃肠动力、修复胃肠黏膜屏障、抑制肠道炎症反应以发挥药效,可为后续进一步作用机制和物质基础研究提供一定的参考。     

基于网络药理学霍山石斛治疗肝损伤作用机制探讨及体内实验验证

目的 基于网络药理学、分子对接及体内实验方法探讨霍山石斛对肝损伤的治疗作用及其机制。方法 运用中国学术期刊全文数据库（CNKI）、PubChem等数据库获取霍山石斛成分,通过 GeneCards数据库筛选肝损伤相关基因,将成分靶点与疾病基因交集后构建蛋白质-蛋白质相互作用（PPI）网络图,进行基因本体（GO）富集分析和京都基因与基因组百科全书（KEGG）通路富集分析,Cytoscape构建“药物-化合物-靶点”网络图,利用 AutoDock Tools对关键活性成分和核心靶点进行分子对接。构建乙醇诱导的肝损伤小鼠模型,通过观察肝脏病理学改变,试剂盒法测定血清中天冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）及白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）水平,通过实时荧光定量逆转录PCR（qRT-PCR）检测酒精性肝损伤小鼠肝脏中相关基因的表达,考察霍山石斛对酒精性肝损伤小鼠的影响。结果 筛选出霍山石斛中 33个活性成分,与肝损伤共有靶点 224个,包含 AKT1、EGFR、SRC等,涉及癌症途径、PI3K/Akt通路等。体内实验结果显示,霍山石斛水提物能够缓解小鼠酒精性肝损伤,并极显著降低AST、ALT和TNF-α、IL-6水平（P＜0.01）,极显著下调IκB激酶（IKK）、核因子-κB（NF-κB）、蛋白激酶B（Akt）mRNA表达水平（P＜0.01）。结论 网络药理学分析及动物实验结果,初步证明霍山石斛水提物可以通过下调Akt、IKK、NF-κB mRNA表达水平,影响NF-κB信号通路上游,参与酒精性肝损伤通路中炎症反应的调控,提示霍山石斛具有改善酒精性肝病的潜在作用,为揭示霍山石斛治疗肝损伤作用机制提供参考。     

蓝萼甲素的药理活性及其机制和毒理作用的研究进展

蓝萼甲素(glaucocalyxin A,GLA)是一种具有对映15-氧-16贝壳杉烯(ent-15-oxo-16-kaurene)骨架结构的二萜类化合物。其具有心血管保护、内皮保护、抗凝血、抗乙肝病毒、抗肿瘤、抑菌、抗炎、耐缺氧、免疫抑制及调节钙离子浓度等多种药理作用,并且有很高的体内安全性。民间常用蓝萼香茶菜治疗肝炎、胃炎、乳腺炎、腹痛、关节痛等疾病,临床上蓝萼香茶菜主要用于治疗冠心病、心绞痛、脑供血不足等缺血、缺氧性心脑血管疾病。本研究将对蓝萼甲素的药理作用及其机制和毒理学作用做一总结。     

Metabolism and hepatotoxicity of N,N-dimethylformamide,N-hydroxymethyl-N-methylformamide,and N-methylformamide in the rat

The metabolism and hepatotoxicity ofN,N-dimethylformamide (DMF) and two of its metabolites,N-hydroxymethyl-N-methylformamide (HMMF) andN-methylformamide (NMF) were evaluated over a 4-day period in rats. DMF toxicity was dose dependent and delayed toxicity after the administration of a high DMF dose (13.7 mmol/kg) in comparison to a lower dose (4.1 mmol/kg) was observed. Treatment of rats with 13.7 mmol/kg DMF, HMMF, or NMF showed i) that DMF is more toxic than HMMF or NMR, and ii) that hepatotoxicity occurs later for DMF than for HMMF or NMF. Analysis of serum and urine samples demonstrated that DMF is first metabolized to HMMF, which is then partially converted to NMF. After HMMF administration, NMF was found both in serum and in urine. The time course of DMF and HMMF toxicity in relation to NMF formation fitted the hypothesis that the hepatotoxicity of DMF and HMMF is mediated via NMF. The degree of hepatotoxicity after HMMF and NMF treatment is similar. However, the degree of DMF hepatotoxicity is much higher than in the case of NMF or HMMF. The role of NMF as an obligatory intermediate in DMF and HMMF hepatotoxicity is discussed.     

