Spatial structure of mitochondria and ER denotes changes in cell physiology of cultured tobacco protoplasts

The structure of mitochondria and of the endoplasmic reticulum (ER) in mesophyll protoplasts and regenerated cells was studied in vivo using the dye DiOC₆(3) (3,3'-dihexyloxacarbocyanine iodide) and confocal laser scanning microscopy (CLSM). The relation to the cell's physiology was investigated using a hormone-based model system for elongation and division. The structure of the mitochondria and of their population depends on the status of the cell. In freshly isolated protoplasts small spherical mitochondria are clustered around the nucleus and the chloroplasts. During the first 4 days of culture they are transformed into long vermiform organelles which distribute evenly throughout the cytoplasm. In a medium containing only auxins, cells then enter a period of expansion. Their mitochondria retain the same structure but increase in quantity. In a medium with auxins and cytokinins cells start dividing. Their mitochondria typically become numerous and very small, and are distributed throughout the cytoplasm. Both types of organization were observed during weeks of ongoing expansion or division. The ER is always present as a network close to the cell membrane. In freshly isolated protoplasts a considerable part of the ER is clustered around the chloroplasts, the remaining part of the network has a reduced complexity and is partly broken. During subsequent protoplast culture the network grows into a complex web with fine meshes incorporating lots of plate-like structures. This is the case in elongating cells as well as in dividing cells. Finally, the ER looks similar to the structure found in epidermal cells of the intact plant.     

Localization of phytochrome in oats by electron microscopy

Phytochrome was localized by immunoelectron microscopy in cells of the coleoptile tip of etiolated and irradiated oat (Avena sativa L., cv. Konata) seedlings. By using ultrathin frozen sections and immunopurified, monospecific antibodies, both the sensitivity and resolution of the immunocytochemical assay were increased. The results with etiolated plants agree with and extend previously published data. A brief red light illumination caused the redistribution of phytochrome from a diffuse to a more particulate appearance. Areas that accumulated phytochrome were identified as small vacuoles into which phytochrome was sequestered following illumination. In seedlings illuminated for several hours and in normal light-grown plants, the cellular distribution of phytochrome is qualitatively similar to that of nonirradiated, dark-grown material, except that in green plants the nucleus shows a positive immunocytochemical reaction.     

Heterogeneity of Rat Brain Acetylcholinesterase: A Study by Gel Filtration and Gradient Centrifugation

Abstract: According to their solubilization properties, two classes of acetyl-cholinesterases (AChE) can be detected in the adult rat brain: a "soluble" species (easily solubilized without detergent), and a membrane-bound species (solubilized only in the presence of detergent). The latter was found to be homogeneous by gel filtration (Stokes radius 8.05 ± 0.35 nm) and sucrose gradient centrifugation (9.75 ± 0.2 S) in the presence of Triton X-100. The "soluble" AChE gives three stable species in the presence of the same detergent with Stokes radii and sedimentation constants of 10.9 ± 0.5 nm and 16 ± 2 S; 6.75 ± 0.30 nm and 10.7 ± 0.4 S; 5.37 ± 0.35 nm and 4.37 ± 0.1 S. Co-chromatography and co-sedimentation or the reduction and alkylation of disulfide bridges show that all the soluble species are different from the membrane-bound AChE. The possibility that soluble and membrane-bound AChE are completely different molecules is discussed.     

Characterization of a Small Auxin-Up RNA (SAUR)-Like Gene Involved in Arabidopsis thaliana Development

The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.     

Biological detection of low radiation doses by combining results of two microarray analysis methods

The accurate determination of the biological effects of low doses of pollutants is a major public health challenge. DNA microarrays are a powerful tool for investigating small intracellular changes. However, the inherent low reliability of this technique, the small number of replicates and the lack of suitable statistical methods for the analysis of such a large number of attributes (genes) impair accurate data interpretation. To overcome this problem, we combined results of two independent analysis methods (ANOVA and RELIEF). We applied this analysis protocol to compare gene expression patterns in Saccharomyces cerevisiae growing in the absence and continuous presence of varying low doses of radiation. Global distribution analysis highlights the importance of mitochondrial membrane functions in the response. We demonstrate that microarrays detect cellular changes induced by irradiation at doses that are 1000-fold lower than the minimal dose associated with mutagenic effects.     

PCC-FISH in skin fibroblasts for local dose assessment: biodosimetric analysis of a victim of the Georgian radiological accident

We propose a new method of biodosimetry that could be applied in cases of localized irradiation. The approach is based on excess chromosome segments determination by the PCC-FISH technique in fibroblasts isolated from skin biopsy. Typically, 0 to 10 Gy ex vivo gamma-irradiated human skin biopsies were dissociated and fibroblasts were isolated and grown for several days. Cells next underwent PCC-FISH painting of whole chromosome 4, and the number of excess chromosome segments per metaphase was determined. An ex vivo reference curve correlating the number of excess chromosome segments per metaphase to the radiation dose was established and used to assess the dose delivered to the skin of one of the victims of the radiological accident that occurred at Lia in Georgia in December 2001. Specifically, the victim suffering from moist desquamation underwent skin excision in Hospital Percy (France). Measurement of excess chromosome segments per metaphase was done in fibroblasts isolated and grown from removed wounded skin and subsequent conversion to radiation doses was performed. The radiation dose map obtained was shown to be in accordance with clinical data and physical dosimetry as well as with conventional biodosimetry. These results demonstrated that PCC-FISH painting applied to skin fibroblasts may be a suitable technique for dose estimation. To assess its worth, this approach needs to be extended to future accidents involving localized radiation exposure.     

Annotated expressed sequence tags for studies of the regulation of reproductive modes in aphids

The damaging effect of aphids to crops is largely determined by the spectacular rate of increase of populational expansion due to their parthenogenetic generations. Despite this, the molecular processes triggering the transition between the parthenogenetic and sexual phases between their annual life cycle have received little attention. Here, we describe a collection of genes from the cereal aphid Rhopalosiphum padi expressed during the switch from parthenogenetic to sexual reproduction. After cDNA cloning and sequencing, 726 expressed sequence tags (EST) were annotated. The R. padi EST collection contained a substantial number (139) of bacterial endosymbiont sequences. The majority of R. padi cDNAs encoded either unknown proteins (56%) or housekeeping polypeptides (38%). The large proportion of sequences without similarities in the databases is related to both their small size and their high GC content, corresponding probably to the presence of 5'-unstranslated regions. Fifteen genes involved in developmental and differentiation events were identified by similarity to known genes. Some of these may be useful candidates for markers of the early steps of sexual differentiation.