Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.     

Binding properties of NADPH-protochlorophyllide oxidoreductase as revealed by detergent and ion treatments of isolated and immobilized prolamellar bodies

Prolamellar bodies were isolated from dark-grown leaves of 6.5-day-old wheat ( Triticum aestivum L. cv. Walde). The prolamellar bodies were immobilized in agarose beads to get a material suitable for studies on pigment and protein release, and to protect the membranes from mechanical breakage. The beads were treated with detergents and salt solutions of different ionic strengths and the eluates collected. Protochlorophyllide in the eluate was determined by fluorescence spectroscopy. Dot-blot tests were used to estimate the amount of released NADPH-protochlorophyllide oxidoreductase (E.C. 1.6.99.1.). Changes in ultrastructure of the treated prolamellar bodies were analysed. Release of both membrane constituents increased by treatment with detergents. With 0.2% (w/v) Triton X-100, 60% of the fluorescence from the immobilized prolamellar bodies was eluted within 30 min. Salt solutions with increasing ionic strength increased the release from 3 to 7%. The detergent treatment resulted in a complete (Triton X-100) or partial ([3-(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, CHAPS; 1-octyl β- d -glucopyranoside, octylglucoside) loss of the highly regular structure of the prolamellar bodies. Immunogold labelling of ultrathin sections revealed the absence of NADPH-protochlorophyllide oxidoreductase when the regular structure was dissolved into single membranes. The regular appearance of the prolamellar bodies was altered by treatment with 0.1 M CaCl₃ and 0.1 M KSCN, respectively, but not with 0.1 M KCl. Immunogold labelling showed that that enzyme was still present in the prolamellar bodies after these treatments. Despite the ultrastructural changes, the spectral properties were unchanged. Thus we conclude that NADPH-protochlorophyllide oxidoreductase is firmly attached to the prolamellar body membranes and that the regular ultrastructure of the prolamellar body is partly controlled by the ionic environment.     

Localization of aggregation substances of Enterococcus faecalis after induction by sex pheromones

The distribution of sex pheromone induced aggregation substance was studied on the cell surface of various Enterococcus faecalis strains. In the accompanying paper we have shown that the aggregation substance appears as a layer of hairlike structures. Using direct and indirect immunogold technique, transmission electron microscopy and high resolution scanning electron microscopy we investigated the appearance and distribution of the aggregation substance. The hairs increase in number with increasing exposure to sex pheromones (maximum density: 1300/m²). We show that these structures are unequally distributed over the cell surface, even if the cells were induced by sex pheromones for a long period of time. Statistical analysis of the unequal distribution indicates that aggregation substance is incorporated into pre-existing old cell-walls and that this incorporation shows a saturation ca. 40 min after addition of sex pheromones.Abbreviations cAD1 sex pheromone specific for plasmid pAD1 - cPD1 sex pheromone specific for plasmid pPD1 - FESEM field emission scanning electron microscope - pAD1 conjugative plasmid specifically transferred in the presence of cAD1 - pPD1 conjugative plasmid specifically transferred in the presence of cPD1 - TEM transmission electron microscope     

Identification and quantification of proteins differentially secreted by a pair of normal and malignant breast‐cancer cell lines

Secreted proteins play a pivotal role in cellular functions. To better understand malignant behavior, we adapted stable isotopic labeling with amino acids in cell culture technology to identify and quantify proteins differentially released into the extracellular media by a pair of normal and malignant breast‐cancer cell lines. Approximately 380 non‐redundant proteins were quantified in serum‐free media. Of the assigned proteins, 62% are classified secreted in protein databases and an additional 25% are designated secreted in the literature. A number of growth factors were found differentially regulated. Tumor necrosis factor, pigment epithelial‐differentiating factor and stem‐cell growth factor precursor showed decreased expression in breast‐cancer cell line, whereas Inhibin beta and macrophage migration inhibitory factor show increased expression. Interestingly, protease inhibitors, including plasma protease (C1) inhibitor, PZP precursor, and SerpinE2 were significantly down‐regulated in cancer cell line as were angiostatic factors from extracellular matrix (ECM) such as endorepillin. Further, the C‐terminal fragment of type XVIII collagen, endostatin, a potent angiostatic factor, was down‐regulated as well whereas extracellular collagens and osteoblast‐specific factor 2 (OSF‐2), were up‐regulated. Differential expression and secretion of SerpinE2 and OSF‐2 were confirmed using Western blotting. These results corroborate models of invasive tumors sustained by elaborate coordination of stromal cells via chemokines and growth factors, while protease inhibitors remodel the ECM to stimulate angiogenesis.     

pH敏感型荧光蛋白及其在植物细胞生物学中的应用

pH敏感型荧光蛋白, 即pHluorin, 是荧光强度及光谱特征随环境pH值的变化而改变的一类荧光蛋白。人们通过对密码子使用偏好和特定剪切位点的修饰, 已使pHluorin及其衍生物成功地在动物、植物和真菌细胞中正常表达, 为测量细胞内微环境pH值的变化, 并研究活细胞内依赖或导致pH变化的生理过程提供了有力工具。该文总结了目前已报道的pH敏感型荧光蛋白的种类及特性, 并对其在细胞生物学, 特别是植物细胞生物学中的应用进行了详细介绍。随着报告基因技术及检测方法的不断改进, pHluorin将在植物科学领域发挥更大的作用。     

