黄土孢粉分析方法的研究

根据黄土的化学成分等特点,采取相应的化学及物理相结合的处理方法,把孢粉离析出来。在黄土最为发育的陕西、甘肃７ 个黄土剖面计９０ 多个样品中均获得丰富的孢粉,观察１—３ 张玻片,孢粉总数可达２０４—５９４ 粒以上,２５０ ｇ 的土样可获得３．９—５５．１ 万粒孢粉     

Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues.     

基于相关分析和偏最小二乘回归的黄绵土土壤全氮和碱解氮含量的高光谱预测

以采取植被恢复措施的陕西省吴起县为研究区,实地采集24个土壤剖面不同层次的黄绵土土样100个,在进行土壤样本全氮（TN）和碱解氮（AHN）含量及实验室反射光谱数据测量和分析的基础上,用相关分析（CA）结合偏最小二乘回归（PLS）方法建立黄绵土土壤TN和AHN含量的校正模型,并用独立样本对校正模型进行验证.结果表明： 利用6种光谱变换方式建立的校正模型中,微分光谱建立的校正模型是预测研究区土壤TN含量的最佳模型,校正和验证R²分别为0.929和0.935,均方根误差（RMSE）分别为0.045和0.047 g·kg^-1,相对预测偏差（RPD）为3.12;而归一化变换建立的校正模型是预测土壤AHN含量的最佳模型,校正和验证R²分别为0.873和0.773,RMSE分别为9.946和16.204 mg·kg^-1,RPD为1.538.所建立的全氮预测模型可以对0～40 cm土层的TN进行有效预测,而碱解氮的预测模型对同一深度只能进行粗略预测.本研究为采取植被恢复措施的退化生态系统区黄绵土土壤全氮的快速预测提供了一种较好的方法,但是对于碱解氮的准确、快速预测,需要进一步研究.     

浑善达克沙地高西马格剖面孢粉分析及植被演化的初步探讨

 为了探讨浑善达克沙地5 000年以来的植被演化过程, 我们对内蒙古正蓝旗境内高西马格湖人工挖掘2.0 m深的剖面进行了较高时间分辨率的孢粉分析,并参考孢粉组合与植被之间的对应关系,得出: 该期的植被和气候演化大致可以划分为3个阶段：阶段 I（约5 010～4 040 aBP）,孢粉组合     

Isolation of human fibroblast heterokaryons with two-colour flow sorting (FACS II)

Red and green fluorescent polystyrene beads were used to label different populations of cultured human skin fibroblasts. After co-cultivation for 24 h no exchange of label could be observed. Twenty-four hours after fusion the population of red-green heterofluorescent cells was sorted out with a FACS II cell sorter. Microscopical examination of the heterofluorescent population 20 h after sorting indicated, that with Sendai-induced fusion, 50–60% of these viable cells contained more than one nucleus. After polyethylene glycol (PEG)-induced fusion, between 70 and 85% bi- or multinucleated cells were present in the sorted and cultured fraction. Autoradiography using [³H]thymidine indicated that the sorted heterofluorescent bikaryons were in fact heterokaryons.     

An attempt to separate mononuclear cells fused with human red blood cell-ghosts from a cell mixture treated with HVJ (Sendai virus) using a fluorescence activated cell sorter (FACS II).

Nucleated cells (Ehrlich ascites tumor cells or L strain cells) and human red blood cells (RBC)-ghosts were mixed and fused by ultraviolet-inactivated HVJ (Sendai virus). The cell mixture was stained with FITC conjugated anti-RBC ghost antiserum and then applied to FACS II apparatus. The apparatus sorted mononuclear cells fused with RBC-ghosts from the cell mixture on the basis of both the light scattering and fluorescence profiles. When the same procedure was carried out on a mixture containing cells and intact human RBC, the cells sorted by this method were cells into which hemoglobin had been injected. The sorted cells were capable of forming colonies in culture. This sorting method may be useful for collecting cells in which macromolecules have been injected artificially by fusion of RBC-ghosts enclosing macromolecules.     

