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.     

A family of Arabidopsis plasma membrane receptors presenting animal beta-integrin domains

A cDNA clone, AtELP1 (Arabidopsis thaliana EGF receptor-like protein) was isolated from an Arabidopsis cDNA library with an oligonucleotide probe corresponding to a highly conserved region of animal beta-integrins. The cloning of this cDNA was previously reported and it has been proposed that AtELP might be a receptor involved in intracellular trafficking. In the present work, using two specific independent sets of anti-peptide antibodies, we show that AtELP1 is mainly located in the plasma membrane, supporting another function for this protein. Structural studies, using methods for secondary structure prediction, indicated the presence of cysteine-rich domains specific to beta-integrins. Database searches revealed that AtELP1 is a member of a multigenic family composed of at least six members in A. thaliana. Northern blot analysis of AtELP1, 2b and 3 was performed on mRNA extracted from cells cultured in normal and stressed conditions, and from several organs and plants submitted to biotic or abiotic stresses. All the genes are expressed at different levels in the same conditions, but preferentially in roots, fruits and leaves in response to water deficit.     

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.     

A molecular phylogeography approach to biological invasions of the New World by parthenogenetic Thiarid snails

The parthenogenetic snail Melanoides tuberculata, present in tropical fresh waters of most of the Old World before 1950, has now invaded the Neotropical area. The phylogeography of this snail was studied to evaluate the pathways and number of such invasions. Because of parthenogenetic reproduction, individuals are structured into genetical clones. Within populations from both the original and invaded areas, several morphologically distinct clones (referred to as morphs) often coexist but the amount of genetic divergence among morphs is unknown. Individuals from 27 morphs and 40 populations world-wide were sequenced at two mitochondrial genes (12S and 16S). Our phylogenetic reconstruction suggests that (i) most of the morphological variation observed in the New World predates invasion, (ii) at least six independent introductions have occurred, and (iii) invasive clones are found throughout most of the phylogenetic tree and do not come from a particular region of the area of origin. Two ideas are discussed in the light of these results. The first lies with the specificities of parthenogenesis in an invasion context. While in sexual species, independently introduced populations eventually merge into a single invasive population, in a parthenogenetic species independently introduced clones have distinct invasion dynamics and possibly exclude each other. Second, although repeated invasions in Melanoides may have an impact on indigenous molluscan faunas, their most likely effect is the world-wide homogenization of the invasive taxon itself.     

Xyloglucan endotransglucosylase action is high in the root elongation zone and in the trichoblasts of all vascular plants from Selaginella to Zea mays

The endotransglucosylase action of the enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was localized in the roots of diverse vascular plants: club-mosses (lycopodiophytes), ferns, gymnosperms, monocots, and dicots. High action was always found in the epidermis cell wall of the elongation zone and in trichoblasts in the differentiation zone. Clearly XTH and its action in root development evolved before the evolutionary divergence of ferns and seed plants and also of the lycopodiophytes and euphyllophytes.     

Evolutionary history and phylogeography of the schistosome-vector freshwater snail Biomphalaria glabrata based on nuclear and mitochondrial DNA sequences

The phylogeography of the freshwater snail Biomphalaria glabrata remains poorly known, although this species is the major vector of schistosomiasis in the New World. It was here investigated in South America and the Lesser Antilles, based on partial mitochondrial large ribosomal subunit (16S rDNA) and nuclear internal transcribed spacer-2 (ITS-2) gene sequences. Sampling included 17 populations from a large part of the current geographic range of the species (Brazil, Venezuela and Lesser Antilles). Substantial variability was detected, as well as a high amount of phylogenetically informative signal. The molecular phylogeny inferred splits B. glabrata into Northern and Southern clades separated by the Amazon river, and may even suggest a supra-specific status for B. glabrata. Brazilian populations were the most diverse and appeared basal to the other populations. Venezuelan haplotypes formed a single clade, albeit not strongly supported. Two Venezuelan haplotypes appear rather similar to Brazilian haplotypes. Similarly, Lesser Antilles haplotypes clustered in the same monophyletic clade, which suggests that the recent colonisation of the Antilles has a northern South American origin. However, the estimated divergence time between Antilles and Venezuelan sequences is extremely large (conservatively higher than 10(5) years). These results are discussed in the light of (i) phylogeographic patterns at South American scale, and (ii) recurrent introduction of molluscs, especially in the Antilles, as a consequence of human activities.