Biotic controls on CO2 and CH4 exchange in wetlands – a closed environment study

Wetlands are significant sources of the important greenhouse gas CH₄. Here we explore the use of an experimental system developed for the determination of continuous fluxes of CO₂ and CH₄ in closed ecosystem monoliths including the capture of ¹⁴CO₂ and ¹⁴CH₄ following pulse labelling with ¹⁴CO₂. We show that, in the ecosystem studied, ebullition (bubble emission) may account for 18 to 50% of the total CH₄ emission, representing fluxes that have been difficult to estimate accurately in the past. Furthermore, using plant removal and ¹⁴C labelling techniques, we use the system to detail the direct influence of vascular plants on CH₄ emission. This influence is observed to be dependent on the amount of vascular plants present. The results that may be produced using the presented experimental set-up have implications for an improved understanding of wetland ecosystem/atmosphere interactions, including possible feedback effects on climate change. In recent years much attention has been devoted to ascertaining and subsequently using the relationship between net ecosystem productivity and CH₄ emission as a basis for extrapolation of fluxes across large areas. The experimental system presented may be used to study the complex relationship between vascular plants and CH₄ emission and here we show examples of how this may vary considerably in nature between and even within ecosystems.     

Quantifying immunogold labelling patterns of cellular compartments when they comprise mixtures of membranes (surface-occupying) and organelles (volume-occupying)

In quantitative immunoelectron microscopy, subcellular compartments that are preferentially labelled with colloidal gold particles can be identified by estimating labelling densities (LDs) and relative labelling indices (RLIs). Hitherto, this approach has been limited to compartments which are either surface occupying (membranes) or volume occupying (organelles) but not a mixture of both (membranes and organelles). However, some antigens are known to translocate between membrane and organelle compartments and the problem then arises of expressing gold particle LDs in a consistent manner (e.g., as number per compartment profile area). Here, we present one possible solution to tackle this problem. With this method, each membrane is treated as a volume-occupying compartment and this is achieved by creating an acceptance zone at a fixed distance on each side of membrane images. Gold signal intensity is then expressed as an LD within the membrane profile area so created and this LD can be compared to LDs found in volume-occupying compartments. Acceptance zone width is determined largely by the expected dispersion of gold labelling. In some cases, the zone can be applied to all visible membrane images but there is a potential problem when image loss occurs due to the fact that membranes are not cut orthogonal to their surface but are tilted within the section. The solution presented here is to select a subset of clear images representing orthogonally sectioned membranes (so-called local vertical windows, LVWs). The fraction of membrane images forming LVWs can be estimated in two ways: goniometrically (by determining the angle at which images become unclear) or stereologically (by counting intersections with test lines). The fraction obtained by either method can then be used to calculate a factor correcting for membrane image loss. In turn, this factor is used to estimate the total gold labelling associated with the acceptance zone of the entire (volume-occupying) membrane. However calculated, the LDs over the chosen (membrane and organelle) compartments are used to obtain observed and expected gold particle counts. The observed distribution is determined simply by counting gold particles associated with each compartment. Next, an expected distribution is created by randomly superimposing test points and counting those hitting each compartment. LDs of the chosen compartments are used to calculate RLI and chi-squared values and these serve to identify those compartments in which there is preferential labelling. The methods are illustrated by synthetic and real data.     

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves

This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-¹⁴C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-¹⁴C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-¹⁴C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process.     

Differences in rhizosphere carbon-partitioning among plant species of different families

Interspecific variations in carbon (C) allocation and partitioning in the rhizosphere were investigated on 12 Mediterranean species belonging to different family groups (grasses, legumes, non-legume forbs) and having different life cycles. Plants grown individually in artificial soil, in a greenhouse and inoculated with rhizosphere microflora were labelled with ¹⁴CO₂ for 3 h at the vegetative stage. Rhizosphere respiration was measured during 6 days after which labelled C partitioning between shoots, roots, soil, root washing solution and respiration was estimated. The percentage of assimilated ¹⁴C allocated below ground differed significantly between species (41 – 76%) but no significant difference was found between grasses, legumes and non-legume forbs. When expressed as percentage of below-ground ¹⁴C, rhizosphere respiration was significantly smaller for non-legume forbs (42%) than for grasses (46%) and legumes (51%). Consequently more ¹⁴C was incorporated into root biomass in the former. Half-life of ¹⁴CO₂ evolution through respiration ranged from 23 h in legumes to 27 h for non-legume forbs and 37 h for grasses. This suggested differences in microbial activities due to quantities and quality of root exuded C. Rhizosphere respiration was positively correlated with the amount of ¹⁴C in the solution used to wash the roots on one hand, and root N concentration on the other hand. This led to a functional hierarchy between plant family groups of the overall rhizosphere activity. It went from non-legume forbs being the less active (except Crepis sancta)in terms of respiration and exudation, to grasses and then legumes, the most active but also the richest in nitrogen.