Conformational transition of DNA in electroreduction studied by in situ UV and CD thin layer spectroelectrochemistry

Electrochemically induced three conformational transitions of calf thymus DNA from B-10.4 to Z(10.2)-DNA and from B-10.2 to B-10.4 and to C-DNA in 10 mM phosphate buffer solution (pH 7.21) at glassy carbon electrode are found and studied by in situ circular dichroism (CD) thin layer spectroelectrochemistry with singular value decomposition least square (SVDLS) analysis. It indicates that the so-called B-10.2 form and the C-form of DNA may be composed of B-10.4 and left-A DNA and of B-10.4 and right-A DNA, respectively. The irreversible electrochemical reduction of adenine and cytosine groups in the DNA molecule is studied by UV-Vis spectroelectrochemistry. Some electrochemical parameters alphan = 0.17, E-0' = -0.70 V (vs. Ag/AgCl), and the standard heterogeneous electron transfer rate constant, k(0) = 1.8 x 10(-5) cm s(-1) are obtained by double logarithmic analysis and non-linear regression. (C) 2000 Elsevier Science B.V. All rights reserved.

Fabrication of layer-by-layer deposited multilayer films containing DNA and its interaction with methyl green

Multilayer films were fabricated by layer-by-layer electrostatic deposition techniques between poly(diallyIdimethylammonium chloride) (PDDA) and calf thymus DNA (CT DNA) on glassy carbon and quartz substrates. Electrochemical impedance spectroscopy (EIS), Fourier transform infrared (FTIR) spectroscopy and UV-vis spectroscopy demonstrated the uniform assembly of PDDA/DNA multilayer films, and X-ray photoelectron spectroscopy confirmed the elemental composition of the films. Moreover, the interaction of DNA in PDDA/DNA films with methyl green was investigated by UV-vis spectroscopy and circular dichroism (CD). (C) 2001 Elsevier Science B.V. All rights reserved.

Interactions of Safranin T with nucleic acids

The interactions of Safranin T (ST) with several nucleic acids have been investigated by electrochemical, UV-visible and CD spectroscopic techniques. The form of the nucleic acid-ST complexes is sensitive to the ratio of the two species. Two electrochemically inactive complexes such as, nucleic acid-ST and nucleic acid-2ST, were formed while ST interacts with nucleic acids. Two processes were obtained from spectral experiments: (1) at the high value of R (R is defined as the ratio of the total concentration of ST to that of nucleic acid), ST is groove-binding with stacking, (2) st the low value of R, ST is groove-binding without stacking. Intrinsic binding constants were obtained by spectral methods. The experiments also show that electrostatic binding plays an important role in the interaction of ST with nucleic acids.

Interaction of brilliant cresyl blue and methylene green with DNA studied by spectrophotometric and voltammetric methods

The binding behavior of two cationic dyes, brilliant cresyl blue (BCB) and methylene green (MG) to calf thymus DNA was studied by spectrophotometric and voltammetric methods. A red shift of the adsorption spectra and hypochromism accompany the binding of BCB and MG to calf thymus DNA. In 5 x 10(-2) mol dm(-3) NaCl, 5 x 10(-3) mol dm(-3) tris-HCl pH 6.87 buffer solution, the apparent binding constants are: K-BCB+ 3.0 x 10(4)M(-1) (N = 4.13) and K-MG+ = 8.8 x 10(4)M(-1) (n = 4.44). Electrochemical studies show that the formal potentials shift negatively upon addition of DNA, indicating that the oxidized forms of the dyes have stronger affinity to DNA than the reduced ones. K-BCB+/K-BCBH and K-MG+/K-MGH are evaluated to be 10.39 and 7.04. respectively. Our investigation suggests that the two cationic dyes interact with DNA predominantly via electrostatic interaction.

Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine

Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P.fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections. (C) 2009 Elsevier Ltd. All rights reserved.

