Pre-column derivatization of amines with 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl chloroformate followed by LC-fluorescence and LC-APCI-MS

A pre-column derivatization method for the sensitive determination of amines using the labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl chloroformate (BCIC-Cl) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives is carried out by high performance liquid chromatography/atmospheric pressure chemical ionization (LC-APCl-MS-MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent is replaced by 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl functional group, which results in a sensitive fluorescence derivatizing reagent BCIC-Cl. BCIC-Cl can easily and quickly label amines. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography and show an intense protonated molecular ion corresponding m/z [MH](+) under APCl in positive-ion mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 260 corresponding to the cleavage of CH2-OCO bond. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3 to 4-fold molar reagent excess. In addition, the detection responses for BCIC derivatives are compared with those obtained using CEOC and FMOC as derivatization reagents. The ratios of l(BCIC)/l(CEOC) and l(BCIC)/l(FMOC) are, respectively, 1.23-3.14 and 1.25-3.08 for fluorescent (FL) responses (here, l is relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C-8 column. Detection limits are calculated from 1.0 pmol injection, at a signal-to-noise ratio of 3, are 10.6-37.8 fmol. The mean interday accuracy ranges from 94 to 105% for fluorescence detection with the largest mean %CV < 7.5. The mean interday precision for all standards is < 6.0% of the expected concentration. Excellent linear responses are observed with coefficients of > 0.9997.

Stability of alpine meadow ecosystem on the Qinghai-Tibetan Plateau

The meadow ecosystem on the Qinghai-Tibetan Plateau is considered to be sensitive to climate change. An understanding of the alpine meadow ecosystem is therefore important for predicting the response of ecosystems to climate change. In this study, we use the coefficients of variation (Cv) and stability (E) obtained from the Haibei Alpine Meadow Ecosystem Research Station to characterize the ecosystem stability. The results suggest that the net primary production of the alpine meadow ecosystem was more stable (Cv = 13.18%) than annual precipitation (Cv = 16.55%) and annual mean air temperature (Cv= 28.82%). The net primary production was insensitive to either the precipitation (E = 0.0782) or air temperature (E = 0.1113). In summary, the alpine meadow ecosystem on the Qinghai-Tibetan Plateau is much stable. Comparison of alpine meadow ecosystem stability with other five natural grassland ecosystems in Israel and southern African indicates that the alpine meadow ecosystem on the Qinghai-Tibetan Plateau is the most stable ecosystem. The alpine meadow ecosystem with relatively simple structure has high stability, which indicates that community stability is not only correlated with biodiversity and community complicity but also with environmental stability. An average oscillation cycles of 3-4 years existed in annual precipitation, annual mean air temperature, net primary production and the population size of consumers at the Haibei natural ecosystem. The high stability of the alpine meadow ecosystem may be resulting also from the adaptation of the ecosystem to the alpine environment.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.

