Innovative analytical system for screening on lectins

Lectins are proteins or glycoproteins from plants, animals or microorganisms, which typically bind specifically to sugar residues, e.g., those located in cell walls or membranes. This reaction might change the physiology of the cell wall and influences the metabolism inside the cell. Some lectins of plants stimulate the immune system by unspecific activation of T-cells or influence cell division; others cause agglutination of cells (e.g., erythrocytes) and are therefore from therapeutic interest. In a new approach, biomolecular interaction analysis (BIA) was utilized for a screening program on lectins. The BIA has been done by a new interferometric biosensor based on spectral-phase interference (SPI). The system can be used either for characterisation of lectin binding domains or for a screening on lectins obtained from natural sources. Several lectin binding surfaces on the basis of SPI have been established.     

Electron Microscopic Identification and Morphologic Preservation of Enriched Populations of Lung Cells Isolated by Laser Flow Cytometry and Cell Sorting: A New Technique

There is increasing need to verify the identities of cell subpopulations enriched by laser flow cytometry and fluorescence-activated cell sorting (FACS). When cell subpopulations isolated from whole organs or tissues have similar characteristics (e.g., size, granularity, staining), light, phase contrast or fluorescence microscopy may not provide sufficient resolution to identify isolated cells accurately and many flow cytometric parameters (e.g., viability, fluorescence) require the cells to be live at the point of analysis where the cell transects the laser beam. In some studies, cells identified by fluorescence microscopy as a highly enriched subpopulation were found by electron microscopy to contain significant populations of other cell types. A technique, fixation-in-flow (FIF), has been developed to increase ability to correlate morphological and laser analyses of cell subpopulations. Sheath fluid is replaced by fixative, permitting fixation to be initiated immediately after laser beam analysis of live cells. This new procedure yields improved cytoarchitectural preservation of recovered cell subpopulation(s) for evaluation by transmission or scanning electron microscopy. This report presents results from applying the methodology to identify more accurately cell subpopulations of the distal lung, specifically type II pneumocytes, Clara cells and pulmonary macrophages. A modification of this procedure was employed to isolate fibroblast subpopulations from murine lung fibroblasts grown in vitro and the procedure is being used to determine the responses of cultured fibroblasts to other permutations (e.g., X-irradiation, cytokines).     

Rapid microfluorimetry of enzyme reactions in single living cells

An optimized photon counting technique allows the microfluorimetric study of NAD⁺ (or NADP⁺) reduction-reoxidation transients in single living cells with a time resolution in the range of 1/50-1/100 sec. The transients resulting from the micro-electrophoretic addition of metabolites (e.g. Glc-6-P or Glc-1-P) can be analyzed in terms of early parameters (e.g. initial lag, rise half time or full rise time) and overall parameters (time of rise and half decay, amplitude, reoxidation time). Both the initial lag and rise half time are considerably longer with Glc-1-P than with Glc-6-P, possibly due to control at the phosphoglucomutase or compartmentation of glycolytic phosphate esters. While glycolytic NAD⁺ (or NADP⁺) reduction proceeds adequately in aerobic EL2 and EAT ascites cells (although ΔNADH/Δt is higher at anaerobiosis), it is critically dependent upon anaerobiosis in L and astrocytoma cells. Thus by rapid microfluorimetry it is possible to resolve the rising phase or other segments of the fluorescence transients into components each corresponding to a particular step in the sequence of intracellular events or control states.     

The site of KCN inhibition in the photosynthetic electron transport pathway

Treatment of chloroplasts with high concentrations of KCN inhibits reactions which involve Photosystem I (e.g. electron transport from water or diaminodurene to methylviologen), but not those assumed to by-pass Photosystem I (e.g. electron transport from water to quinonediimides). The spectrophotometric experiments described in this paper showed that KCN inhibits the oxidation of cytochrome f by far-red light without blocking its reduction by red light. Both optical and EPR experiments indicated that KCN does not inhibit the photooxidation of P700 but markedly slows down the subsequent dark decay (reduction). Reduction of P700 by Photosystem II is prevented by KCN. It is concluded that KCN blocks electron transfer between cytochrome f and P700, i.e. the reaction step which is believed to be mediated by plastocyanin. In KCN-poisoned chloroplasts the slow dark reduction of P700 following photooxidation is greatly accelerated by reduced 2,6-dichlorophenolindophenol or by reduced N-methylphenazonium methosulfate (PMS), but not by diaminodurene. It appears that the reduced indophenol dye and reduced PMS are capable of donating electrons directly to P700, at least partially by-passing the KCN block.     