三种海水经济鱼类早期发育生物学的研究

海水经济鱼类的养殖在我国已经形成第四次海水养殖浪潮，经济效益显著，有力地推动了我国海水养殖的产业结构调整和可持续发展。然而在海水养殖发展过程中也存在着诸多问题，尤其是早期发育阶段的高死亡率，严重制约了我国海水养殖产业的稳定和健康发展。 海水鱼类养殖的关键为高质量，高存活率苗种的生产和培育，由于鱼类种类繁多，生物多样性丰富，对应实际的繁育技术，尤其是新品种的开发，必须要做出相应的调整。这就要求我们必须对每一种鱼类早期发育有所了解，并将形态和组织上的数据用于指导生产。 本文通过显微观察和组织学研究，主要描述和研究了我国北方三种重要的海水经济鱼类(条斑星鲽、杂交鲆、条石鲷)的早期发育生物学，并结合实际生产进一步阐明关键期的产生原因，机理以及采用相应的对策。具体结果如下： 1.条斑星鲽：作为冷温性鲆鲽鱼类，条斑星鲽早期发育过程的特征主要有： ① 条斑星鲽受精卵无油球，卵子呈半浮性；不同步卵裂现象提前，发生在第三次卵裂；卵裂期裂球大小差异大。孵化过程较长，在水温8 ± 0.3℃，盐度33的条件下，经9 d孵化。条斑星鲽胚胎发育的不同时期对温度的敏感性不同，其中原肠期对温度比较敏感。 ②在8-10℃，盐度33的条件下，8-9 dph开口摄食。且开口时，其吻前端出现有一点状黑褐色素，构成了条斑星鲽仔鱼“开口期”的重要标志。卵黄囊于消失。在后期仔鱼末期，背鳍和臀鳍上形成特有的黑褐色条斑带。 ③杯状细胞首先出现在咽腔后部和食道前段，胃腺和幽门盲囊出现于29 dph，变态期始于30dph。在条斑星鲽早期发育过程中，观察到其直肠粘膜层细胞质出现大量嗜伊红颗粒，为仔鱼肠道上皮吸收的蛋白质。 ④首先淋巴化的免疫器官是头肾，然后是胸腺和脾脏，这与大部分硬骨鱼类不同。条斑星鲽除头肾和脾脏外，胸腺实质也形成MMCs。其中以脾脏形成MMCs最为丰富，形态多样。 2. 杂交鲆：为同属的牙鲆和夏鲆间的远缘杂交种，其发育过程的特点为： ① 在温度为15.4～16.0℃，杂交鲆胚胎从受精到孵化所需的时间为76 h左右，胚孔关闭前期，胚胎先出现视囊及克氏囊，而后形成体节。孵出前胚体在卵膜内环绕不到1周。 ② 孵化后消失。杂交鲆群体变态间隔长(34-60 dph)，且变态高峰期出现的冠状幼鳍不明显(与母本牙鲆相比)，数量为7-8根。 ③组织学观察发现，其消化系统中胃腺出现较晚，且胃腺发育过程缓慢(与母本牙鲆相比)。甲状腺滤泡增生不明显，颜色较浅，数量较少。杂交鲆在早期发育过程中，并没有出现鳔原基。 3. 条石鲷作为岩礁性的暖水性鱼类，早期发育过程也较为特殊，包括外形以及内部的器官结构。主要特点有： ① 受精卵：受精卵卵黄上具有龟裂结构，为鱼卵的分类特征之一。 ② 初孵仔鱼：初孵仔鱼背鳍膜上的黑色素，从体背面向背鳍膜边缘移动，到3dph仔鱼基本消失，此为本种仔鱼发育所特有的特点。 ③ 后期仔鱼和稚鱼：肠道肌肉层加厚明显，仔稚鱼胃肠排空率急剧上升，死亡率增加，通过改善常规的投饵方式部分解决了这个死亡高峰的问题。在幼鱼初期，牙齿融合为骨喙，为石鲷科鱼类的特征。 ④胸腺上皮分泌细胞：类似的现象同样在虹鳟鱼中发现，但是虹鳟鱼胸腺上皮分泌细胞不如条石鲷的丰富，同样也不如条石鲷的排列整齐，而是零星分布在胸腺上皮与咽腔接触的表面。除了正常的造血器官—脾脏和头肾外，肝脏、胰腺和鳔等多种组织等也出现MMCs，此现象在硬骨鱼类不多见，一般发生在软骨鱼类。

The antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-(2,3-dibromo-4,5-dihydroxybenzyl)-2,3-dibromo-4,5-dihydroxy benzyl methyl ether (1), (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroisobenzofuran (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethyl-pyrocatechol (3), 2,2',3,3'-tetrabromo-4,4',5,5'-tetrahydroxy-diphenylmethane (4), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (5), 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxymethyldiphenylmethane (6) were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTT assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402 and HCT-8. Their inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana (EELN) was evaluated on S-180-bearing mice. All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC50 below 10 mu g/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4%, respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenol derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer.

