鼎湖山南亚热带森林细根分解干物质损失和元素动态

鼎湖山南亚热带森林细根分解干物质损失和元素动态温达志（中国科学院华南植物研究所,广州５１０６５０）魏平张佑昌（中国科学院华南植物研究所鼎湖山树木园,肇庆５２６０７０）孔国辉（中国科学院华南植物研究所,广州５１０６５０）ＤｒｙＭａｓＬｏｓａｎｄＣｈｅｍ...     

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases ¹. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression ¹. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer''s disease and multiple sclerosis ¹. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. ² In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.Download video file.^{(134M, mp4)}     

Nuclear Dry Mass and Area in Epidermal Cells of Senescing Soybean Cotyledons

The nuclear dry mass and area of epidermal cells of senescingsoybean cotyledons were measured with an interference microscopeand found to decrease until cotyledon abscission. Decapitationafter germination prevented yellowing and abscission of cotyledonsand the decrease of both nuclear dry mass and area. The meansof nuclear dry mass and area for normal and decapitated cotyledonswere highly correlated in two experiments, r=0.0.76 and 0.90respectively.     

The Nature and Origin of Chemical Shift for Intracellular Water Nuclei in Artemia Cysts

We investigated the possible existence of chemical shift of water nuclei in Artemia cysts using high resolution nuclear magnetic resonance (NMR) methods. The results conducted at 60, 200, and 500 MHz revealed an unusually large chemical shift for intracellular water protons. After correcting for bulk susceptibility effects, a residual downfield chemical shift of 0.11 ppm was observed in fully hydrated cysts. Similar results have been observed for the deuterium and ¹⁷O nuclei.

We have ruled out unusual intracellular pH, diamagnetic susceptibility of intracellular water, or interaction of water molecules with lipids, glycerol, and/or trehalose as possible origins of the residual chemical shift. We conclude that the residual chemical shift observed for water nuclei (¹H, ²H, and ¹⁷O) is due to significant water-macromolecular interactions.

     

Radioluminescence Microscopy: Measuring the Heterogeneous Uptake of Radiotracers in Single Living Cells

Radiotracers play an important role in interrogating molecular processes both in vitro and in vivo. However, current methods are limited to measuring average radiotracer uptake in large cell populations and, as a result, lack the ability to quantify cell-to-cell variations. Here we apply a new technique, termed radioluminescence microscopy, to visualize radiotracer uptake in single living cells, in a standard fluorescence microscopy environment. In this technique, live cells are cultured sparsely on a thin scintillator plate and incubated with a radiotracer. Light produced following beta decay is measured using a highly sensitive microscope. Radioluminescence microscopy revealed strong heterogeneity in the uptake of [¹⁸F]fluoro-deoxyglucose (FDG) in single cells, which was found consistent with fluorescence imaging of a glucose analog. We also verified that dynamic uptake of FDG in single cells followed the standard two-tissue compartmental model. Last, we transfected cells with a fusion PET/fluorescence reporter gene and found that uptake of FHBG (a PET radiotracer for transgene expression) coincided with expression of the fluorescent protein. Together, these results indicate that radioluminescence microscopy can visualize radiotracer uptake with single-cell resolution, which may find a use in the precise characterization of radiotracers.     

Microbial Water Relations: Features of the Intracellular Composition of Sugar-Tolerant Yeasts

Several factors contributed to differences in intracellular composition between sugar-tolerant (osmophilic) and nontolerant species of yeast. One such factor was the difference in accumulation of those nonelectrolytes whose uptake was not dominated by vigorous metabolism. In such cases (lactose and glycerol), the sugar-tolerant species had a much lower capacity for the solute than did the nontolerant species. Sucrose uptake was consistently different between all sugar-tolerant strains on the one hand and all nontolerant strains on the other. The difference was attributable in part to metabolism of sucrose by the nontolerant yeasts. The major difference between the two types of yeast, however, was the presence of one or more polyhydric alcohols at high concentrations within each of the sugar-tolerant strains but none of the nontolerant strains. In most cases the major polyol was arabitol. The solute concentration (and, hence, water availability) of the growth medium affected both the amount of arabitol produced by Saccharomyces rouxii and the proportion retained by the yeast after brief washing with water at 0 C. When the yeast was suspended in a buffer at 30 C, the polyol leaked out at a slow, constant, reproducible rate. The polyene antibiotic amphotericin B caused rapid release of polyol by the yeast, the rate being proportional to amphotericin concentration. Contact of the yeast with glucose (1 mM) caused an extremely rapid ejection of polyol which lasted less than 40 s. Some implications of these results are discussed, as is the role of the polyol as a compatible solute in determining the water relations of the yeast.     

