中药青蒿萜类代谢物谱分析及青蒿素生物合成的研究

青蒿素是从中药青蒿，学名黄花蒿（Artemisia annua L.）植物地上部分分离出的抗疟疾有效单体，为一种倍半萜内酯类化合物，其生物合成途径属于植物类异戊二烯代谢途径。青蒿素生物合成途径及其调控机制仍不完全清楚，本论文采用GC-MS 和GC×GC-TOFMS 方法对青蒿萜类代谢物谱进行检测，用多维统计学方法对检测结果进行整理和比较分析，研究青蒿素生物合成及其与青蒿中其他萜类代谢的关系，取得了以下结果： 一、通过GC×GC-TOFMS 方法对青蒿挥发油成分进行分析，共鉴定出303 种组分。其中挥发油中相对百分含量大于1%的10 种组分中有9 种为萜类化合物，含量接近总挥发油的50%。在相对百分含量大于0.1%的49 种成分中，有30 种萜类化合物。有27 种相对百分含量大于0.1%的成分首次在青蒿挥发油中报道，其中包括10 种萜类化合物。 二、利用GC-MS 方法分析了青蒿001 和SP18 两个青蒿素高产株系不同生长时期萜类代谢物谱，结果表明：青蒿中萜类化合物在不同时期合成和积累是动态变化的，萜类化合物的种类和数量在营养生长期随生长时间的延长而提高，在营养生长后期和现蕾前期达到最高水平，进入生殖生长后随生长时间的延长而迅速降低。通过多维统计PLS-DA（Partial Leasted Square Discriminant Analysis） 分析，确定001 中有17 个化合物的含量在不同生长时期有明显变化，其中15 个为萜类化合物。SP18 中有18 个化合物的含量在不同生长时期有明显变化，其中16 个为萜类化合物。青蒿素，青蒿酸，二氢青蒿酸，青蒿素B 都是含量变化明显的标记物。其中青蒿酸和二氢青蒿酸含量在营养生长后期达到最高水平，进入生殖生长后迅速下降，而青蒿素和青蒿素B 在整个检测时期含量变化相对较小，在营养生长时期含量已经较高，在现蕾前期含量稍有上升，进入现蕾期后有所下降，本研究确定现蕾前期为代谢物谱分析最佳取样时期，并为药材采收提供指导。 三、不同基因型青蒿代谢物谱研究表明，青蒿素高产株系SP18 和001 代谢物表现出一定的差异，通过多维统计PLS-DA 分析，共找出了22 种在两种基因型中差异明显的化合物，其中包括倍半萜化合物12 种，单萜化合物3 种，三萜化合物4 种。SP18 特征化合物为樟脑和两个未鉴定倍半萜化合物，而001 特征化合物是龙脑和β-法呢烯。另外两种基因型中青蒿素及相关前体化合物的积累模式差异明显，SP18 中二氢青蒿酸和青蒿素含量高，而青蒿酸和青蒿素B 含量极低；001 中二氢青蒿酸和青蒿素含量相对SP18 要低，但青蒿素B 和青蒿酸含量比SP18 要高。该结果表明在青蒿素高产株系中，青蒿素含量与二氢青蒿酸的含量呈正相关，结合Brown 等的活体标记研究结果分析，从二氢青蒿酸到青蒿素的转化可能是青蒿素合成的限速步骤。 四、利用GC×GC-TOFMS 方法对转基因青蒿萜类代谢物谱进行了分析，共对200 个左右化合物峰进行PLS-DA 和OSC-PLS (Orthogonal Signal Correction–Partial leasted Square)多维统计分析，结果表明：青蒿萜类代谢物谱在外源基因转入后发生显著变化，与对照株系相比均呈现显著差异。其中过量表达Amorpha-4,11-diene 合酶基因（ads）株系中青蒿素及相关化合物变化最明显，而过量表达FPP 合酶基因（fps）株系中青蒿素及相关化合物变化相对较小，在受到调控而成为差异标记物的化合物中，70%是倍半萜类化合物。 五、考察了外源茉莉酸甲酯对青蒿素生物合成的影响，结果表明：300 μM 外源茉莉酸甲酯能提高青蒿素含量，在处理后第8 天青蒿素含量提高38%。青蒿萜类代谢物谱研究表明，茉莉酸甲酯不仅可以诱导青蒿中青蒿素的合成，还能诱导很多化合物，特别是倍半萜和三萜类的合成。OSC-PLS 分析结果找出了9 个处理后含量明显提高的标记物，其中6 个倍半萜化合物，3 个三萜化合物。标记物鲨烯含量提高了67%，另一个未鉴定出结构的倍半萜提高了60%，这些化合物可能与青蒿素有着类似的调控机制，而外源喷洒茉莉酸甲酯可以作为提高青蒿素产量的有效途径之一。