贵阳市不同土地利用方式土壤有机碳地球化学研究

本研究根据贵阳市区域内不同土地利用方式，选择了自然土壤、农业土壤和城市土壤为主要研究对象。对表层土壤实行多样点的统计分析、以及典型剖面有机碳迁移过程分析，同时结合同位素地球化学示踪原理，探讨了贵阳市区域内土地利用方式变化对土壤有机碳的影响，以及不同土地利用方式间土壤有机碳的来源和降解过程的差异。主要结论如下： (1) 自然土壤转化为农业土壤后，表层(0～10cm) SOC有较为明显的降低趋势。其中，相对于自然土壤(黄壤) 表层SOC(平均值)而言，玉米地、水田、果园下降了40%左右，菜地下降了15%左右。然而，不同菜地土壤间耕种强度存在较大差异，其SOC变异程度高于其它几类农业土壤(CV���57.07%)。与之相反，农业土壤表层DOC(18.86～48.20mg•L-1)接近或超过自然土壤(10.74～36.30 mg•L-1)，且ƒDOC占SOC的比例明显大于自然土壤。其中，玉米地DOC最高(平均值：48.20mg•L-1)，菜地次之(平均值：30.00mg•L-1)，果园第三(平均值：29.87mg•L-1)，水田最低(平均值：18.86mg•L-1)，但水田由于干湿交替的影响，DOC的变异程度最大(CV���128.57%)。据此推断，在相同气候条件下，自然土壤转化为农业土壤后，由于表层DOC数量和比例的增加，提高了SOC的迁移性，进而加速了碳素在土壤中的迁移转化进程。 (2) 自然土壤转化为农业土壤后，剖面内部(>10cm)多数层次SOC相对于黄壤和黄色石灰土有明显的增加趋势。且通过对不同类型农业土壤人为干扰强度的调查表明：人为干扰强度越强，剖面中一定深度内SOC增加幅度越大。即：离城市较近的菜地2增长最为突出，果园其次，水田和玉米地相当。离城市较远的菜地1由于受人为干扰层次较浅，且出现了犁底层，剖面内SOC的含量水平与黄色石灰土相当。 (3) 原始土壤经搬运重组后形成城市公路绿化带土壤，表层SOC和DOC变幅较宽、离散程度较大，且没有随时间或植被类型等因素的变化而呈现明显的变化趋势。其中，SOC变异程度依次为新成公路绿化带2(CV���58.0％)、老成公路绿化带(CV���55.5％)、新成公路绿化带1(CV���34.1％)。DOC变异程度依次为新成公路绿化带1(CV���93.8％)、新成公路绿化带2(CV���85.7％)、老成公路绿化带(CV���78.0％)。 (4) 在自然土壤、农业土壤和城市绿化带土壤表层中，DOC与SOC、N、C/N、NO3-、NH4-，以及粘粒含量等的相关性均未达到显著水平。另据方差分析显示：果园、水田、菜地和玉米地表层土壤间DOC、SOC含量均无显著差异，说明农业土壤利用方式不是决定土壤表层SOC和DOC含量的绝对因素；新成公路绿化带1，2和老成公路绿化带表层土壤间DOC、SOC含量均无显著差异，说明植被类别和形成时间不是决定土壤表层SOC和DOC含量的绝对因素。 (5) 自然土壤中，枯枝落叶转化为表层(0～5cm)土壤有机质后，δ13CSOC值升高了1~4‰。通过不同碳源间δ13Corg相互关系的判断，在具备枯枝落叶覆盖的表层土壤中，DOC主要来源于枯枝落叶；而在土壤剖面内，随土壤剖面深度的增加，来自于土壤腐殖类物质的DOC占土壤DOC总量的比例呈增加趋势。在黄壤和黄色石灰土中(>5cm土层)，土壤剖面中大多数层次DOC比SOC更富13C。 (6) 大多数农业土壤有机碳δ13C值显示其有机肥源中存在C4-C源。且农业土壤中受碳源多样性的影响，菜地、果园、水田和玉米地表层土壤中δ13CSOC与δ13CDOC的相关性均未达到显著水平。其次，除玉米地土壤剖面外，其它供试农业土壤剖面大多数层次δ13CDOC值比δ13CSOC偏负，说明菜地、果园、水田土壤DOC主要为外源的加入。 (7) C3植被转化为C4植被(林-农生态系统转化)后，玉米地剖面中SOC有2.55％～20.80％源于C4-C，随剖面层次的加深有降低趋势，但表现为“之”字形反复；DOC中C4-C的比例在剖面0～40cm处较为相近(25.94％～34.54％)，40cm以下则急剧下降(3.18％～15.65％)。说明玉米地剖面 DOC主要来源于土壤腐殖类物质的转化。与林-农生态系统转变过程中的变化趋势相反，洼地农业土壤退耕弃荒一段时间(林-农-林生态系统转化)后，土壤剖面内C4-C占SOC的比例随土壤层次的加深逐渐增加，变化范围在5.77～26.76％。 (8) 在C3植被转向C4植被(林-农生态系统转化)后，玉米地δ13CSOC值与C4-C、C3-C相关系数(r)分别为0.372和-0.102，δ13CDOC值与C4-C、C3-C相关系数分别为0.131和-0.339，其相关性均未达到显著水平。而再从C4植被转回C3植被后，土壤δ13CSOC与C3-C之间呈显著相关性（r=0.88，n＝7），说明退耕弃荒后新加入的C3-C对土壤δ13CSOC值影响较大。其SOC的主要来源于洼地周边坡面土壤的侵蚀堆积物和新生草本植被残体。结合当前SOC降解过程的研究成果，本研究认为：洼地土壤退耕弃荒后一段时间里，土壤SOC可能处于累积大于损失状态。这有利于土壤性状向良性方向发展。 (9) 根据同位素值的相互关系和有机碳的来源调查，判断公路绿化带土壤中C4-C为原始土壤所带来。大气颗粒物和雨水中的DOC是表层土壤DOC的主要来源。公路绿化带土壤剖面中，随着时间的增加，土壤腐殖类物质与DOC的相互转化逐渐加强。 上述结论可为人类认识城市区域(特别是有强烈酸性沉降历史的喀斯特城市区域)土地利用方式改变对土壤碳循环的影响，以及不同土地利用方式间土壤有机碳迁移转化过程提供科学依据，也可为正确评估城市区域土壤与其他圈层间碳循环的源、汇关系提供基础资料。

Enhanced anthocyanin production by repeated-batch culture of strawberry cells with medium shift

