多根乌头生物碱成分的研究

从新疆产多根乌头AconitumkarakolicumRap.块根中分得五个二萜生物碱,经理化常数测定和光谱分析,分别鉴定为：乌头碱、脱氧乌头碱、新乌碱、准噶尔乌头碱和12-表-欧乌碱。其中12-表-欧乌碱系首次从该植物中获得。     

药用植物准噶尔乌头的研究

对准噶尔乌头的生物学特性,有效成分的鉴别、分离等方面进行了研究报,从准噶尔乌头中首次分得１２－表－欧乌碱及１２－表－脱氢欧乌碱,且准噶尔头碱及新乌碱含量较高,为准噶尔乌头药用资源的开发利用提供了依据。     

HPLC和TLC法测定准噶尔乌头中的生物碱成分

采用高效液相色谱和薄层色谱对准噶尔乌头中生物碱进行分析,建立了HPLC法测定其中各生物碱含量的方法。结果确定准噶尔乌头中主要含有12-表-欧乌碱、宋果灵、尼奥灵、准噶尔碱和欧乌碱,其中准噶尔碱和欧乌碱为首次在该植物中发现;其疗效与川乌和草乌相近但化学成分差异较大。色谱条件为:Inertsil NH2柱(150 mm×4.6 mm,5μm),流动相正己烷∶乙醇(92∶8),流速1.0 mL/min,检测波长270 nm,进样量10μL。本方法操作简便,结果可靠,重现性好,为准噶尔乌头的质量标准研究提供了实验依据。     

草乌中生物碱的化学研究

从草乌－北乌头（Ａｃｏｎｉｔｕｍ ｋｕｓｎｅｚｏｆｆｉｉ Ｒｅｉｃｈｂ．）的块根－中分离得到１６个单体成分,据波谱方法分别鉴定为：乌头碱（ａｃｏｎｉｔｉｎｅ,１）、３－脱氧乌头碱（３－ｄｅｏｘｙａｃｏｎｉｔｉｎｅ,２）、中乌头碱（ｍｅｓａｃｏｎｉｔｉｎｅ,３）、北乌碱（ｂｅｉｗｕｔｉｎｅ,４）、次乌头碱（ｈｙｐａｃｏｎｉｔｉｎｅ,５）、１０－羟基乌头碱（ａｃｏｎｉｆｉｎｅ,６）、１４－苯甲酰乌头原     

瓜叶乌头中生物碱成分的研究

从瓜叶乌头 (AconitumhemsleyanumPritz)根中分得 12个已知C19 二萜生物碱 ;氨茴酰牛扁碱(anthranoyllycoctonine) 1、牛扁碱 (lycoctonine) 2、8 去乙酰滇乌碱 (8 deacetylyunaconitine) 3、伪乌头宁(pseudaconine) 4、sachaconitine 5、尼奥宁 (neoline) 6、senbusineA 7、6 表弗斯生 (6 epiforesticine) 8、滇乌碱(yunaconitine) 9、印乌碱 (indaconitine) 10、查斯曼宁 (chasmanine) 11、和塔拉萨敏 (talatisamine) 12。其中化合物 1～ 8是首次从该植物中分离得到。应用光谱法和对照TLC鉴定了所有化合物的结构。     

黑顺片的二萜生物碱成分

常用中药川乌、附子为毛莨科乌头属植物乌头(Aconitum carmichaeli Debx.)的母根和子根.从附子的加工炮制品黑顺片中分离鉴定了5种C19乌头碱型二萜生物碱和1个C20纳哌啉型二萜生物碱.通过MS、NMR、IR等波谱分析和已知化合物数据对照,分别鉴定为次乌头碱(1)、尼奥宁(2)、塔拉地萨敏(3)、多根乌头碱(4)、异塔拉萨定(5)和去氢松果灵(6).     

膝瓣乌头中生物碱成分的研究

从膝瓣乌头（Ａｃｏｎｉｔｕｍ ｇｅｎｉｃｕｌａｔｕｍ）根中共分得１４单体成分,我谱法分别鉴定为,黄草乌碱甲、丙（ｖｉｌｍｏｒｒｉａｎｉｎｅｓ,Ａ,Ｃ）１和２、乌碱（ｙｕｎｎａｃｏｎｉｔｉｎｅ）３、南乌碱乙（ＡｕｓｔｒｏｃｏｎｉｔｉｎｅＢ）４、印乌碱（ｉｎｄａｃｏｎｉｔｉｎｅ）５,８－乙酰－１４－苯甲酰尼奥宁（８－ａｃｅｔｙｌ－１４－ｅｎｚｏｙｉｎｅｏｌｉｎｅ）８、塔拉萨敏（ｔａｌａｔｉｚｍｉｎｉｅ     