Enzymatic Lysis of Living Candida Cells in the Presence of Chemical Agents and Antibiotics

The enzyme preparation from Streptomyces No. 8 displayed β-1-3-glucanase, chitinase and protease activities, and attacked heat-treated cells of Candida albicans. The preparation, however, attacked native cells of the same strain and other Candida strains when supplemented with appropriate concentration of sodium dodecyl sulfate or variotin. Neither of the agents attacked native cells when used alone. Usefulness of the combinational use of cell wall lytic enzymes and antibiotics for therapeutic or preventative purpose is discussed.     

单细胞内Ca2+时空变化的激光共聚焦显微测定

应用激光扫描共聚焦显微系统(LSCM)和Fluo-3/AM荧光探剂标记技术, 测定了单个活细胞胞内游离Ca²⁺的动态变化与立体分布影像. 结果显示, 在37℃, Fluo-3/AM终浓度为6μmol/L的条件下, C57BL/6J小鼠巨噬细胞负载1h左右即可获得良好的标记效果. 相反, 若探剂浓度太高或负载时间太长, 胞内荧光强度太强, 影响在共聚焦显微镜镜下分辨细胞内结构. 因此用LSCM研究细胞内游离Ca²⁺变化时, 荧光探剂的负载应以获得最适荧光信号而不是以最大荧光强度为标准. 上述方法在其他如平滑肌细胞、卵母细胞中的测定亦获得满意的结果, 这对进一步研究各种生理和病理条件下细胞内Ca²⁺信号的动态变化、与跨膜Ca²⁺梯差的关系及对活细胞功能活动的调节提供了一种可行的、直观的研究手段.     

Cation Exchange Capacity and Chemical Composition of the Floral Parts of Antirrhinum and Lilium

Flowers of Antirrhinum majus L. were separated and the componentparts analysed for cation exchange capacity (CEC), uronic acid(UA), and mineral composition. The potassium content fell, andboth the calcium content and Ca/K ratio increased in the orderstigma style ovary. These changes in composition in the differentparts of the flower are in keeping with the variation expectedfrom, and found for, their CEC values. Lilium tigrinum Ker-Gawl.did not show the above effects.     

Cation Exchange Capacity, Chemical Composition and the Balance of Carboxylic Acids in the Floral Parts of Various Plant Species

Floral and other parts of antirrhinum, fuchsia, daffodil, andmaize have been analysed for cation-exchange capacity (CEC),major and trace-element constituents, and carboxylic acids.The two dicot species, but not the monocot species, have a gradientof CEC, Ca, and Ca/K increasing along the style and into theovary. Trace-element levels in anthers and ovaries are oftenhigher than in the adjacent filaments and styles. The floralparts of daffodil, with the exception of ovary, ovary wall,and anthers, have closely related K/Ca and citric/malic acidratios which are comparable with those for the rest of the plant.     

Changes in the Chemical Composition and Decay Resistance of Thermally-Modified Hevea brasiliensis Wood

In this study the effect of thermal treatment on the equilibrium moisture content, chemical composition and biological resistance to decay fungi of juvenile and mature Hevea brasiliensis wood (rubber wood) was evaluated. Samples were taken from a 53-year-old rubber wood plantation located in Tabapuã, Sao Paulo, Brazil. The samples were thermally-modified at 180°C, 200°C and 220°C. Results indicate that the thermal modification caused: (1) a significant increase in the extractive content and proportional increase in the lignin content at 220°C; (2) a significant decrease in the equilibrium moisture content, holocelluloses, arabinose, galactose and xylose content, but no change in glucose content; and (3) a significant increase in wood decay resistance against both Pycnoporus sanguineus (L.) Murrill and Gloeophyllum trabeum (Pers.) Murrill decay fungi. The greatest decay resistance was achieved from treatment at 220°C which resulted in a change in wood decay resistance class from moderately resistant to resistant. Finally, this study also demonstrated that the influence of thermal treatment in mature wood was lower than in juvenile wood.     

Chromobodies to Quantify Changes of Endogenous Protein Concentration in Living Cells

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Highlights

• •Chromobodies are stabilized by antigen binding in live cells.
• •Monitoring changes of endogenous protein levels in living cells with chromobodies.
• •Broadly applicable system to generate turnover-accelerated chromobodies.
• •Quantification of time- and dose-dependent compound effects.