AM真菌摩西管柄囊霉对干旱胁迫下枳抗氧化酶基因表达的影响

测定分析了接种丛枝菌根（AM）真菌摩西管柄囊霉Funneliformis mosseae对正常供水与干旱处理的盆栽枳Poncirus trifoliata实生苗生长、活性氧代谢及抗氧化酶基因表达量的影响。结果表明,7周干旱处理显著降低了根系菌根侵染率。接种摩西管柄囊霉显著促进了干旱处理的枳植株生长,增加了根系体积和叶片相对含水量,显著降低了叶片脯氨酸含量,同时也上调了干旱处理的枳叶片精氨酸脱羧酶基因（PtADC1和PtADC2）和超氧化物歧化酶基因（PtFe-SOD和PtMn-SOD）、过氧化物酶基因（PtPOD）和过氧化氢酶基因（PtCAT1）的表达,因而维持了一个相对更低的活性氧水平（如过氧化氢）,有利于增强植株的抗旱性。     

Venetian Turpentine as an Aid in Squashing and Concomitant Production of Durable Chromosome Mounts

Root tips are hydrolyzed in 1 N HCl at 60 C for 10-12 min, Feulgen stained, and macerated in a minimal amount of propiono-carmine. A drop of Venetian turpentine mounting medium (Harleco brand was used) is mixed with the carmine stain, the cover slip applied, the tissue pressed gently while observed under a dissecting microscope to spread the chromosomes, and finally firmly squashed. High quality slides of over 1 yr durability are obtained which are well suited to morphological studies, photographing under oil, or routine screening in polyploidy studies. The carmine stain in conjunction with the Feulgen aids to give contrast to chromosomes which are difficult to stain (e.g. hops) but it may be omitted for other species.     

Research advancement in the processes and mechanisms of transporting methane by emerged herbaceous plants and hygrophytes

Methane (CH₄) is an important greenhouse gas, and is involved in atmospheric chemical reactions. Aquatic and hydric environments are important sources of atmospheric CH₄. Majority of CH₄ are transported and released to atmosphere by emerged herbaceous plants and hygrophytes in aquatic and hydric environments. In recent decades, there has been increasing attention on how plants transport CH₄. During CH₄ transportation processes, several interfaces of CH₄ exchange play important roles. First, the tips of lateral roots are primary locations (hotspots) for CH₄ entering the root systems and regulate the gross CH₄ transportation. Then, the diaphragms in the aerenchyma and the root collar impose great resistances for the overall CH₄ transportation processes. In early studies, it was controversial that whether CH₄ emission from plants to atmosphere was controlled by stomas or micropores (small cracks and holes in aboveground part of plant except the blade). Recent studies have confirmed the dominant role of micropores for CH₄ transportation and emission. The dead and damaged stems are widely considered to have positive effects on CH₄ transportation. Diffusion and convection are the two main transporting mechanisms of CH₄, with the efficiency of convection being generally higher than that of diffusion. Both biological (e.g. biomass and photosynthesis) and environmental (e.g. light, temperature and humidity) factors regulate the CH₄ transportation. Many studies have contributed to understanding the CH₄ transportation processes and mechanisms by emerged herbaceous plants and hygrophytes. However, there are still some questions needing further investigations. Issues of consideration may include the operational efficiency in the critical interfaces of CH₄ exchange, the plant parts that play a decisive role in the entire CH₄ transportation, the underlying roles of diffusion and convection on CH₄ interfaces exchanges and entire long distance transports, the combined and coupling effects and mechanisms of biotic and abiotic factors, and the similarities and differences of CH₄ transporting processes and mechanisms among plant species.     

Application and prospects of high-throughput screening for in vitro neurogenesis

Over the past few decades, high-throughput screening (HTS) has made great contributions to new drug discovery. HTS technology is equipped with higher throughput, minimized platforms, more automated and computerized operating systems, more efficient and sensitive detection devices, and rapid data processing systems. At the same time, in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes. However, challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional (3D) in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions. Here, we review the applications of HTS technologies on in vitro neurogenesis, especially aiming at identifying the essential genes, chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids. We also discuss the developmental tendency of HTS technology, e.g., so-called next-generation screening, which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.     