Repeated-batch cultures of strawberry cells (Fragaria ananassa cv. Shikinari) subjected to four medium-shift procedures (constant LS medium, constant B5 medium, alternation between LS and B5 starting from LS and alternation between LS and B5 starting from B5) were investigated for the enhanced anthocyanin productivity. To determine the optimum period for repeated batch cultures, two medium-shift periods of 9 and 14 days were studied, which represent the end of the exponential growth phase and the stationary phase. By comparison with the corresponding batch cultures, higher anthocyanin productivity was achieved for all the repeated-batch cultures at a 9-day medium-shift period. The average anthocyanin productivity was enhanced 1.7-and 1.76-fold by repeated-batch cultures in constant LS and constant B5 medium at a 9-day shift period for 45 days, respectively. No further improvement was observed when the medium was alternated between LS (the growth medium) and B5 (the production medium). Anthocyanin production was unstable at a 14-day shift period regardless of the medium-shift procedures. The results show that it is feasible to improve anthocyanin production by a repeated-batch culture of strawberry cells.

社会形势预警的几种建模方法比较

Based on social survey data conducted by local research group in some counties executed in the nearly past five years in China, the author proposed and solved two kernel problems in the field of social situation forecasting: i) How can the attitudes’ data on individual level be integrated with social situation data on macrolevel; ii) How can the powers of forecasting models’ constructed by different statistic methods be compared? Five integrative statistics were applied to the research: 1) algorithm average (MEAN); 2) standard deviation (SD); 3) coefficient variability (CV); 4) mixed secondary moment (M2); 5) Tendency (TD). To solve the former problem, the five statistics were taken to synthesize the individual and mocrolevel data of social situations on the levels of counties’ regions, and form novel integrative datasets, from the basis of which, the latter problem was accomplished by the author: modeling methods such as Multiple Regression Analysis (MRA), Discriminant Analysis (DA) and Support Vector Machine (SVM) were used to construct several forecasting models. Meanwhile, on the dimensions of stepwise vs. enter, short-term vs. long-term forecasting and different integrative (statistic) models, meta-analysis and power analysis were taken to compare the predicting power of each model within and among modeling methods. Finally, it can be concluded from the research of the dissertation: 1) Exactly significant difference exists among different integrative (statistic) models, in which, tendency (TD) integrative models have the highest power, but coefficient variability (CV) ones have the lowest; 2) There is no significant difference of the power between stepwise and enter models as well as short-term and long-term forecasting models; 3) There is significant difference among models constructed by different methods, of which, support vector machine (SVM) has the highest statistic power. This research founded basis in all facets for exploring the optimal forecasting models of social situation’s more deeply, further more, it is the first time methods of meta-analysis and power analysis were immersed into the assessments of such forecasting models.

中学生强迫症状及其心理、环境危险因素和整合模型研究

Obsessive-Compulsive Symptoms, which are one of common factors effected on mental health of secondary school students, have been rarely studied at home and abroad. In accordance with the problems existed in these literature up till now, the thesis had mainly studied the measured tool, contents and structure, developmental features, psychosocial risk factors and integrated model of obsessive-compulsive symptoms from them by means of investigation with quetionnaires. The entire research was divided into three phases. 3185 students(age 14.68±1.75 years) were firstly measured with the 20-item Leyton Obsessional Inventory-Child Version(LOI-CV) at four secondary schools including six grades in Beijing city, which was applied to revise LOI-CV, and to study the structure and contents, developmental features and screen of obsessive-compulsive symptoms. Then, 216 subjects with obsessive-compulsive symptoms, paired with controls in the light of school, grade and gender, were investigated with 10 self-rating scales on obsessive-compulsive symptoms, anxiety, depression, personality, coping and attributional style, negative life events, parent's rearing style, family environment and life adaptation in school, and with an inventory on social demography. The results were used to explore psychosocial risk factors and integrated model of obsessive-compulsive symptoms. The third survey was only carried out, about two months after the second, among 264 subjects with obsessive-compulsive symptoms through MMOCI and Negative Life Event Scale for Adolescents, in order to probe into the integrated model. The research had mainly found: (1) LOI-CV can be used as a screen tool for obsessive-compulsive symptoms in urban adolescents in China; (2) Total screening-out ratio of obsessive-compulsive symptoms was 13.6% (male:15.0%, female:12.2%). The most common manifestations of obsessive-compulsive symptoms were hating dirt and contamination, doing things in exact manner, angry if someone messes desk, bad conscience but no one else, worry about cleanliness, repeated thoughts or words; the least were favorite or special number, spending extra time on homework, special number or words to avoid, talking or moving to avoid bad luck, fussy about hands. The checking and repetition, cleanliness and tidiness, general obsessions were more common forms than numbers-luck; (3) No differences were existed in serious degree of obsessive-compulsive symptoms, but the screening-out ratio in male was higher than it in female; (4) No differences were detected in the serious degree of obsessive-compulsive symptoms except the scores of cleanliness and tidiness among grades, but the screening-out ratio of the grades justly entering secondary school or going to graduate were higher than other ones; (5) The main psychosocial risk factors for obsessive-compulsive symptoms included anxiety, mother's over-protecting and over-interfering, fantasy, flexibility, self-actualization, peers relationship, sense of responsibility, negative life events, mother's occupation, help-seeking, and (6) The integrated model on psychosocial risk factors suggested that the possible developed and sustained mechanism of obsessive-compulsive symptoms was that personality, coping and attributional styles constructed the developmental diathesis foundation of obsessive-compulsive symptoms; negative life events were promoting factors of them. There was a dynamic interaction between personality and environmental factors. Negative emotion played a core role in the developmental process of them. The continued existence of obsessive-compulsive symptoms was related to pre-existed obsessive-compulsive symptoms and negative life events experienced by an individual. Therefore, this research not only let us get a deeper understanding of obsessive-compulsive symptoms and more entirely find out psychosocial risk factors, firstly applied diathesis-stress theory to comprehend the psychological mechanism of obsessive-compulsive symptoms and, moreover, elaborate and expand it, but also has more important practice significance of treatment, prevent and education for obsessive-compulsive symptoms in secondary school students.