花亭乌头中的三个新二萜生物碱

从花亭乌头(Aconitum scaposum Franch.)根中分到三个新二萜生物碱——花亭乌头碱(scaconineⅠ)、N-去乙酰花亭乌头碱(N-deacetyl scaconitineⅡ)及花亭乌     

展毛短柄乌头中的一个新二萜生物碱

从展毛短柄乌头(Aconitum brachypodum var.laxiflorum Fletcher et Lauener)根中分离鉴定了五个二萜生物碱,其中四个为已知成分,分别为乌头碱(aconitine)、3-去氧乌头碱(3-deoxyaconitine)、3-乙酰乌头碱(3-acetylaconitine)、雪乌碱(penduline),另一个为新成分,命名为丽鲁碱(laxiconitine),其结构通过光谱分析及化学反应测定如(1)。     

敦化乌头的二萜生物碱成份

从敦化乌头(Aconitum dunhuaense S.H.Li)的根中分得6个单体二萜生物碱成份,经光谱分析及同标准品对照,鉴定它们分别为乌头碱(aconitine,1)、下乌头碱(hypaconitine,2)、尼奥灵(nepline,3)、去氧乌头碱(3-deoxyaconitine,4)、中乌头碱(mesaconitine,5)和阿康诺辛(aconosine,6)。     

多根乌头中二萜生物碱成分

为研究多根乌头(AconitumkarakolicumRapaics)中二萜生物碱成分,本研究采用正反相硅胶柱和高效液相等色谱分离方法,从中分离得到15个二萜生物碱;通过多种波谱手段以及文献对比的方法鉴定其结构分别为aconitine(1),3-deoxyaconitine(2),16-epipyroaconine(3),neoline(4),indaconitine(5),14-benzoyl-8-O-methylaconitine(6),spicatineA(7),15-α-hydroxyneoline(8),taurenine(9),14-benzoylaconine(10),14-benzoylaconine-8-oleate(11),lappaconitine(12),beiwudine(13),13-hydroxyfranchetine(14)和8-O-linoleoyl-14-benzoylaconine(15),化合物3~15为首次从该植物中分离得到。采用MTT法和叶碟法分别考察了部分化合物的抗肿瘤和拒食活性,化合物14-benzoylaconine-8-oleate(11)对人乳腺癌MCF-7细胞、人肺癌H460细胞、肝癌HepG2细胞的IC50值分别为11.9、27.6和31.8μM。乌头碱型的二萜生物碱aconitine(1)、3-deoxyaconitine(2)、indaconitine(5)和beiwudine(13)表现出一定的拒食活性的活性(EC50<2mg/cm^2)。     

Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras,protein kinase C,and protein kinase A in neuronal cells

The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.     

NGF-Dependent neurite outgrowth in PC12 cells overexpressing the Src homology 2-domain protein shb requires activation of the Rap1 pathway

The Src homology 2 (SH2) domain adaptor protein Shb has been shown to transmit NGF- and FGF-2-dependent differentiation signals in PC12 cells. To study if this involves signaling through the small GTPase Rap1, Rap1 activity was assessed in Shb-overexpressing PC12 cells. We demonstrate that NGF and EGF induce Rap1 activation in PC12-Shb cells, while FGF-2 fails to do so. However, PC12 cells expressing Shb with an inactivated SH2 domain do not respond to NGF stimulation with Rap1 activation. The CrkII SH2 domain interacts with Shb and a 130- to 135-kDa phosphotyrosine protein present mainly in PC12-Shb cells and these interactions may thus relate to the effect of Shb on Rap1 activation. Transient expression of RalGDS-RBD or Rap1GAP to block the Rap1 pathway reduces the NGF-dependent neurite outgrowth in PC12-Shb cells. These results suggest a role of Shb in NGF-dependent Rap1 signaling and this pathway may be of significance for neurite outgrowth under certain conditions.     