Molecular evolution of CXCR1, a G protein-coupled receptor involved in signal transduction of neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1.

Identification of differentially expressed immune-relevant genes in Chinese soft-shelled turtle (Trionyx sinensis) infected with Aeromonas hydrophila

Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and profiling gene expression. Aeromonas hydrophila, a ubiquitous waterborne bacterium, is one of the most frequent pathogens isolated from diseased aquatic organisms. In order to understand the molecular mechanism of anti-bacteria immune response in reptile, we have investigated the differentially expressed genes in Chinese soft-shelled turtle (Trionyx sinensis) experimentally infected with A. hydrophila by suppression subtractive hybridization (SSH). Forty-two genes were identified from more than 200 clones, of which 25 genes are found for the first time in reptiles, and classified into 6 categories: 18 in defense/immunity. 4 in catalysis, 2 in retrotransposon; 2 in cell signal transduction, 5 in cell metabolism, 10 in protein expression, and 1 in cell structure. Of the 42 differentially expressed genes, 6 genes, IL-8, serum amyloid A (SAA), CD9, CD59, activating transcription factor 4 (ATF4) and cathepsin L genes, were further observed to be up-regulated in the infected turtles by virtual Northern hybridization and RT-PCR assays. (C) 2008 Elsevier B.V. All rights reserved.

Molecular characterization and IFN signal pathway analysis of Carassius auratus CaSTAT1 identified from the cultured cells in response to virus infection

Type I interferon (IFN) exerts its pleiotropic effects mainly through the JAK-STAT signaling pathway, which is presently best described in mammals. By subtractive suppression hybridization, two fish signaling factors, JAK1 and STAT1, had been identified in the IFN-induced crucian carp Carassius auratus L. blastulae embryonic (CAB) cells after treatment with UV-inactivated grass carp hemorrhagic virus (GCHV). Further, the full-length cDNA of STAT1, termed CaSTAT1, was obtained. It contains 2926 bp and encodes a protein of 718 aa. CaSTAT1 is most similar to rat STAT1 with 59% identity overall and displays all highly conserved domains that the STAT family possesses. Like human STAT1beta, it lacks the C-terminus acting as transcriptional activation domain in mammals. By contrast, only a single transcript was detected in virus-induced CAB cells. Expression analysis showed that CaSTAT1 could be activated by stimulation of CAB cells with poly I:C, active GCHV, UV-inactivated GCHV or CAB IFN, and displayed diverse expression patterns similar to that of mammalian STATI. Additionally, the expression of an antiviral gene CaMx1 was also induced under the same conditions, and expression difference between CaSTAT1 and CaMx1 was revealed by induction of CAB IFN. These results provide molecular evidence supporting the notion that the fish IFN signaling transduction pathway is similar to that in mammals. Fish IFN exerts its multiple functions, at least antiviral action, through a JAK-STAT pathway. (C) 2004 Elsevier Ltd. All rights reserved.

Lineshape Analysis of the Beat Signal Between Optical Carrier and Delayed Sidebands

This paper presents the lineshape analysis of the beat signal between the optical carrier and the shifted and delayed side-bands produced by sinusoidal amplitude modulation. It is shown that the beat signal has a typical lineshape with a very narrow delta-peak superposed on a quasi-Lorentzian profile. Theoretical explanation for the appearance of this peak has been given based on optical spectral structure constructed by a large number of optical wave trains. It is predicted that the delta-peak is originated from the beat between the wave trains in the carrier and those in the delayed sidebands when their average coherence length is longer than the delay line. Experiments carried out using different delay lines clearly show that the delta-peak is always located at the modulation frequency and decreases with the increasing delay line. Our analysis explicitly indicates that the linewidth is related to the observation time. It is also suggested that the disappearance of the delta-peak can be used as the criterion of coherence elimination.

