半夏种内居群形态变异的模糊聚类分析

在同一栽培条件下,对主要引自长江中下游地区的１５个半夏〔Ｐｉｎｅｌｉａｔｅｒｎａｔａ（Ｔｈｕｎｂ．）Ｂｒｅｉｔ．〕居群的１６个主要形态性状进行模糊聚类分析,在置信水平λ＝０．６７６４时,将１５个居群划分成４个类型：（１）双珠芽类型：主要形态特征为叶柄上均具双珠芽,但叶型和块茎形状变异较小;（２）普通类型：主要形态特征为叶柄上均只着生单珠芽,但叶型和块茎形状变异较大;（３）长茎类型：主要形态特征为叶柄上具单珠芽,但着生位置较低,块茎呈矩圆形;（４）复合类型：主要形态特征为叶柄上具单珠芽,但居群内常有双珠芽个体出现。     

日本榧树果实的化学成分研究

本文研究了日本榧树果实的化学成分。原植物用甲醇提取后经硅胶柱色谱、制备薄层色谱和制备HPLC色谱进行分离纯化,从日本榧树果实中分离得到了4个化合物,用质谱、红外、紫外、核磁等波谱方法鉴定为18-羟基弥罗松酚（1）、kayadiol（2）、花柏酚（3）和落叶松脂素（4）,化合物1．3、4为首次从该植物果实中分离得到。用MTT法检测了化合物1～3的细胞毒性,在浓度100μmol／L时lisa细胞生存率分别为100．00％、26．30％和59．74％。     

黄褐毛忍冬生药学鉴定研究

黄褐毛忍冬具有消炎、抗菌、免疫调节等多种功效,但容易与忍冬属其他物种混淆,通过生药学研究将为黄褐毛忍冬的鉴定工作和药材标准的制定提供科学依据。该文采用植物学鉴定、显微镜观察、薄层色谱鉴定和分子鉴定的方法从植物学性状、药材显微特征、薄层色谱和ITS(internal transcribed spacer)序列特征等方面对黄褐毛忍冬进行了专属性特征鉴定。结果表明：(1)黄褐毛忍冬花朵横切面分泌细胞较多,花瓣外表面有黄褐色腺毛;花冠表皮上层细胞为多角形,蚌形花粉囊对开,花粉粒形状规则,有三角形和卵圆形两种,油室椭圆形。(2)粉末显微检测发现中柱鞘纤维呈短棱形;木栓细胞棱角明显,呈浅黄色;木纤维呈粗短的棱形,偶见有弯曲;网纹导管较多,细胞腔密布草酸钙方晶。(3)薄层色谱显示黄褐毛忍冬花中三柰酚含量较高,三柰酚可作为黄褐毛忍冬的检视成分。(4)基于ITS序列系统聚类结果显示,ITS序列可以作为DNA条形码将黄褐毛忍冬、灰毡毛忍冬、华南忍冬和金银花准确地区分。上述结果表明黄褐毛忍冬生药学特征明显,该研究为黄褐毛忍冬药材鉴定、成分分析和质量标准制定提供了参考。     

滴水珠珠芽发育过程研究

珠芽是滴水珠(Pinellia cordata)的营养繁殖结构,为揭示滴水珠珠芽发育过程中的特征,该研究以野外采集材料进行盆栽观察试验,通过形态观察法和石蜡切片方法,探索滴水珠叶片和叶柄处珠芽发育过程中的形态学和解剖学结构特征。结果表明:滴水珠珠芽发育过程在形态上分为叶柄无现象时期、早期珠芽结构分化和珠芽膨大成熟时期三个时期;相应的解剖学发育时期为珠芽原基启动期和形成期、原基分化期和膨大成熟期四个阶段。叶片和叶柄珠芽起始细胞均由叶柄腹面表皮下层薄壁细胞组织脱分化形成,起始细胞不断分裂形成细胞团最后发育成珠芽原基;在原基分化期的特征是生长点形成,最终分化形成芽原基和鳞片叶;膨大成熟期的特征是珠芽结构不断生长,生长点侧生分化鳞片叶增多。叶片和叶柄处珠芽成熟脱落时期为灰色椭圆形结构,测得平均直径分别为(2.79±0.40)mm和(2.69±0.36)mm,外部有鳞片包裹,内部含大量营养物质。在环境适宜条件下,珠芽遇土萌发,萌发率达75%。这表明滴水珠珠芽与同属植物半夏珠芽发育过程差别不大,都是无性克隆营养繁殖体结构。     

