金钱白花蛇及其伪品的Cyt b基因片段序列分析和PCR鉴别研究

对金钱白花蛇及其伪、混品药材和原动物的Cyt b基因片段的序列分析发现,该基因片段在金钱白花蛇及其伪品间的差异远远大于金钱白花蛇种内个体间的差异,是理想的用于鉴别金钱白花蛇及其伪混品的分子遗传标记。在对Cyt b基因片段序列分析的基础上,设计了金钱白花蛇PCR鉴别的一对高度特异性引物BuL-1和BuH-1。结果表明,该对引物在对金钱白花蛇的PCR鉴别中,用60℃～65℃的复性温度,可以100%检出金钱白花蛇,误检率和漏检率为0,并能在混合的药材粉末中检测出被检样品中是否含有金钱白花蛇组份。本研究还表明PCR鉴别将有可能成为中成药复方组分鉴别的一种新手段。     

鹿类中药材的位点特异性PCR鉴定研究

目的　建立一种简便、准确的鹿类中药材鹿茸、鹿鞭、鹿筋、鹿胎的DNA分子标记鉴定方法。方法　在对鹿类中药材的正品原动物梅花鹿、马鹿及其混伪品原动物的Cyt b 基因全序列分析的基础上,设计了一对专用于鉴定正品鹿类药材的位点特异性鉴别引物ILu01-L和ILu01-H。结果　在6 4℃的复性温度下,用鉴别引物对原动物样品进行鉴别PCR ,仅正品能得到约365bp阳性扩增带;对鹿茸、鹿鞭及鹿筋正、伪品药材进行PCR鉴定,结果表明:3批鹿茸仅一批为正品,2批鹿鞭皆为伪品,鹿筋正、伪品药材PCR鉴定与形态鉴定结果一致。随机选取2枚鹿茸及一个原动物做Cyt b 基因片段序列分析,其结果与PCR鉴定完全一致。结论　对市售鹿类商品药材需加强质量监督和管理。所设计的鉴别引物对梅花鹿、马鹿有高度特异性,可应用于以其为原动物的鹿类中药材的鉴定。     

鹿类中药材的位点特异性PCR鉴定研究

目的　建立一种简便、准确的鹿类中药材鹿茸、鹿鞭、鹿筋、鹿胎的DNA分子标记鉴定方法。方法　在对鹿类中药材的正品原动物梅花鹿、马鹿及其混伪品原动物的Cytb基因全序列分析的基础上 ,设计了一对专用于鉴定正品鹿类药材的位点特异性鉴别引物ILu0 1 L和ILu0 1 H。结果　在 6 4℃的复性温度下 ,用鉴别引物对原动物样品进行鉴别PCR ,仅正品能得到约 36 5bp阳性扩增带 ;对鹿茸、鹿鞭及鹿筋正、伪品药材进行PCR鉴定 ,结果表明 :3批鹿茸仅一批为正品 ,2批鹿鞭皆为伪品 ,鹿筋正、伪品药材PCR鉴定与形态鉴定结果一致。随机选取 2枚鹿茸及一个原动物做Cytb基因片段序列分析 ,其结果与PCR鉴定完全一致。结论　对市售鹿类商品药材需加强质量监督和管理。所设计的鉴别引物对梅花鹿、马鹿有高度特异性 ,可应用于以其为原动物的鹿类中药材的鉴定。     

基于Cytb基因对药用昆虫地鳖虫炮制品的分子鉴别研究

目的:建立基于线粒体DNA细胞色素氧化酶b(Cytb)基因的分子标记技术对地鳖虫(地鳖、冀地鳖)及其混伪品炮制药材进行分子鉴定的方法。方法:采用改良的饱和氯化钠法对地鳖虫及其混伪品炮制药材的总DNA进行提取。通过通用引物REVCB2H、REVCBJ对所有样品的Cytb基因进行聚合酶链反应(PCR)扩增;用MEGA 5.1软件对所有样品的Cytb基因序列采用邻接(NJ)法构建系统发育树;并利用DNAMAN软件对得到的所有样品的Cytb基因序列进行比对,分析正品、混淆品间序列差异,在差异较大区域设计特异性引物Esin-F和Esin-R进行分子鉴定。结果:以提取的DNA为模板均能成功扩增出相应的Cytb基因片段;构建的系统发育树与其亲缘关系一致;设计的特异性引物Esin-F和Esin-R在同样的条件下,只有地鳖虫得到了扩增条带。结论:建立了地鳖虫及其混伪品药材炮制品的DNA提取方法;设计的特异性引物对地鳖虫有高度的特异性,能够有效鉴别地鳖虫及其混伪品。     