Human ferritin light chain gene sequences mapped to several sorted chromosomes

Summary The iron storage ferritin light-chain gene exhibits multiple restriction enzyme fragments which have been mapped by analyzing sorted human chromosomes. A dual laser chromosome sorter was used to construct spot-blot filter panels representing 22 chromosome fractions. Hybridization of radiolabeled human ferritin-L gene probe to spot-blot panels revealed the ferritin-L gene on more than one chromosome. Miniaturized restriction enzyme analysis was used to map each of the ferritin-L restriction fragments uniquely to one of three chromosomes. This combination of sorted chromosome analyses provides a rapid method to map homologous DNA sequences located on more than one chromosome.     

Flow cytometry and sorting of meiotic prophase cells of female rabbits

We present a new, flow cytometric method by which cells in various stages of the meiotic prophase can be quantitated and sorted in partly enriched fractions. Ovarian cells of 3-16-day-old rabbits were mechanically dispersed and fixed in ethanol and aldehydes. The cell suspension was stained with the DNA fluorochrome mithramycin and analysed and sorted in a FACS IV cell sorter according to the fluorescence and forward light scatter distribution. Cells sorted onto slides were stained with haematoxylin and eosin and differentially counted in the microscope. In the diploid fraction, preleptotene cells were more fluorescent than somatic cells. Leptotene cells were found throughout the S fraction and the tetraploid fraction. Zygotene and pachytene cells caused a major peak in the tetraploid region with 10-25% more fluorescence than somatic cells. Cells in diplotene had 5-15% more fluorescence than somatic cells. Mitotic cells were 20-40% more fluorescent than somatic cells and scattered the light more intensely than did meiotic cells with the same fluorescence.     

Viable sorting of intact multicellular spheroids by flow cytometry

A flow cytometric method has been developed for sorting viable, intact multicellular spheroids in order to obtain uniformly-sized populations with diameters in the range of 50-100 microns. A FACS II instrument was modified for this purpose by installing a 200-microns-diameter exit orifice and by making adjustments in the sheath flow, oscillator frequency, and number of droplets sorted. Polystyrene microspheres (44 and 88 microns diameter) and 41-96-microns-diameter spheroids could be sorted and recovered with 70-100% efficiency, an improvement over previous reports. Unstained, viable spheroids were simultaneously analyzed for small-angle forward light scatter, 90 degree light scatter, and autofluorescence using a 488-nm laser operating at 100 mW. Analysis of the data demonstrated a considerable variation in both the 90 degrees light scatter and the autofluorescence signals for a given forward angle light scattering signal. By setting narrow sort windows on the forward angle light scattering signal and either the 90 degree light scatter or autofluorescence signals, uniformly spherical spheroid populations could be recovered. These sorted populations had coefficients of variation of the mean diameter in the range of 5-9%. This represents a variation of less than one cell diameter, and is a major improvement over any other technique. There was no significant difference in the subsequent growth rates of sorted spheroids compared to the unsorted spheroids. This technique will apply when uniform populations of small spheroids are required, such as investigations of the contact effect or in the initiation of growth curve studies.     

Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates

Stable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost-effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their alpha- and epsilon-amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical "shotgun" proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 microg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.     

High-throughput fluorescence-based isolation of live C. elegans larvae

For the nematode Caenorhabditis elegans, automated selection of animals of specific genotypes from a mixed pool has become essential for genetic interaction or chemical screens. To date, such selection has been accomplished using specialized instruments. However, access to such dedicated equipment is not common. Here we describe live animal fluorescence-activated cell sorting (laFACS), a protocol for automatic selection of live first larval stage (L1) animals using a standard FACS system. We show that FACS can be used for the precise identification of GFP-expressing and non-GFP-expressing subpopulations and can accomplish high-speed sorting of live animals. We have routinely collected 100,000 or more homozygotes from a mixed starting population within 2 h, and with greater than 99% purity. The sorted animals continue to develop normally, making this protocol ideally suited for the isolation of terminal mutants for use in genetic interaction or chemical genetic screens.     

Routine monitoring of Cryptosporidium oocysts in water using flow cytometry

A flow cytometric method for the routine analysis of environmental water samples for the presence of Cryptosporidium oocysts has been developed. It uses a Coulter Epics Elite flow cytometer to examine water samples and to separate oocysts from contaminating debris by cell sorting. The sorted particles are then rapidly screened by microscopy. The method has been evaluated and compared with direct epifluorescence microscopy on 325 river, reservoir and drinking water samples. The technique was found to be more sensitive, faster and easier to perform than conventional epifluorescent microscopy for the routine examination of water samples for Cryptosporidium.     

Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein

The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that colocalized by immunostaining with endogenous pro-SP-C were scattered throughout the parenchyma. EGFP was not detected in Clara cell secretory protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence, because EGFP is not secreted. Type II cells could also be purified from single-cell suspensions of lung homogenates using fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared with >95% of the sorted population. As expected for type II cells, ultrastructural analysis revealed that the sorted cells contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected in the sorted population, but T1alpha and CD31 (platelet endothelial cell adhesion molecule) were not, indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.     

Penetration of metham and mylone into soil columns as measured by their effect on viability of microsclerotia ofVerticillium dahliae

Summary The penetration of metham into loess or heavy soils was studied by measuring the viability of microsclerotia (MS) ofV. dahliae placed at various depths in soil columns. Equal quantities of metham were applied in three ways; concentrated, dilute and combined application (i.e. concentrated and dilute application successively). Penetration was best in both soils when a combined application of the fungicide was used. In dilute application metham killed MS through the top 21 cm, wheres in concentrated application the fungicide killed MS placed between 26–38 cm. The combined application however, killed MS placed from 0–38 cm. It was found that using the combined application method, metham could kill MS in loess soil to a depth of 74 cm depending on depth of irrigation and amount of fungicide used. With mylone in granular form surface application was found to be better for MS control than incorporated or combined applications. Activities of metham and mylone againstV. dahliae MS increased with the increase of incubation temperatures; the highest acitivities of the fungicides were observed at 35°C. Determinations of lethal curves of MS treated with metham or mylone showed that in both heavy and loess soils activity of metham against MS was higher than that of mylone in granular form. In heavy soil the amount of metham or mylone required to achieve ED 50 was about 25% more than in loess soil.Contribution from the Agricultural Research Organisation, The Volcani Center, Bet Dagan, Israel. No. 212-E, 1978 Series.     

The potential role of life cycle assessment in regulation of chemicals in the European union

Scope and Background  This paper presents the preliminary results from an ongoing feasibility study, investigating potential application of elements from the life cycle assessment (LCA) framework in European chemicals’ policy. Many policy areas affect manufacturing, marketing and use of chemicals. This article focuses on the general chemical legislation, especially issues related to regulatory risk assessment and subsequent decisions on risk reduction measures. Method  Current and upcoming chemical regulation has been reviewed and empirical knowledge has been gained from an ongoing case study and from dialogues with various stakeholders. Results and Discussion  LCAs are comparative and more holistic in view as compared to chemical risk assessments for regulatory purposes¹. LCAs may therefore potentially improve the basis for decisions between alternatives in cases where a risk assessment calls for risk reduction. In this process, LCA results might feed into a socio-economic analysis having similar objectives, but some methodological aspects related to system boundaries need to be sorted out. Life cycle impact assessment (LCIA) of toxic effects has traditionally been inspired by the more regulatory-orientated risk assessment approaches. However, the increasing need for regulatory priority setting and comparative/ cumulative assessments might in the future convey LCIA principles into the regulatory framework. The same underlying databases on inherent properties of chemicals are already applied in both types of assessment. Similarly, data on the use and exposure of chemicals are needed within both risk assessments and LCA, and the methodologies might therefore benefit from a joint ‘inventory’ database. Outlook  The final outcome of the feasibility study will be an implementation plan suggesting incorporation of core findings in future chemical regulation and related policy areas.     

Sorting out of normal and virus-transformed cells in cellular aggregates

The sorting-out behavior (self-segregation of two cell types from mixtures of the two) of five different established cell lines was studied. Eight of the ten possible binary combinations of these lines, cultured as cellular aggregates, were examined. Mouse BALB/c 3T3 cells sorted out internally to the corresponding malignant SV40 virus-transformed 3T3 cells. The transformed 3T3 line (SVT-2) did not sort out from a revertant line selected from SVT-2 cells by resistance to concanavalin A (con A). The revertant cells sorted out externally to the parent BALB/c 3T3 cells, although segregation was generally incomplete. BALB/c 3T3 cells did not sort out from another contact-inhibited line of 3T3 cells derived from Swiss albino mice (Swiss 3T3). Both BALB/c 3T3 and Swiss 3T3 cells sorted out from cells of the contact-inhibited hamster line, NIL B. Instead of a two-layered sphere, however, a three-layered structure was observed with most of the NIL B cells external to the 3T3 cells, and a few NIL B cells comprising the center of the sphere. On the other hand, NIL B cells did not consistently sort out from either the SVT-2 or con A cells. In general, sorting out between pairs of these five lines are slower and less complete than is generally observed between the more extensively studied chick embryonic tissue cells, suggesting that the cultured cells may be more closely related in their adhesive properties. The internal segregation of BALB/c 3T3 cells relative to SVT-2 cells is consistent with the hypothesis that transformed cells are less adhesive than their nontransformed counterparts.     