Inhibition of respiration in yeast by light

Irradiation of starved cultures of Saccharomyces cerevisiae with blue light under aerobic conditions inhibited the capacity of the yeast cells to respire added substrates (e.g., ethanol) and stimulated endogenous respiration. Spectroscopic examination of the cells showed that the irradiation destroyed both cytochrome a and a₃ components of cytochrome oxidase and a part of the cytochrome b. Irradiation under anaerobic conditions had no effect on the respiratory capacity or the cytochrome content of the cells. Under aerobic conditions cytochrome a₃ was protected against photodestruction when complexed with cyanide and cytochrome a was protected when complexed with azide.     

A recent retrotransposon insertion of J caused E6 locus facilitating soybean adaptation into low latitude

Soybean (Glycine max) is an important legume crop that was domesticated in temperate regions. Soybean varieties from these regions generally mature early and exhibit extremely low yield when grown under inductive short-day (SD) conditions at low latitudes. The long-juvenile (LJ) trait, which is characterized by delayed flowering and maturity, and improved yield under SD conditions, allowed the cultivation of soybean to expand to lower latitudes. Two major loci control the LJ trait: J and E6. In the current study, positional cloning, sequence analysis, and transgenic complementation confirmed that E6 is a novel allele of J, the ortholog of Arabidopsis thaliana EARLY FLOWERING 3 (ELF3). The mutant allele e6^PG, which carries a Ty1/Copia-like retrotransposon insertion, does not suppress the legume-specific flowering repressor E1, allowing E1 to inhibit Flowering Locus T (FT) expression and thus delaying flowering and increasing yields under SD conditions. The e6^PG allele is a rare allele that has not been incorporated into modern breeding programs. The dysfunction of J might have greatly facilitated the adaptation of soybean to low latitudes. Our findings increase our understanding of the molecular mechanisms underlying the LJ trait and provide valuable resources for soybean breeding.     

莱州湾滨海柽柳林湿地植被碳储量的分布特征及其影响因素

莱州湾南岸的柽柳林湿地是我国北方现存面积最大的柽柳林滨海湿地,也是我国“南红北柳”生态工程的重要组成部分.本文基于2014年8月在昌邑海洋生态特别保护区的调查资料,研究了该湿地植被生物量、碳储量的空间分布特征及其影响因素.结果表明: 该湿地植被生物量为949.0 g·m^-2,植被碳储量为393.1 g·m^-2,基本呈中部高、东西部低的分布特征;植被各部分生物量、碳储量均表现为:地上部>地下部>凋落物.该区域主要有柽柳、碱蓬2个单优群落和4个混生群落,植被碳储量以柽柳群落最高,混生群落居中,碱蓬群落最低.受北部潮间带防潮坝的影响,表层土壤的含水量和电导率均不高,土壤盐分不是植被碳储量的主控因子,植被碳储量主要受土壤的营养盐状况(全氮和全磷)和粒径结构(粉粒含量)的影响.土壤水文条件的改变造成了植被群落的演替,在由耐盐群落(盐地碱蓬群落)向轻耐盐群落(茵陈蒿群落、狗尾草群落等)的演替过程中,植被碳储量增大.     

A Method for Improved Resolution for Fluorescence Microscopy Using Plastic-Embedded Material Subjected to Resin Extraction

A protocol is given that uses NaOH, benzene, acetone and methanol to extract epoxy resins from semithin sections. Such sections appear superior to paraffin or unsectioned materials for fluorescence microscopic observations. Use of ultrarapid films (e.g., Kodak T-Max P3200) at ISO 3200 minimizes fading without use of antifading agents and without introducing unacceptable photographic grain size.     

Nickel Grids Cause Astigmatism in the Transmission Electron Microscope

Nickel grids are used in various methods (e.g., in immunocytochemistry) where chemically inert grids are required. Recently (Neiss 1983) we have used formvar-coated nickel grids when removing osmium from mounted ultrathin sections with 10% periodic acid (Lewis and Knight 1977). This is possible because nickel, unlike copper, adequately withstands the oxidation necessary for osmium removal. Handling sections on nickel grids avoids the disadvantages of free-floating sections, whose use for osmium removal has been recommended by Lewis and Knight (1977, chapter 2.2.3).     