Activation of Gz attenuates Rap1-mediated differentiation of PC12 cells

We previously identified a specific activation-dependent interaction between the alpha subunit of the heterotrimeric G protein, G(z), and a regulator of Rap1 signaling, Rap1GAP (Meng, J., Glick, J. L., Polakis, P., and Casey, P. J. (1999) J. Biol. Chem. 274, 36663-36669). We now demonstrate that activated forms of Galpha(z) are able to recruit Rap1GAP from a cytosolic location to the membrane. Using PC12 cells as a model for neuronal differentiation, the influence of G(z) activation on Rap1-mediated cell differentiation was examined. Introduction of constitutively-activated Galpha(z) into PC12 cells markedly attenuated the differentiation process of these cells induced by a cAMP analogue. Treatment of PC12 cells expressing wild type Galpha(z) with a specific agonist to the alpha(2A)-adrenergic receptor also attenuated cAMP-induced PC12 cell differentiation, demonstrating that receptor-mediated activation of G(z) was also effective in this regard. Furthermore, activation of G(z) decreased the ability of the cAMP analogue to trigger both Rap1 and extracellular-regulated kinase (ERK) activation. Differentiation of PC12 cells induced by nerve growth factor (NGF) is also thought to be a Rap1-mediated process, and G(z) activation was found to attenuate this process as well. Rap1 activation, ERK phosphorylation, and PC12 cell differentation induced by NGF treatment were all significantly attenuated by either transfection of constitutively activated Galpha(z) or receptor-mediated G(z) activation. Based on these findings, a model is proposed in which activation of G(z) results in recruitment of Rap1GAP to the membrane where it can effectively down-regulate Rap1 signaling. The implications of these findings in regard to a possible role for G(z) in neuronal development are discussed.     

A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway

Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.     

Rap1-mediated lymphocyte function-associated antigen-1 activation by the T cell antigen receptor is dependent on phospholipase C-gamma1

The small GTPase, Rap1, is a potent activator of leukocyte integrins and enhances the adhesive activity of lymphocyte function-associated antigen-1 (LFA-1) when stimulated by the T cell receptor (TCR) or chemokines. However, the mechanism by which Rap1 is activated remains unclear. Here, we demonstrate that phospholipase C (PLC)-gamma1 plays a critical role in the signaling pathway leading to Rap1 activation triggered by the TCR. In Jurkat T cells, TCR cross-linking triggered persistent Rap1 activation, and SDF-1 (CXCL12) activated Rap1 transiently. A phospholipase C inhibitor, U73122, abrogated Rap1 activation triggered by both the TCR and SDF-1 (CXCL12). PLC-gamma1-deficient Jurkat T cells showed a marked reduction of TCR-triggered Rap1 activation and adhesion to intercellular adhesion molecule-1 (ICAM-1) mediated by LFA-1. In contrast, SDF-1-triggered Rap1 activation and adhesion were not affected in these cells. Transfection of these cells with an expression plasmid encoding PLC-gamma1 restored Rap1 activation by the TCR and the ability to adhere to ICAM-1, accompanied by polarized LFA-1 surface clustering colocalized with regulator of adhesion and polarization enriched in lymphoid tissues (RAPL). Furthermore, when expressed in Jurkat cells, CalDAG-GEFI, a calcium and diacylglycerol-responsive Rap1 exchange factor, associated with Rap1, and resulted in enhanced Rap1 activation and adhesion triggered by the TCR. Our results demonstrate that TCR activation of Rap1 depends on PLC-gamma1. This activity is likely to be mediated by CalDAG-GEFI, which is required to activate LFA-1.     

国产12种乌头属和18种翠雀属植物的细胞学研究

研究了12种乌头属Aconitum L．和18种翠雀属Delphinium L．植物的染色体。在12种乌头属植物中,除粗花乌头A．crassiflorum为四倍体(2n=4x=32)外,其他种类都为二倍体(2n=2x＝16),中甸乌头 A.piepunense中有B染色体存在,牛扁亚属Aconitum subgen．Lycoctonum的二倍体植物与乌头亚属Aconitum subgen．Aconitum 植物的染色体在大小和形态上有明显区别;所有18种翠雀属植物都为二倍体(2n＝2x=16),其染色体在大小和形态上极为相似,但与乌头亚属的染色体易于区别。翠雀属植物的核型不对称性程度明显高于乌头属植物,因此从染色体证据来看,翠雀属要比乌头属进化。     

Nitric oxide activates Rap1 and Ral in a Ras-independent manner

Rap1 and Ral, the small GTPases belonging to the Ras superfamily, have recently attracted much attention; Ral because of Ral-specific guanine nucleotide exchange factors which are regulated by direct binding to Ras and Rap1 because of its proposed role as an antagonist of Ras signaling. We have previously demonstrated that nitric oxide (NO) activates Ras and proposed the structural basis of interaction between NO and Ras. In the present study we have shown that NO activates Rap1 and Ral in a time- and concentration-dependent manner. Using activation-specific probes for Rap1 and Ral, it was found that the NO-generating compounds SNP and SNAP could activate both Rap1 and Ral in Jurkat and PC12 cell lines. To investigate the involvement of Ras in NO mediated activation of Rap1 and Ral, we used PC12 cell lines expressing either the Ras mutant C118S (Cys118 mutated to Ser) or N17 (GDP-locked and inactive). We had previously shown that NO fails to activate Ras in these mutant cell lines. However, here it was found that Rap1 and Ral were activated by NO in these cell lines. The evidence presented in this study unambiguously demonstrates the existence of Ras-independent pathways for NO mediated activation of Rap1 and Ral.