Phylogenetic origins of the Himalayan endemic Dolomiaea, Diplazoptilon and Xanthopappus (Asteraceae : Cardueae) based on three DNA regions

Background and Aims It is an enduring question as to the mechanisms leading to the high diversity and the processes producing endemics with unusual morphologies in the Himalayan alpine region. In the present study, the phylogenetic relationships and origins of three such endemic genera were analysed, Dolomiaea, Diplazoptilon and Xanthopappus, all in the tribe Cardueae of Asteraceae.Methods The nuclear rDNA internal transcribed spacer (ITS) and plastid trnL-F and psbA-trnH regions of these three genera were sequenced. The same regions for other related genera in Cardueae were also sequenced or downloaded from GenBank. Phylogenetic trees were constructed from individual and combined data sets of the three types of sequences using maximum parsimony, maximum likelihood and Bayesian analyses.Key Results The phylogenetic tree obtained allowed earlier hypotheses concerning the relationships of these three endemic genera based on gross morphology to be rejected. Frolovia and Saussurea costus were deeply nested within Dolomiaea, and the strong statistical support for the Dolomiaea-Frolovia clade suggested that circumscription of Dolomiaea should be more broadly redefined. Diplazoptilon was resolved as sister to Himalaiella, and these two together are sister to Lipschitziella. The clade comprising these three genera is sister to Jurinea, and together these four genera are sister to the Dolomiaea-Frolovia clade. Xanthopappus, previously hypothesized to be closely related to Carduus, was found to be nested within a well-supported but not fully resolved Onopordum group with Alfredia, Ancathia, Lamyropappus, Olgaea, Synurus and Syreitschikovia, rather than the Cardinis group. The crude dating based on ITS sequence divergence revealed that the divergence time of Dolomiaea-Frolovia from its sister group probably occurred 13.6-12.2 million years ago (Ma), and the divergence times of the other two genera, Xanthopappus and Diplazoptilon, from their close relatives around 5.7-4.7 Ma and 2.0-1.6 Ma, respectively.Conclusions The findings provide an improved understanding of the intergeneric relationships in Cardueae. The crude calibration of lineages indicates that the uplifts of the Qiinghai -Tibetan Plateau since the Miocene might have served as a continuous stimulus for the production of these morphologically aberrant endemic elements of the Himalayan flora.

Origin of the Qinghai-Tibetan Plateau endemic Milula (Liliaceae): further insights from karyological comparisons with Allium

Milula, a monotypic genus endemic to the Qinghai-Tibetan Plateau, was found to be nested deeply within Allium by the molecular phylogeny despite the aberrant morphology. It remains unknown what had contributed to the rapid evolution of morphology and origin of this exceptional species. In contrast to a previous report of its karyotypes with 2n = 16 = 8M+8SM (2SAT), similar to most species of Allium, a rather different karyotype, 2n = 20 = 4M +10SM+6T (2SAT), was found in examined 31 individuals from 6 populations of M. spicata distributed in the central Tibet. Karyotypes of 7 Allium species occurring in the Qinghai-Tibetan Plateau were further reported. The basic number x = 8 was confirmed for all of them and their karyotypes consist mainly of metacentric and submetacentric chromosomes with rare subterminal and terminal chromosomes. The karyotype of M. spicata is distinctly different from that of most Allium species occurring in the plateau through a complete comparison of all available species in this region and adjacent areas. However, the same chromosome number and similar karyotypic structure were found in A. fasciculatum of Sect. Bromatorrhiza, indicating a possible close relationship between them. But this similarity is contradictory to the preliminary molecular phylogenetic analysis that Milula was closely related to A. cyathophorum of Sect. Bromatorrhiza with x=8, but the other species with x=10 and 11 in this section were clearly placed in the other clade. We therefore suggested that the paralleling evolution from x=8 to x=9, 10 and 11 with increasing asymmetry of karyotype possibly due to the chromosomal Robertsonian translocation might occur separately in the two recognized phylogenetic lineages of Allium. In addition to aneuploidy and following change of the chromosomal structures, the habitat isolation due to the recent uplift of the Qinghai-Tibetan Plateau and the Quaternary climatic oscillation, plays a greater role in origin of Milula and other endemic species (genera) with aberrant morphology from their progenitors.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same speciticity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients. (C) 2004 Elsevier B.V. All rights reserved.

Screening and analysis of bioactive compounds with biofingerprinting chromatogram analysis of traditional Chinese medicines targeting DNA by microdialysis/HPLC

Biofingerprinting chromatogram, analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein. cell. etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophoraflavescens Ait. (Sophora) to bind to ct-DNA. respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated. © 2005 Elsevier B.V. All rights reserved.