中药材龟甲及原动物的高特异性PCR鉴定研究

目的:建立一种简便、实用的龟甲药材DNA 分子鉴定方法。方法:根据22 种亚洲产龟类的线粒体12SrRNA 基因片段序列,设计一对专用于鉴定中药材龟甲原动物乌龟的鉴别引物,用该对引物扩增从乌龟和其他18 种龟共48 个样品的DNA 模板。结果:在72℃的复性温度下进行PCR,4 个乌龟的模板DNA 均得到约180 bp 的阳性扩增带,而其他各龟的模板DNA,在同样条件下无扩增产物,用这对鉴别引物经一次PCR 反应便可准确地鉴定受试原动物是否为乌龟。同法对江苏省药品检验所提供的17 块样品龟甲进行了鉴定,结果表明只有4 块样品为正品,其余皆为伪品,与性状鉴定和DNA序列分析鉴定结果完全一致。结论:所设计的鉴别引物对乌龟有高度特异性,所配制的龟甲药材鉴定试剂盒可在龟甲药材鉴定中使用。     

鹿鞭线粒体DNA指纹鉴定

目的探讨鹿鞭及牛鞭线粒体细胞色素b基因部分序列片段特征,建立中药材鹿鞭敏感、特异的DNA分子标记学鉴定方法。方法碱变性法提取新鲜鹿鞭及牛鞭,采用引物设计软件PrimerPremier5.0对鹿鞭及牛鞭的细胞色素b基因序列分析,用于鉴定正品鹿鞭的特异性引物,经聚合酶链式反应(PCR)扩增,对鹿鞭进行DNA指纹鉴定。结果对鹿鞭进行mtDNA鉴定图谱显示,真品鹿鞭有306bp条带,伪品没有。结论用此方法对鹿鞭进行鉴定,可准确辨别正品与伪品。重现性、稳定性好,简便准确而可行。鹿鞭mtDNA具有特异性指纹特征,所得鹿鞭DNA指纹特征图谱可用于鹿鞭的鉴定。     

基于Cyt b基因的PCR技术对牛鞭药材的鉴定方法研究

目的:建立对牛鞭药材的聚合酶链式反应(PCR)鉴定方法,从分子水平上对牛鞭药材的真伪进行鉴定。方法:提取牛鞭及其伪品(驴鞭、猪鞭、羊鞭)的基因组DNA,并对其完整性、纯度和浓度进行检测。利用GenBank相关信息,以牛鞭的线粒体细胞色素b基因(Cyt b)为靶基因,应用Primer-BLAST在线软件设计特异性引物,并对不同物种鞭类样品进行PCR扩增,对产物进行电泳分析;对获得的牛鞭样本PCR产物进行克隆和DNA测序验证。对所建立的PCR鉴定方法进行特异性和重复性考察验证。结果:提取所得的牛鞭及其伪品DNA的纯度较高,无蛋白质或RNA污染;1.5%琼脂糖凝胶电泳结果显示,牛鞭样本在200～300 bp之间均出现明显的目的基因条带,而其他伪品未见相应条带出现。DNA测序结果证实,所获牛鞭样本基因片段的核苷酸序列与GeneBank中牛鞭物种相似性均为100%。方法学验证结果显示所建方法特异性、重复性均良好。结论:本研究所建基于Cyt b基因的PCR鉴定方法简单、快速,准确性、特异性、重复性均良好,可满足牛鞭及其伪品的分析鉴定需求。     

海马类药材的分子遗传标记鉴定研究

应用古DNA研究技术从5种海马药材中提取DNA,用PCR技术扩增约450bp的12SrRNA基因片段和约490 bp的细胞色素b基因片段。对扩增产物进行了限制性片段长度多态性(RFLP)分析和DNA序列分析。用RFLP分析方法可以鉴别2种海马,用DNA序列分析方法得到的分子遗传标记可以鉴别所有5种海马。对其它动物类药材的鉴定有一定参考价值。     

市售鹿鞭的DNA指纹鉴定研究

目的：探讨鹿鞭及牛鞭线粒体细胞色素b基因（Cytb）部分序列片段特征,建立中药材鹿鞭敏感、特异的DNA分子标记学鉴定方法。方法对市售鹿鞭进行样本处理,模板制备,采用鹿鞭特异性引物进行PCR反应,对市售鹿鞭样本采取DNA指纹鉴定。结果对市售鹿鞭中药材进行mtDNA鉴定,有三个样本为正品。结论此方法对市售鹿鞭鉴定,可准确辨别正品与伪品。重现性、稳定性好,简便准确而可行,说明鹿鞭mtDNA具有特异性指纹特征,所得鹿鞭DNA指纹特征图谱可用于市售鹿鞭的鉴定。     