Prevalence and characterization of the mechanisms of macrolide, lincosamide and streptogramin resistance in viridans group streptococci

The presence of erm genes conferring constitutive and inducible resistance, as well as that of the mefA gene conferring only constitutive resistance, was investigated using PCR in 70 erythromycin resistant (MIC≥1 mg/l) strains of viridans group streptococci (VGS) (18 Streptococcus mitis biotype 1, 16 S. mitis biotype 2, 15 S. oralis, 12 S. salivarius and nine S. sanguis) isolated from the oropharynx of healthy Greek children. All of the 56 isolates belonging to resistance phenotype M harbored the mefA gene. All of the 14 isolates constitutively resistant to macrolides and lincosamides (phenotype CR) harbored the ermB gene. Co-presence of both genes was not observed, whereas class A erm gene (previously known as ermTR) was not detected. Our results are consistent with a possible role of VGS as a reservoir of resistance genes now prevalent in pathogenic species of streptococci.     

Experimental studies on the effects of toxic Microcystis aeruginosa PCC7820 on the survival and reproduction of two freshwater rotifers Brachionus calyciflorus and Brachionus rubens

Blooms of Microcystis aeruginosa frequently occur in many eutrophic lakes in China, however, there is very little experimental study on the relationship between Microcystis and rotifers from Chinese waters. The effects of different concentrations of toxic M. aeruginosa PCC7820 on two common freshwater rotifers Brachionus calyciflorus and B. rubens were investigated in laboratory experiments. B. calyciflorus was able to utilize this strain of M. aeruginosa as a food source. However, M. aeruginosa suppressed the survival and reproduction of B. calyciflorus at the highest concentration (10⁶ cells/ml) probably due to the inadequate nutrition. B. rubens was inhibited by toxic M. aeruginosa PCC7820 and the inhibition increased with the increasing Microcystis concentration. Our study indicates that the two rotifers have different sensitivities to toxic M. aeruginosa and that toxic cyanobacteria may affect zooplankton community structure by differentially inhibiting the different zooplankton taxa.     

A comparison of the kinetics of d- and l-amphetamine in the brain of isolated and aggregated rats

The kinetics of d- and l-amphetamine were investigated in isolated and aggregated male rats. The i.p. injection of 15 mg/kg of d- or l-¹⁴C-amphetamine was followed by the determination of drug concentrations in the cerebral cortex, hypothalamus, medulla oblongata-pons, cerebellum, striatum, hippocampus, and the whole brain after solvent extraction.In isolated rats, the disappearance curves of the labelled amphetamines were monoexponential. The half-lives of d- and l-amphetamine in whole brain were 1.2±0.1 and 1.3±0.1 h, respectively. In whole brains of aggregated rats the disappearance curves were biexponential (half-lives: d-amphetamine, 0.9±0.1 and 2.3±0.2 h; l-amphetamine, 0.7±0.1 and 2.3±0.1 h).For the brain areas of isolated rats, the elimination curves of amphetamines were monoexponential and the half-lives of d- and l-amphetamine were almost identical. In aggregated rats certain differences were observed. d-Amphetamine was eliminated from the striatum and hippocampus as a biexponential function, and l-amphetamine elimination was biphasic only in the striatum. In other brain regions both d- and l-amphetamine were eliminated as a monoexponential function. The half-lives of d-amphetamine in different brain areas were, however, shorter than those of the l-isomer.The observed differences between d- and l-amphetamine kinetics in brain areas of isolated and aggregated rats may explain certain aspects of the pharmacological activities of these drugs.     