Instability of anthocyanin accumulation in Vitis vinifera L. var. Gamay Freaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, in Vitis vinifera cell cultures. Therefore, four cell line suspensions of Vitis vinitera L. var. Gamay Freaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73 +/- 0.15, 1.45 +/- 0.04, 0.77 +/- 0.024 and 0.27 +/- 0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% for V vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities to L-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line, showed greater potential in enhanced the anthocyanin production.

Preparation of supported PtRu/C electrocatalyst for direct methanol fuel cells

In this work, high-surface supported PtRu/C were prepared with Ru(NO)(NO3)(3) and [Pt(H2NCH2CH2NH2)(2)]Cl-2 as the precursors and hydrogen as a reducing agent. XRD and TEM analyses showed that the PtRu/C catalysts with different loadings possessed small and homogeneous metal particles. Even at high metal loading (40 wt.% Pt, 20 wt.% Ru) the mean metal particle size is less than 4 nm. Meanwhile, the calculated Pt crystalline lattice parameter and Pt (220) peak position indicated that the geometric structure of Pt was modified by Ru atoms. Among the prepared catalysts, the lattice parameter of 40-20 wt.% PtRu/C contract most. Cyclic voltammetry (CV), chronoamperometry (CA), CO stripping and single direct methanol fuel cell tests jointly suggested that the 40-20 wt.% PtRu/C catalyst has the highest electrochemical activity for methanol oxidation. (c) 2004 Elsevier Ltd. All rights reserved.

Structure and chemical composition of supported Pt-Sn electrocatalysts for ethanol oxidation

Carbon supported PtSn alloy and PtSnOx particles with nominal Pt:Sn ratios of 3:1 were prepared by a modified polyol method. High resolution transmission electron microscopy (HRTEM) and X-ray microchemical analysis were used to characterize the composition, size, distribution, and morphology of PtSn particles. The particles are predominantly single nanocrystals with diameters in the order of 2.0-3.0 nm. According to the XRD results, the lattice constant of Pt in the PtSn alloy is dilated due to Sn atoms penetrating into the Pt crystalline lattice. While for PtSnOx nanoparticles, the lattice constant of Pt only changed a little. HRTEM micrograph of PtSnOx clearly shows that the change of the spacing of Pt (111) plane is neglectable, meanwhile, SnO2 nanoparticles, characterized with the nominal 0.264 nm spacing of SnO2 (10 1) plane, were found in the vicinity of Pt particles. In contrast, the HRTEM micrograph of PtSn alloy shows that the spacing of Pt (111) plane extends to 0.234 nm from the original 0.226 nm. High resolution energy dispersive X-ray spectroscopy (HR-EDS) analyses show that all investigated particles in the two PtSn catalysts represent uniform Pt/Sn compositions very close to the nominal one. Cyclic voltammograms (CV) in sulfuric acid show that the hydrogen ad/desorption was inhibited on the surface of PtSn alloy compared to that on the surface of the PtSnOx catalyst. PtSnOx catalyst showed higher catalytic activity for ethanol electro-oxidation than PtSn alloy from the results of chronoamperometry (CA) analysis and the performance of direct ethanol fuel cells (DEFCs). It is deduced that the unchanged lattice parameter of Pt in the PtSnOx catalyst is favorable to ethanol adsorption and meanwhile, tin oxide in the vicinity of Pt nanoparticles could offer oxygen species conveniently to remove the CO-like species of ethanolic residues to free Pt active sites. (C) 2005 Elsevier Ltd. All rights reserved.