Small-Signal Equivalent-Circuit Model and Characterization of 1.55-mu m Buried Tunnel Junction Vertical-Cavity Surface-Emitting Lasers

The work was supported in part by the National Natural Science Foundation of China under Grant 60536010, Grant 60606019, Grant 60777029, and Grant 60820106004, and in part by the National Basic Research Program of China under Grant 2006CB604902, Grant 2006CB302806, and Grant 2006dfa11880.

Subtraction of scattering parameters for small-signal modulation characteristics of laser diode

An extended subtraction method of scattering parameters for characterizing laser diode is proposed in this paper. The intrinsic response is extracted from the measured transmission coefficients of laser diode, and the parasitics of packaging net-work laser chip are determined from the measured reflection coefficient of laser diode simultaneously. It is shown that the theories agree well with the experimental results.

Measurement of small-signal and large-signal responses of packaged laser modules at high temperature

In this paper, the pulsed injection method is extended to measure the chip temperature of various packaged laser modules, such as the DFB laser modules, the FP laser modules, and the EML laser modules. An optimal injection condition is obtained by investigating the dependence of the lasing wavelength on the width and period of the injection pulse in a relatively wide temperature range. The small-signal frequency responses and large-signal performances of packaged laser modules at different chip temperature are measured. The adiabatic small-signal modulation characteristics of packaged LD are first extracted. In the large-signal measurement, the effects of chip temperature, bias current and driving signal on the performances of the laser modules are discussed. It has been found that the large-signal performances of the EML modules depend on the different red-shift speeds of the DFB and EAM sections as chip temperature varying, and the optimal characteristics may be achieved at higher temperature.

Small-signal power measuring technique for measuring the frequency response of electroabsorption modulators

We propose and demonstrate measurement of the frequency response of an electroabsorption (EA) modulator using an extended small-signal power measuring technique. In this technique, the modulator is driven by a microwave carrier amplitude modulated by a low-frequency signal, and the modulator frequency response is obtained without the need of a high-speed photodetector. Based upon the nonlinear characteristics of the EA modulator and the underlying principle of the present method, equations have been derived. A measurement scheme using a network analyzer and a low-speed photodetector has been proposed and constructed, and the experimental results confirm that our proposed method is as accurate as the swept-frequency measurement using a network analyzer directly.

XPS depth profiling study of n/TCO interfaces for p-i-n amorphous silicon solar cells

Detailed X-ray photoelectron spectroscopy (XPS) depth profiling measurements were performed across the back n-layer/transparent conducting oxide (n/TCO) inter-faces for superstrate p-i-n solar cells to examine differences between amorphous silicon (a-Si:H) and microcrystalline silicon (mu c-Si:H) n-layer materials as well as TCO materials ZnO and ITO in the chemical, microstructural and diffusion properties of the back interfaces. No chemical reduction of TCO was found for all variations of n-layer/TCO interfaces. We found that n-a-Si:H interfaces better with ITO, while n-mu c-Si:H, with ZnO. A cross-comparison shows that the n-a-Si:H/ITO interface is superior to the n-mu c-Si:H/ZnO interface, as evidenced by the absence of oxygen segregation and less oxidized Si atoms observed near the interface together with much less diffusion of TCO into the n-layer. The results suggest that the n/TCO interface properties are correlated with the characteristics of both the n-layer and the TCO layer. Combined with the results reported on the device performance using similar back n/TCO contacts, we found the overall device performance may depend on both interface and bulk effects related to the back n/TCO contacts. (c) 2006 Elsevier B.V. All rights reserved.

A small signal equivalent circuit model for resonant tunnelling diode

We report a resonant tunneling diode (RTD) small signal equivalent circuit model consisting of quantum capacitance and quantum inductance. The model is verified through the actual InAs/In0.53Ga0.47As/AlAs RTD fabricated on an InP substrate. Model parameters are extracted by fitting the equivalent circuit model with ac measurement data in three different regions of RTD current-voltage (I-V) characteristics. The electron lifetime, representing the average time that the carriers remain in the quasibound states during the tunneling process, is also calculated to be 2.09 ps.