Pre- and postsynaptic effects of p-tyramine and p-octopamine in the prostatic portion of the rat vas deferens

Summary The effects of p-tyramine and p-octopamine on the twitch responses of the prostatic portion of the rat vas deferens to electrical stimulation (0.025 Hz) were compared with the effects of noradrenaline. In tissues with normal monoamine oxidase (MAO) activity, the three amines increased the height and duration of the twitch contractions. When MAO activity was inhibited by pargyline (10 mol/l), p-tyramine and p-octopamine had mixed excitatory-inhibitory effects on the twitches, while noradrenaline had mostly excitatory effects along the whole range of concentrations assayed (0.158–15.8 mol/l). Selective blockade of ₁- and ₂-adrenoceptors, by corynanthine and yohimbine, respectively, showed that the excitatory effect of the amines depended on the activation of ₁-adrenoceptor and that the inhibitory action was related to the activation of ₂-adrenoceptors. Pretreatment with reserpine (5 mg/kg, 24 h; 2.5 mg/kg, 2 h before the experiment) largely prevented the effects of p-tyramine and p-octopamine, but the amines still modified the twitch responses to field stimulation. The addition of corynanthine and yohimbine to the bathing fluid revealed a considerable activation of ₁-excitatory and ₂-inhibitory adrenoceptors. Cocaine (10 mol/l) did not antagonize, but rather enhanced the inhibitory effects of p-tyramine and p-octopamine in tissues with normal contents of noradrenaline. Moreover, cocaine did not antagonize the inhibition caused by p-tyramine, and enhanced the inhibition induced by p-octopamine in the prostatic portion of the vasa deferentia from reserpine-pretreated animals. These results suggest that in this tissue, at least when MAO activity is inhibited, p-tyramine and p-octopamine behave similarly. The effects of both amines on the twitch contractions depend on the noradrenaline-releasing action of the compounds and, in addition, the compounds seem to activate adrenoceptors directly. Send offprint requests to S. M. Celuch at the above addressCareer Investigator on leave of absence from the Consejo de Investigaciones Científicas y Técnicas, ININFA, Junín 956, 5°P, RA-1113 Buenos Aires, Argentina     

Reassessment of the toxin profile of Cylindrospermopsis raciborskii T3 and function of putative sulfotransferases in synthesis of sulfated and sulfonated PSP toxins

The toxigenic freshwater cyanobacterium Cylindrospermopsis raciborskii T3 has been used as a model to study and elucidate the biosynthetic pathway of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP). There are nevertheless several inconsistencies and contradictions in the toxin profile of this strain as published by different research groups, and claimed to include carbamoyl (STX, NEO, GTX2/3), decarbamoyl (dcSTX), and N-sulfocarbamoyl (C1/2, B1) derivatives. Our analysis of the complete genome of another PSP toxin-producing cyanobacterium, Raphidiopsis brookii D9, which is closely related to C. raciborskii T3, resolved many issues regarding the correlation between biosynthetic pathways, corresponding genes and the T3 toxin profile. The putative sxt gene cluster in R. brookii D9 has a high synteny with the T3 sxt cluster, with 100% nucleotide identity among the shared genes. We also compared the PSP toxin profile of the strains by liquid chromatography coupled to mass spectrometry (LC-MS/MS). In contrast to published reports, our reassessment of the PSP toxin profile of T3 confirmed production of only STX, NEO and dcNEO. We gained significant insights via correlation between specific sxt genes and their role in PSP toxin synthesis in both D9 and T3 strains. In particular, analysis of sulfotransferase functions for SxtN (N-sulfotransferase) and SxtSUL (O-sulfotransferase) enzymes allowed us to propose an extension of the PSP toxin biosynthetic pathway from STX to the production of the derivatives GTX2/3, C1/2 and B1. This is a significantly revised view of the genetic mechanisms underlying synthesis of sulfated and sulfonated STX analogues in toxigenic cyanobacteria.     