Demonstration of Phenolic Compounds in Plant Tissues by An Osmium-Iodide Postfixation Procedure

A simple procedure to stain phenols in plant tissues is described, Postfixation with an aqueous solution prepared by mixing 2 cc of 2% osmium tetroxide and 8 cc of 3% potassium iodide yields brilliant visualization of phenol-containing vacuoles in different tissues of plants (e.g. coffee, oak, tobacco and spruce) bearing high concentration of phenolic compounds. Areas bearing phenols become dark gray to black. Chemical experiments demonstrate that osmium-potassium iodide (Os-KI) mixture reacts rapidly with several naturally occurring plant phenols, developing black solutions from which black solids precipitate. Phenols containing o-dihydroxy groups react with Os-KI solution more rapidly than other structurally different phenols. Therefore, o-dihydroxy units in an aromatic ring seem to function as primary sites of reactivity with the osmium-iodide complexes.     

Improved Methods for Permanent Chromosome Preparations: Cellophane Squashes and Citric Acid Dissociation

Normally, squash preparations from prestained acetic acid softened or HCl macerated tissues are made by pressing the tissue under a coverglass (e.g. Walker 1973). When permanent slides are wanted the coverglass has to be removed sooner or later, which is only possible by hardening the squashed tissue by freezing, for example in carbon dioxide snow, or by slow diffusion of fixatives into the space between the slide and the coverglass. These methods are either expensive or time-consuming, and upon removal of the coverglass, many cells and chromosomes either are lost or are poorly preserved.     

A Teflon Disk for Processing Loose Serial Celloidin Sections

Celloidin sections are routinely used for Nissl, Golgi, or Golgi-Cox staining (e.g., Glaser and Van der Loos 1981) when sections thicker than 30 μm are required. In spite of the advantages of the celloidin method (see Voogd and Feirabend 1981, Buschke 1979), processing free-floating serial sections of celloidin embedded material, which may often be preferred, is not very convenient.     

Membrane potential generation by two reconstituted mitochondrial systems: Liposomes inlayed with cytochrome oxidase or with ATPase

Formation of a membrane potential in two types of liposomes, one inlayed with cytochrome c + cytochrome oxidase, and another, with oligomycin-sensitive ATPase, has been demonstrated. To detect a membrane potential, phenyl dicarbaundecaborane (PCB⁻), a penetrating anion probe, was used.

The first type of liposome was reconstituted from a solution of purified cytochrome oxidase, mitochondrial phospholipids and cytochrome c, the latter being enclosed inside liposomes. Cytochrome c bound to the outer surface of the liposome membrane was removed by washing with NaCl. Such liposomes catalyzed oxidation of ascorbate by oxygen in the presence of phenazine methosulfate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The oxidation was found to support the PCB⁻ uptake by liposomes. The PCB⁻ response was prevented and reversed by cyanide, protonophorous uncouplers and external cytochrome c.

Liposomes of the second type were prepared from a solution of mitochondrial phospholipids, coupling factors F₁and F_c, and the hydrophobic proteins of the oligomycin-sensitive ATPase. These liposomes catalyzed ATP hydrolysis coupled with the PCB⁻ uptake. The latter effect was prevented and reversed by oligomycin and uncouplers.

The conclusion is made that membrane potential can be independently formed by enzymic reactions of two different kinds: (1) redox (e.g. cytochrome c oxidase) and (2) hydrolytic (ATPase).     

干扰对动物传播森林植物种子有效性影响的研究进展

干扰在森林生态系统中普遍存在,并影响森林的更新和演替.动物传播种子是种子更新的必经阶段,其对森林干扰的响应在一定程度上能够预测未来的森林群落组成和结构变化,对于明确森林演替方向具有重要意义.本文论述了森林干扰对动物传播种子有效性(包括动物传播种子的数量和质量)影响研究的生态学意义,全面揭示了自然干扰(火干扰、林窗干扰等)和人为干扰(生境破碎化、狩猎、采伐等)对动物传播种子数量、传播距离以及传播后幼苗更新影响的研究进展,指出干扰通过影响动物种群动态,进而造成动物传播种子数量发生了改变,动物传播种子的距离对干扰的响应基本表现出轻微负相关;干扰对传播后幼苗更新的影响结果因干扰类型的不同而复杂多变,干扰迹地环境因子的变化也影响着传播后的种子萌发和幼苗更新.干扰对动物传播种子有效性影响研究中存在的问题,主要表现为火干扰迹地恢复过程、增益性的干扰(如抚育、间伐、林窗)等对种子传播有效性影响研究的匮乏,以及忽略了温带森林内的干扰对动物传播种子的影响等.今后,应开展干扰对种子传播有效性的长期研究;对于干扰多发地带的森林,应高度重视增益性干扰影响动物传播植物种子的研究.