黄连ISSR反应条件优化的研究

以黄连(味连,Coptis Chinensis Franch.)基因组DNA为模板,通过单因子、双因子实验研究了ISSR反应体系中主要成分（Mg²⁺、dNTP、引物、模板、Taq DNA聚合酶）以及热循环参数（退火温度、循环数、变性时间、退火时间、延伸时间）对扩增结果的影响,并找出各自的最适条件,建立了适合黄连ISSR分析的反应体系和扩增程序,即在25μL反应体系中,内含1×PCR buffer、1.5mmol·L^-1 Mg²⁺、200μmol·L^-1 dNTP、0.3 μmol·L^-1引物、40 ng模板、1 U TaqDNA聚合酶。扩增程序为94℃预变性5 min,然后进行35个循环：94℃变性30 s,（据不同引物的退火温度）复性1 min,72℃延伸1.5 min,循环结束后72℃延伸7 min,-4℃保存。这一优化系统的建立为今后利用ISSR标记技术进行黄连鉴定及种质遗传多样性分析提供了一个标准化程序。     

The retention properties of nucleobases in alkyl C8-/C18- and IAM-chromatographic systems in relation to log Pow

In order to evaluate the differences in the partition properties of 35 structurally congeneric nucleobases of biological interests in octanol-water biphasic, alkyl C(8)/C(18), and IAM systems, a comparative chromatographic study was performed. Comparing with the reversed-phase C(8)/C(18) retention data, most of the purines possessed weaker IAM retention except for those with specific H-bond and/or electrostatic interactions. Quantitative correlations between the experimental log P(ow) literature values and the IAM, C(8), and C(18) log k were evaluated (R(2)=0.943, 0.794, and 0.767, respectively). Although IAM retention correlated significantly better (larger R(2) value) with the log P(ow) values statistically, the latter was revealed apparently behaving more like (slope approaching unity) alkyl C(8)/C(18) retention and hence also has the same shortcoming in under-representing analytes capable of forming short-term H-bond/electrostatic interactions with polar head-groups of phospholipids. A chemically meaningful structure-retention model (q(2)=0.824 and R(2)=0.968) was derived, in which the hydrophobic interaction is identified as the underlying factor for the retention of purines in IAM system modulated non-trivially by H-bond/electrostatic interactions.     

Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS

Malonyl-CoA is a key intermediate involved in lipid synthesis and lipid oxidation. Here, we report on a novel method for the quantification of malonyl-CoA and seven other short-chain acyl-CoAs in various rat and mouse tissues using ion-pairing reversed-phase HPLC/MS. This method is capable of measuring malonyl-CoA, free coenzyme A (CoASH), acetyl-CoA, beta-hydroxyl-butyryl-CoA (HB-CoA), 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), propionyl-CoA, succinyl-CoA, and isobutyryl-CoA simultaneously with a dynamic linear range over two orders of magnitude in a 7.0 min HPLC gradient run. The lower limit of quantification (LLOQ) was 0.225 pmol for all acyl-CoAs studied, except for HMG-CoA which had a higher LLOQ of 0.90 pmol. The interference of HB-CoA on the quantification of malonyl-CoA in animal tissues was also explored for the first time.     

An effective sample preparation approach for screening the anticancer compound piceatannol using HPLC coupled with UV and fluorescence detection

Piceatannol, compared with the renowned resveratrol, is a better anticancer agent and a superior agent with other biological activities. However, as there are only few plants reported to contain minute quantity of piceatannol, the scarcity of sources greatly impedes the piceatannol-related researches. To explore new sources of piceatannol, we established a sample preparation approach for screening the piceatannol in plants using HPLC-UV-fluorescence detection. When the HPLC is coupled with UV and fluorescence detectors, the decrease of signals in interferences and increase of signal in piceatannol in the fluorescence chromatogram mark clearly the position of the piceatannol peak; ultimately, it allows identification without standards. In this study, we systematically evaluated the factors affecting the extraction efficiency of piceatannol in sample preparation. Of the sample preparation strategies studied, direct solvent extraction and liquid nitrogen treatment followed by solvent extraction gave satisfactory recoveries for both piceatannol and resveratrol. These approaches avoided time-consuming lyophilization procedure. In addition, all procedures must be done in the dark to avoid negative impact of irradiation from fluorescence light on the recoveries of piceatannol and resveratrol. With the present method, we re-examined the plants previously claimed to contain only resveratrol for their piceatannol contents. The species examined included Polygonum cuspidatum, Arachis hypogaea, Vitis thunbergii, and Ampelopsis brevipedunculaata. The results showed, for the first time, all these plants contain piceatannol. The finding implies that the resveratrol-containing plants may also contain piceatannol. The results demonstrate the feasibility of these sample preparation approaches and may further the understanding for the distribution of piceatannol in plants.     

Quantification of valproic acid and its metabolite 2-propyl-4-pentenoic acid in human plasma using HPLC-MS/MS

A specific and sensitive HPLC-MS/MS method for the quantitative determination of valproic acid (VPA) and its metabolite, 2-propyl-4-pentenoic acid in human plasma has been developed, using VPA-d15 as the internal standard. The method was based on pre-column derivatization using 4-dimethylaminobenzylamine dihydrochloride. The derivatives were separated with a gradient elution and quantified by positive electrospray ionization with multiple reaction monitoring. The assay provides routine quantification limits of 200 ng/mL for VPA and 20 ng/mL for 4-ene VPA with within- and between-day coefficients of variation of <10%. This method has been applied to the analysis of plasma samples obtained from patients treated with this drug.     

Two new antibacterial sesquiterpenoids from Centipeda minima

Two new guaiane-type sesquiterpene lactones, compounds 1 and 2, along with three known guaianolide- or pseudoguaianolides, were isolated from Centipeda minima (whole plant). Their structures were identified by spectroscopic and mass-spectrometric analyses. The configuration at C5 of the guaiane framework of 1 was rationalized by quantum-mechanical calculations (Table 2). All compounds were found to be active against eight different microbial pathogens (Table 3), with MIC values in the range of 6.25-100 microg/ml.     

Cellular-automata models of solid-liquid interfaces

A series of cellular-automata (CA) models have been created, simulating relationships between water (or aqueous solutions) and solid surfaces of differing hydropathic (i.e., hydrophilic or hydrophobic) nature. Both equilibrium- and dynamic-flow models were examined, employing simple breaking and joining rules to simulate the hydropathic interactions. The CA simulations show that water accumulates near hydrophilic surfaces and avoids hydrophobic surfaces, forming concave-up and concave-down meniscuses, resp., under equilibrium conditions. In the dynamic-flow simulations, the flow rate of water was found to increase past a wall surface as the surface became less hydrophilic, reaching a maximum rate when the solid surface was of intermediate hydropathic state, and then declining with further increase in the hydrophobicity of the surface. Solution simulations show that non-polar solutes tend to achieve higher concentrations near hydrophobic-wall surfaces, whereas other hydrophobic/hydrophilic combinations of solutes and surfaces do not show such accumulations. Physical interpretations of the results are presented, as are some possible biological consequences.     

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.