A New Extraction Method of Loess Shoulder-Line Based on Marr-Hildreth Operator and Terrain Mask

Loess shoulder-lines are significant structural lines which divide the complicated loess landform into loess interfluves and gully-slope lands. Existing extraction algorithms for shoulder-lines mainly are based on local maximum of terrain features. These algorithms are sensitive to noise for complicated loess surface and the extraction parameters are difficult to be determined, making the extraction results usually inaccurate. This paper presents a new extraction approach for loess shoulder-lines, in which Marr-Hildreth edge operator is employed to construct initial shoulder-lines. Then the terrain mask for confining the boundary of shoulder-lines is proposed based on slope degree classification and morphology methods, avoiding interference from non-valley area and modify the initial loess shoulder-lines. A case study is conducted in Yijun located in the northern Shanxi Loess Plateau of China. The Digital Elevation Models with a grid size of 5 m is applied as original data. To obtain optimal scale parameters, the Euclidean Distance Offset Percentages between shoulder-lines is calculated by the Marr-Hildreth operator and the manual delineations. The experimental results show that the new method could achieve the highest extraction accuracy when σ = 5 in Gaussian smoothing. According to the accuracy assessment, the average extraction accuracy is about 88.5%, which indicates that the proposed method is applicable for the extraction of loess shoulder-lines in the loess hilly and gully areas.     

A combination method of chemical with enzyme reactions for identification of membrane proteins

A simple method for effective analysis of various proteins has been developed, including membrane proteins, with LC-MS/MS, using CNBr and acetic acid cleavage in one reaction for the digestion of both the M/ and /D/ positions within the target proteins. This dual chemical reaction has been compared with traditional CNBr or an acid cleavage method using a rat kidney membrane fraction and it showed an advantage of the dual reaction with respect to a high number of peptides detected and a high protein recovery. Furthermore, when this dual chemical reaction was combined with trypsin digestion, the number of proteins surprisingly increased approximately 3.0 times more than in the cases with the trypsin digestion only. It was also 1.9 times more than in cases dealing with Tube-Gel trypsin digestion, which is one of the most efficient digestion methods. In addition, it was shown that this dual chemical reaction could be applied to an in-gel digestion. Using the combination of the chemical and enzyme reaction, 172 proteins including 95 membrane proteins were identified. This indicated that this method is one of the efficient systems in single MS/MS analysis. In particular, many membrane proteins identified in this study were detected by a new combination, but not by a traditional trypsin digestion method.     

西北黄土区石油污染土壤原位微生物生态修复试验研究

通过对西北黄土石油开采区石油污染土壤生物强化原位微生物生态修复方法的试验研究,充分利用强化原位微生物菌群辅以物理和化学方法与土壤环境相结合的微生物生态技术,进行了土壤中石油的降解与修复试验研究,试验结果显示,土壤中平均石油含量在2754mg/kg时,经过lld～32d强化原位微生物生态修复技术的修复,土壤中石油含量降解可达40.92%～80.37%,验证了微生物生态修复技术在西北黄土区土壤石油污染修复的有效性,探索了推广应用的可行性.     

THE PHYTOTOXICITY OF MINERAL OILS AND HYDROCARBONS

Mineral oils are being increasingly used as herbicides, and there is little doubt that they could be used more effectively if the phytotoxic properties of any particular fraction could be predicted from knowledge of its physical and chemical properties without the need for extensive testing in the field. This paper reviews some of the work that has been done on the correlation of toxicity with physical and chemical properties of oils, and presents some of the results obtained in an investigation into the toxicity of mineral oils and hydrocarbons to plants, being carried out by the Agricultural Research Council Unit of Experimental Agronomy.