     

Mag-indo1 Affinity for Ca, Compartmentalization and Binding to Proteins: The Challenge of Measuring Mg Concentrations in Living Cells

A physicochemical study of the Mag-indo1 binding to Ca²⁺ in solution showed that: (i) the characteristic fluorescence spectra of Ca²⁺-bound and Mg²⁺-bound Mag-indo1 are identical; (ii) two successive equilibria occur for increasing Ca²⁺ concentrations; and (iii) the value of the dissociation constant of the first one, as determined by using a probe dilution protocol, amounts to 780 nM. In order to investigate the fluorescence level of Mag-indo1 trapped in cell organelles, fluorescence spectra of Mag-indo1-loaded fibroblasts were recorded before and after a digitonin permeabilization. Their resolution into cation-bound, protein-bound, and free Mag-indo1 characteristic spectra allowed measurement of the fluorescence intensities of these species. The intensities emitted from whole cells were compared to those emitted from organelles (assumed to be endoplasmic reticulum according to a DiOC₆ loading). The cation-bound Mag-indo1 fluorescence resulted partially (20 to 50%) from the cytosol for 30% of the cells, and totally from compartments for 70% of the cells. We found a concentration value of 500 nM for compartmentalized Ca²⁺ and concluded that the Mag-indo1 binding to Ca²⁺ is likely to affect drastically the Mg²⁺ concentration measurements in cells. Moreover, we showed that the amount variation of protein-bound Mag-indo1 also affects Mg²⁺ measurements when using the two-wavelength ratio method.     

Cell Walls of Tobacco Cells and Changes in Composition Associated with Reduced Growth upon Adaptation to Water and Saline Stress

The relative mass of the cell walls of tobacco (Nicotiana tabacum L.) cells adapted to grow in medium containing 30% polyethylene glycol 8000 or 428 millimolar NaCl was reduced to about 50% of that of the walls of unadapted cells. Cellulose synthesis was inhibited substantially in adapted cells. The proportions of total pectin in walls of unadapted and adapted cells were about the same, but substantial amount of uronic acid-rich material from walls of cells adapted to either NaCl or polyethylene glycol was more easily extracted with cold sodium ethylenediamine tetraacetic acid solutions (NM Iraki et al. [1989] Plant Physiol. 91: 39-47). We examined the linkage composition of the pectic and hemicellulosic polysaccharides to ascertain chemical factors that may explain this difference in physical behavior. Adaptation to stress resulted in the formation of a loosely bound shell of polygalacturonic acid and rhamnogalacturonan. Pectins extracted from walls of adapted cells by either cold sodium ethylenediamine tetraacetic acid or hot ammonium oxalate were particularly enriched in rhamnose. Compared to pectins of unadapted cells, rhamnosyl units of the rhamnogalacturonans of adapted cells were more highly substituted with polymers containing arabinose and galactose, but the side groups were of greatly reduced molecular size. Possible functional roles of these modifications in cell wall metabolism related to adaptation to osmotic stress are discussed.     

The Utility of Spectral Indices from Landsat ETM+ for Measuring the Structure and Composition of Tropical Dry Forests1

There is a growing emphasis on developing methods for quantifying the structure and composition of tropical forests that can be applied over large landscapes, especially for tropical dry forests that are severely fragmented and have a high conservation priority. This study investigates the relationships between various measures of forest structure (annual woody increment, canopy closure, stand density, stand basal area) and composition (tree species diversity, tree community composition) measured in semi‐deciduous tropical dry forests on islands in Lago Guri, Venezuela and three spectral indices derived from Landsat ETM+: Normalized Difference Vegetation Index (NDVI), Infrared Index (IRI), and Mid‐Infrared Index (MIRI). Even though there were significant autocorrelations among spectral indices, there were significant differences in the relationships between spectral indices and forest attributes. IRI was not significantly correlated with any of the structural variables while MIRI was correlated with canopy closure and NDVI values were correlated with canopy closure as well as annual woody increment. NDVI and MIRI were both related to relative tree diversity and all three indices were associated with aspects of tree species composition. Based on the results of this study, it appears that spectral indices, and in particular NDVI, may be useful indicators of forest attributes in tropical dry forest habitats. Further research needs to be undertaken to identify if the results of this study can be applied to other tropical dry forests at a global spatial scale.