Investigation of the prevalence of tetQ, tetX and tetX1 genes in Bacteroides strains with elevated tigecycline minimum inhibitory concentrations

In this study, the antibiotic susceptibilities to tigecycline and tetracycline of 35 selected Bacteroides fragilis group strains were determined by Etest, and the presence of tetQ, tetX, tetX1 and ermF genes was investigated by polymerase chain reaction (PCR). tetQ was detected in all 12 B. fragilis group isolates (100%) exhibiting elevated tigecycline minimum inhibitory concentrations (MICs) (≥8 μg/mL) as well as the 8 strains (100%) with a tigecycline MIC of 4 μg/mL, whilst tetX and tetX1 were present in 15% and 75% of these strains, respectively. All of these strains were fully resistant to tetracycline (MIC ≥ 16 μg/mL). On the other hand, amongst the group of strains with tigecycline MICs < 4 μg/mL (15 isolates), tetQ, tetX and tetX1 were found less frequently (73.3%, 13.3% and 46.7%, respectively). All but two strains harbouring the tetQ gene in this group were non-susceptible to tetracycline, with a MIC > 4 μg/mL. These data suggest that in most cases tigecycline overcomes the tetracycline resistance mechanisms frequently observed in Bacteroides strains. However, the presence of tetX and tetX1 genes in some of the strains exhibiting elevated MICs for tigecycline draws attention to the possible development and spread of resistance to this antibiotic agent amongst Bacteroides strains. The common occurrence of ermF, tetX, tetX1 and tetQ genes together predicted the presence of the CTnDOT-like Bacteroides conjugative transposon in this collection of Bacteroides strains.     

Toxicity of Lithium to Three Freshwater Organisms and the Antagonistic Effect of Sodium

Lithium (Li) is the lightest metal and occurs primarily in stable minerals and salts. Concentrations of Li in surface water are typically <0.04mg l^–1 but can be elevated in contaminated streams. Because of the general lack of information concerning the toxicity of Li to common toxicity test organisms, we evaluated the toxicity of Li to Pimephales promelas (fathead minnow), Ceriodaphnia dubia, and a freshwater snail (Elimia clavaeformis). In the laboratory, the concentration of Li that inhibited P. promelas growth or C. dubia reproduction by 25% (IC25) was dependant upon the dilution water. In laboratory control water containing little sodium (2.8mg l^–1), the IC25s were 0.38 and 0.32mg Li l^–1 and in ambient stream water containing 17mg Na l^–1, the IC25s were 1.99 and 3.33, respectively. A Li concentration of 0.15mgl^–1 inhibited the feeding of E. clavaeformis in laboratory tests. Toxicity tests conducted to evaluate the effect of sodium on the toxicity of Li were conducted with fathead minnows and C. dubia. The presence of sodium greatly affected the toxicity of Li. Fathead minnows and Ceriodaphnia, for example, tolerated concentrations of Li as great as 6mg l^–1 when sufficient Na was present. The interaction of Li and Na on the reproduction of Ceriodaphnia was investigated in depth and can be described using an exponential model. The model predicts that C. dubia reproduction would not be affected when animals are exposed to combinations of lithium and sodium with a log ratio of mmol Na to mmol Li equal to at least 1.63. The results of this study indicate that for most natural waters, the presence of sodium is sufficient to prevent Li toxicity. However, in areas of historical disposal or heavy processing or use, an evaluation of Li from a water quality perspective would be warranted.     

Constituents of the essential oil of theCinnamomum cassia stem bark and the Biological Properties

GC-MS analysis on the essential oil (CC-oil) of Cinnamomum cassia stem bark led to the identification of cinnamaldehyde (CNA, 1), 2-hydroxycinnamaldehyde (2-CNA), coumarin (2), and cinnamyl acetate. The major volatile flavor in CC-oil was found to be 2-CNA. Coumarin was first isolated from this plant by phytochemical isolation and spectroscopic analysis. CNA and CC-oil showed potent cytotoxicity, which was effectively prevented by N-acetyl-L-cysteine (NAC) treatment. Intraperitoneal administration with CNA considerably decreased malondialdehyde (MDA) formation and glutathione S-transferase activity in rats. These results suggest that CC-oil and CNA can regulate the triggering of hepatic drug-metabolizing enzymes by the formation of a glutathione-conjugate.