弥勒魔芋珠芽发育过程的形态学和解剖学研究

珠芽是弥勒魔芋的一种重要营养繁殖器官。以弥勒魔芋为材料,通过形态学观察以及解剖学研究,揭示了弥勒魔芋珠芽形成过程中的一系列特征。结果显示:弥勒魔芋珠芽生长于植株叶柄分叉以及裂叶分叉处,由主干茎或主干叶柄表皮下的薄壁细胞重新获得分裂能力后分化而来。珠芽的发育过程可分为珠芽原基启动形成期,珠芽膨大期和珠芽成熟期。珠芽的生长终止于植株衰老倒伏。研究表明,弥勒魔芋的珠芽生长位点明显区别于其他常见植物,珠芽繁殖是魔芋适应生态环境的一种重要机制。     

土木香化学成分的研究

采用硅胶柱色谱、制备薄层色谱、制备高效液相、葡聚糖凝胶SephadexLH-20等方法进行分离纯化,根据光谱数据进行结构鉴定。从乙醇提取物中分离得到了8个化合物,分别鉴定为：异土木香内酯（1）,11,13-二氢异土木香内酯（2）,土木香内酯（3）,β-谷甾醇（4）,3-hydroxy-11,13-dihydroisoalantolactone（5）,macrophyllilae-toneE（6）,HIS-12-en-18-H-3-O-β-D-glucopyranoside（7）,caffeic acid anhydride（8）。化合物7和8系首次从该属植物中分离得到。     

尼日利亚螫毛果(Cnestis ferruginca)中一种具有清除自由基的活性化合物的分离鉴定

用二苯基苦基苯肼自由基薄层实验法（DPPH-TLC-ASSAY）,从尼日利亚螫毛果（Cnestis ferruginca)植物的甲醇提取物中发现了一种具有很强的清除自由基活性的物质;用柱色谱及反相制备柱色谱（RP-LPPLC）,制备出了该活性物质的纯品;用质谱（HR ESI-MS）,核磁共振氢谱（^1H0,碳谱（^13C,DEPT)。紫外光谱及化学方法对该物质进行了结构鉴定,鉴定结果为对苯酚基-6-O-反式咖啡酸基-β-D-吡喃葡萄糖甙。     

瓦松(Orostachys fimbriatus)的形态解剖学研究

利用光学显微镜及扫描电子显微镜,全面观察了一种传统的东方药草——瓦松的茎内部结构、气孔器类型、大小、气孔指数、角质膜纹饰等叶表皮特征及花粉形态和外壁超微结构,并将瓦松的上述结构与景天属植物进行了比较。研究结果表明：瓦松的茎与景天属植物的茎构造基本一致,均由表皮(一层)、皮层(数层)及维管柱三部分组成,但两者在皮层薄壁细胞腔的大小、异细胞含量及维管柱中木质部有无间隔等方面差异明显。叶表皮方面,两者在气孔器类型及分布等方面是一致的,区别在于表皮细胞形状及垂周壁式样、气孔凹陷的程度,以及瓦松的角质层纹饰为十分致密的细小块(片)状,而景天属植物为较典型的条网纹饰等。孢粉学方面,瓦松与景天属植物各项特征亦基本一致,如均为近球形—长球形花粉,角萌发孔,三孔沟明显,外壁2层互内、外层几乎等厚,不同之处在于花粉粒的大小及外壁厚度上。建议将一些差异显著的特征,如角质层纹饰等,作为景天科属、种划分的重要依据。综合瓦松的形态解剖学研究结果,我们认为在系统发育方面,瓦松比景天属植物进化。     

黔产小叶黄杨中环维黄杨星D的鉴别及不同部位含量测定

对黔产小叶黄杨中环维黄杨星D进行薄层色谱研究,并比较了小叶黄杨不同部位环维黄杨星D的含量,为合理开发小叶黄杨的药用资源提供科学依据。采用薄层色谱法对小叶黄杨中环维黄杨星D进行薄层色谱研究,采用高效液相色谱电喷雾检测器法对小叶黄杨不同部位环维黄杨星D进行含量测定。结果显示:薄层色谱中环维黄杨星D斑点清晰;高效液相色谱中小叶黄杨不同部位环维黄杨星D的含量差异很大,粗茎含量最高,叶子含量最低。该方法可用于小叶黄杨的薄层鉴别和含量测定,为了合理开发和利用黔产小叶黄杨,其药用部位以粗茎最好。     