市售鹿鞭的DNA指纹鉴定研究

目的探讨鹿鞭及牛鞭线粒体细胞色素b基因(Cytb)部分序列片段特征,建立中药材鹿鞭敏感、特异的DNA分子标记学鉴定方法。方法对市售鹿鞭进行样本处理,模板制备,采用鹿鞭特异性引物进行PCR反应,对市售鹿鞭样本采取DNA指纹鉴定。结果对市售鹿鞭中药材进行mtDNA鉴定,有三个样本为正品。结论此方法对市售鹿鞭鉴定,可准确辨别正品与伪品。重现性、稳定性好,简便准确而可行,说明鹿鞭mtDNA具有特异性指纹特征,所得鹿鞭DNA指纹特征图谱可用于市售鹿鞭的鉴定。     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Nachweis einiger Heilmittel in der Kniegelenksynovialflüssigkeit

Zusammenfassung Mittels Gaschromatographie und Dünschichtchromatographie wiesen die Autoren 11 Substanzen nach, welche durch Injektion oder nach Verabreichung per os in die Kniegelenksynovialflüssigkeit eindrangen. In ihrer Aufstellung konnten sie eine direkte Beziehung zwischen Struktur sowie chemischphysikalischen Eigenschaften der Substanz und ihrer Fähigkeit, aus dem Blut in die Kniegelenksynovialflüssigkeit einzudringen, nicht nachweisen, außer der Tatsache, daß Substanzen mit starker Affinität zu Eiweißstoffen erst in höheren Dosen nachweisbar waren.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

Gender-related symptoms and correlates of alcohol dependence among men and women with a lifetime diagnosis of alcohol use disorders

This study explored gender-related symptoms and correlates of alcohol dependence in a crosssectional study of 150 men and 150 women with a lifetime diagnosis of alcohol use disorders (AUD). Participants were recruited in equal numbers from treatment settings, correctional centres and the general community. Standardized measures were used to determine participants' use of substances, history of psychiatric disorders and psychosocial stress, their sensation seeking and family history of substance use and mental health disorders. Multivariate analyses were used to detect patterns of variables associated with gender and the lifetime severity of AUD. Men had a longer history of severe AUD than women. Women had similar levels of alcohol dependence and medical and psychological sequelae as men, despite 6 fewer years of AUD. More women than men had a history of severe psychosocial stress, severe dependence on other substances and antecedent mental health problems, especially mood and anxiety disorders. There were differences in family history of alcohol-related problems approximating same-gender aggregation. The severity of a lifetime AUD was predicted by its earlier age at onset and the occurrence of other disorders, especially anxiety, among both men and women. The limitations in the generalizability of these findings due to sample idiosyncrasies are discussed.     

Biochemical and molecular characterisation of cubozoan protein toxins

Class Cubozoa includes several species of box jellyfish that are harmful to humans. The venoms of box jellyfish are stored and discharged by nematocysts and contain a variety of bioactive proteins that are cytolytic, cytotoxic, inflammatory or lethal. Although cubozoan venoms generally share similar biological activities, the diverse range and severity of effects caused by different species indicate that their venoms vary in protein composition, activity and potency. To date, few individual venom proteins have been thoroughly characterised, however, accumulating evidence suggests that cubozoan jellyfish produce at least one group of homologous bioactive proteins that are labile, basic, haemolytic and similar in molecular mass (42-46 kDa). The novel box jellyfish toxins are also potentially lethal and the cause of cutaneous pain, inflammation and necrosis, similar to that observed in envenomed humans. Secondary structure analysis and remote protein homology predictions suggest that the box jellyfish toxins may act as α-pore-forming toxins. However, more research is required to elucidate their structures and investigate their mechanism(s) of action. The biological, biochemical and molecular characteristics of cubozoan venoms and their bioactive protein components are reviewed, with particular focus on cubozoan cytolysins and the newly emerging family of box jellyfish toxins.     

Dose tolerability of chronically inhaled voriconazole solution in rodents

Invasive pulmonary aspergillosis (IPA) is a fungal disease of the lung associated with high mortality rates in immunosuppressed patients despite treatment. Targeted drug delivery of aqueous voriconazole solutions has been shown in previous studies to produce high tissue and plasma drug concentrations as well as improved survival in a murine model of IPA. In the present study, rats were exposed to 20 min nebulizations of normal saline (control group) or aerosolized aqueous solutions of voriconazole at 15.625 mg (low dose group) or 31.25 mg (high dose group). Peak voriconazole concentrations in rat lung tissue and plasma after 3 days of twice daily dosing in the high dose group were 0.85 ± 0.63 μg/g wet lung weight and 0.58 ± 0.30 μg/mL, with low dose group lung and plasma concentrations of 0.38 ± 0.01 μg/g wet lung weight and 0.09 ± 0.06 μg/mL, respectively. Trough plasma concentrations were low but demonstrated some drug accumulation over 21 days of inhaled voriconazole administered twice daily. Following multiple inhaled doses, statistically significant but clinically irrelevant abnormalities in laboratory values were observed. Histopathology also revealed an increase in the number of alveolar macrophages but without inflammation or ulceration of the airway, interstitial changes, or edema. Inhaled voriconazole was well tolerated in a rat model of drug inhalation.