超积累和非超积累生态型东南景天茎、叶内生细菌多样性分析

【目的】探究和比较超积累和非超积累生态型东南景天茎、叶微生物群落结构的异同。【方法】采用高通量测序技术研究野外两种生态型东南景天茎和叶片的内生细菌群落结构。【结果】4个样品总共得到366 783条有效序列和39 948个OTU(97%相似度)。从Shannon指数得知:两种生态型东南景天叶片内生菌的多样性均高于茎;超积累生态型东南景天叶片内生菌的多样性高于非超积累生态型,但非超积累生态型东南景天茎组织中内生菌多样性高于超积累生态型东南景天。超积累生态型东南景天的叶片和茎中的内生菌分别包括26和21个门,123和117个科;非超积累生态型东南景天叶片和茎中的内生菌分别包括43和22个门,175和83个科,4个样品的优势菌群均为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和蓝藻细菌门(Cyanobacteria)。在属分类水平上,超累积生态型东南景天叶片和茎第一优势菌属分别为Synechococcus和Plesiomonas;非超积累生态型东南景天叶片和茎组织第一优势菌群分别为Pseudomonas和Dechloromonas。【结论】两种生态型东南景天的叶片和茎中均具有丰富的内生细菌,但超积累生态型东南景天叶片内生菌多样性最大,且存在一些独有的功能菌群。     

"6 markers/5 colors" extended white blood cell differential by flow cytometry.

Electronic white blood cell (WBC) differential by standard cytology (hematology analyzer and visual inspection of blood smears) is limited to five types and identification of abnormal cells is only qualitative, often problematic, poorly reproducible, and labour costing. We present our results on WBC differential by flow cytometry (FCM) with a 6 markers, 5 colors CD36-FITC/CD2-PE+CRTH2-PE/CD19-ECD/CD16-Cy5/CD45-Cy7 combination, on 379 subjects, with detection of 12 different circulating cell types, among them 11 were quantified. Detection of quantitative abnormalities of whole leucocytes, neutrophils, eosinophils, basophils, monocytes, or lymphocytes was comparable by FCM and by standard cytology in terms of sensitivity and specificity. FCM was better than standard cytology in detection and quantification of circulating blast cells or immature granulocytes, with a first lineage orientation in the former case. All cases of lymphocytosis, with lineage assignment, were detected by FCM. FCM identified a group of patients with excess of CD16pos monocytes as those having an inflammatory syndrome. WBC differential by FCM is at least as reliable as by standard cytology. FCM superiority consists in identification and systematic quantification of parameters that cannot be assessed by standard cytology such as lineage orientation of blast cells or lymphocytes, and expression of markers of interest such as CD16 on inflammatory monocytes.     

Combination of quantification and observation methods for study of "Side Population" cells in their "in vitro" microenvironment.

BACKGROUND: Qualitative and quantitative analyses of the rare phenotypic variants in in vitro culture systems is necessary for the understanding of cell differentiation in cell culture of primary cells or cell lines. Slide-based cytometry combines image acquisition and data treatment, and associates the power of flow cytometry (FCM) and the resolution of the microscopic studies making it suitable for the analysis of cells with rare phenotype. In this paper we develop a method that applies these principles to a particularly hot problem in cell biology, the study of stem cell like cells in cultures of primary cells, cancer cells, and various cell lines. METHODS: The adherent cells were labeled by the fluorescent dye Hoechst 33342. The images of cell populations were collected by a two-photon microscope and processed by a software developed by us. The software allows the automated segmentation of the nuclei in a very dense cell environment, the measurement of the fluorescence intensity of each nucleus and the recording of their position in the plate. The cells with a given fluorescence intensity can then be located easily on the recorded image of the culture plate for further analysis. RESULTS: The potential of our method is illustrated by the identification and localization of SP cells in the cultures of the C2C12 cell line. Although these cells represent only about 1% of the total population as calculated by flow cytometry, they can be identified in the culture plate with high precision by microscopy. CONCLUSION: Cells with the rare stem-cell like phenotype can be efficiently identified in the undisturbed cultures. Since the fluorescence intensity of rare events and the position of thousands of surrounding cells are recorded at the same time, the method associates the advantage of the FCM analysis and the microscopic observation.     

Ultrastructure of "villose cells" in the early stages of their formation in the process of chemical carcinogenesis in the CNS]

Early stages of redistribution of cellular elements around the pill of carcinogenic agent DMBA introduced into the right hemisphere of the brain of female SHK albino rats were studied. Precursors of the ciliated cells were established to appear in the perivascular tissue within 12 h. Within 24 h they accumulate around the pill bed, and within 48 h a cellular edging is formed around DMBA. The cytoplasm of ciliated cells become richer in organelles. The main marker in identification of cells in all periods of experiment were lipoid inclusions in the cytoplasm which are different from lipids of usual macrophages occurring both in experiments and in control, in a polygonal shape and sinuous contour. Within 48 h the cytoplasm of ciliated cells form long lancet-shaped spiculae with upright walls. The cytoplasm of macrophages gives only short, somewhat sinuous processes.     

S-100 alpha-like immunoreactivity in tubules of rat kidney. A clue to the function of a "brain-specific" protein

The localization of the alpha and beta subunits of S-100 protein was studied in normal tissue where the identification of three subclasses of S-100 containing cells was derived: i) cells that contain both alpha and beta subunits; ii) cells that contain only the alpha subunit; and iii) cells that contain only the beta subunit. In this study monospecific antibodies against the S-100 alpha and beta subunits were used to characterize the S-100-like immunoreactivity in the rat kidney: Certain cells in the distal nephron, i.e., the connecting piece, collecting ducts, and the thin limb of Henle's loop, contained S-100 alpha immunoreactivity. Proximal tubules, the thick ascending limb of Henle's loop, the distal tubules, and the juxtaglomerular apparatus were negative. No S-100 beta immunoreactivity was found in kidney tubules. However, Schwann cells of renal pelvic nerves contained S-100 beta immunoreactivity. The presence of S-100 alpha antigen in certain cells of the kidney gives further support to the assumption that the alpha subunit of S-100a is related to cells that are highly involved in pH, electrolyte, and water regulation.     

Global proteomic assessment of the classical protein-tyrosine phosphatome and "Redoxome"

Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H(2)O(2). Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.     

Primary culture of medaka (<Emphasis Type="Italic">Oryzias latipes</Emphasis>) testis: a test system for the analysis of cell proliferation and differentiation

An in vitro test system using primary testis cells of the medaka (Oryzias latipes) was established that provides quantitative data on cell proliferation and spermatocyte differentiation. The primary cultures were characterised over a period of 2 days with respect to cell viability and the distribution of adherent and suspended cells. These two cell populations were maintained at a dynamic equilibrium in vitro for several days. The proliferating cells were predominantly present amongst the clusters of suspended cells as determined by BrdU labelling (cytological identification and quantification by ELISA). Based on cytological criteria the proliferating cells were mostly spermatogonia and preleptotene spermatocytes. Differentiation of spermatocytes to spermatids or spermatozoa was also observed mainly amongst suspended cells. Quantification of cell proliferation and cell differentiation by flow cytometry was achieved by labelling the primary cells with carboxyfluorescein diacetate N-succinimidyl ester, which allowed the identification and quantification of meiotically or mitotically dividing primary cells. Addition of the flavonoid genistein (10 µg/ml) to the primary cultures inhibited both cell proliferation and cell differentiation significantly. The test system is suitable for the study of the effect of substances which interfere with spermatogenesis in the vertebrate medaka model.     

Visible and "cryptic" segregation of parental chromosomes in embryonic stem hybrid cells

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.     

Proteomic analysis of the Mexican lime tree response to "Candidatus Phytoplasma aurantifolia" infection

"Candidatus Phytoplasma aurantifolia" is the causative agent of witches' broom disease in the Mexican lime tree (Citrus aurantifolia L.), and is responsible for major tree losses in Southern Iran and Oman. The pathogen is strictly biotrophic, and, therefore, completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We applied a proteomics approach to analyse gene expression in Mexican limes infected with "Ca. Phytoplasma aurantifolia". Leaf samples were collected from healthy and infected plants and were analysed using 2-DE coupled with MS. Among 800 leaf proteins that were detected reproducibly in eight biological replicates of healthy and eight biological replicates of infected plants, 55 showed a significant response to the disease. MS resulted in identification of 39 regulated proteins, which included proteins that were involved in oxidative stress defence, photosynthesis, metabolism, and the stress response. Our results provide the first proteomic view of the molecular basis of the infection process and identify genes that could help inhibit the effects of the pathogen.     

Human bone marrow: a reservoir for "enhanced effector memory" CD8+ T cells with potent recall function

The role of human bone marrow (BM) CD8+ T cells in the immune response to viral Ags is poorly defined. We report here the identification and characterization of a functionally enhanced effector memory CD8+ T cell population (TEM) in the BM of patients undergoing total joint replacement for osteoarthritis. These BM-derived TEM differ strikingly from correlate cells in peripheral blood (PB), expressing elevated levels of CD27, HLA-DR, CD38, CD69, and unique patterns of chemokine receptors. Interestingly, while BM TEM have low levels of resting perforin and granzyme B, these molecules evidence profound up-regulation in response to TCR stimulation resulting in enhanced cytotoxic potential. Moreover, compared with the TEM subset in PB, BM CD8+ TEM cells demonstrate a more vigorous recall response to pooled viral Ags. Our results reveal that human BM serves as a repository for viral Ag-specific TEM with great therapeutic potential in vaccine development.