GABAergic interneuron to astrocyte signalling: a neglected form of cell communication in the brain

GABAergic interneurons represent a minority of all cortical neurons and yet they efficiently control neural network activities in all brain areas. In parallel, glial cell astrocytes exert a broad control of brain tissue homeostasis and metabolism, modulate synaptic transmission and contribute to brain information processing in a dynamic interaction with neurons that is finely regulated in time and space. As most studies have focused on glutamatergic neurons and excitatory transmission, our knowledge of functional interactions between GABAergic interneurons and astrocytes is largely defective. Here, we critically discuss the currently available literature that hints at a potential relevance of this specific signalling in brain function. Astrocytes can respond to GABA through different mechanisms that include GABA receptors and transporters. GABA-activated astrocytes can, in turn, modulate local neuronal activity by releasing gliotransmitters including glutamate and ATP. In addition, astrocyte activation by different signals can modulate GABAergic neurotransmission. Full clarification of the reciprocal signalling between different GABAergic interneurons and astrocytes will improve our understanding of brain network complexity and has the potential to unveil novel therapeutic strategies for brain disorders.     

Diversity and Activity of Cellulose-Decomposing Bacteria,Isolated from a Sandy and a Loamy Soil after Long-Term Manure Application

The community of culturable cellulolytic bacteria was analyzed in two long-term experimental field sites on Albic Luvisol (silty sand) and Haplic Phaeozem (loam), with and without farmyard manure treatment. Against the backdrop of significant differences in soil properties, the bacterial community structure differed clearly between sites and was affected by manure application as analyzed by T-RFLP of 16S rDNA. The population densities of cellulolytic bacteria were significantly increased by manure application in Phaeozem. Cellulose decomposing potentials of 537 isolates were tested on soluble, colloidal, and crystalline cellulose. The results showed some evidence of a greater proportion of isolates with high decomposition activity in Luvisol, but no impact from manure application could be observed in both soils. Restriction analysis and sequencing of 16S rDNA of isolates revealed a rather simple community composition that was dominated by Streptomyces (67%). The composition of the RFLP groups was affected by manure application, which was most evident in Luvisol, whereas an effect of the soil type could not be found. Although abundant RFLP groups were assigned to phylogenetically different bacterial classes (Actinobacteria, Betaproteobacteria, and Gammaproteobacteria), cellulolytic activity could not consistently be differentiated. All in all, cellulolytic capabilities of the isolates were highly variable and did not map to phylogenetic affiliation.     

Fluorescence immunolocalization of bound atrazine residues in plant tissue

Bound atrazine was detected inElodea canadensis by an improved immunohistochemical fluorescence procedure using anti-triazine antibodies from rabbits, biotin-labelled anti-rabbit immunoglobulin G and streptavidin-phycoerythrin conjugate. Whereas no labelling was found in control plants grown in charcoal-filtered, atrazine-free water, the labelling of plants obtained from their natural habitat and grown in tap water was sometimes nearly as high as in samples loaded with atrazine. The efficiency of the immunofluorescence procedure was compared using several antisera obtained by immunizing with different hapten conjugates and purified by various purification methods. The best results were observed with the atrazine analogue ametryn sulfoxide, which was coupled to bovine serum albumin for immunization and to Sepharose for immunoaffinity chromatography. The procedure described in this paper may serve as a general tool for detecting bound pesticide residues in plant material. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration

The model bryophyte Physcomitrella patens exhibits high frequencies of gene targeting when transformed with DNA constructs containing sequences homologous with genomic loci. ‘Targeted gene replacement’ (TGR) resulting from homologous recombination (HR) between each end of a targeting construct and the targeted locus occurs when either single or multiple targeting vectors are delivered. In the latter instance simultaneous, multiple, independent integration of different transgenes occurs at the targeted loci. In both single gene and ‘batch’ transformations, DNA can also be found to undergo ‘targeted insertion’ (TI), integrating at one end of the targeted locus by HR with one flanking sequence of the vector accompanied by an apparent non-homologous end-joining (NHEJ) event at the other. Untargeted integration at nonhomologous sites also occurs, but at a lower frequency. Molecular analysis of TI at a single locus shows that this occurs as a consequence of concatenation of the transforming DNA, in planta, prior to integration, followed by HR between a single site in the genomic target and two of its repeated homologues in the concatenated vector. This reinforces the view that HR is the major pathway by which transforming DNA is integrated in Physcomitrella.     

A promising 31P NMR-multivariate analysis approach for the identification of milk phosphorylated metabolites and for rapid authentication of milk samples

A fast and reliable method for the identification of milk from different mammalians was developed by using ³¹P NMR metabolite profile of milk serum coupled to multivariate analysis (PCA and classification models UNEQ, SIMCA and K-NN). Ten milk samples from six different mammalians, relevant to human nutrition (human, cow, donkey, mare, goat, sheep), were analyzed and eight monophosphorylated components were identified and quantified: phosphocreatine (PCr), glycerophosphorylcholine (GPC), glycerophosphorylethanolamine (GPE), N-acetylglucosamine-1-phosphate (NAcGlu-1P), lactose-1-phosphate (Lac-1P), galactose-1-phosphate (Gal-1P), phosphorylcholine (PC), glucose-6-phosphate (Glu-6P). PCA showed interesting clustering based on the animal genus. K-NN can be successfully used to discriminate between donkey and cow samples while UNEQ class-modeling resulted more suitable for compliance verification. Results confirm the natural variability of milk samples among different species. These data highlight the great potentials of NMR/multivariate analysis combined method in the rapid analysis of phosphorylated milk serum metabolites for milk origin assessment and milk adulteration detection.     

High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a K_i of approximately 4 μM. Uptake studies with [1,2-dimethyl-¹⁴C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a K_m value of 4 ± 1 μM and a maximum rate of transport (V_max) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance ¹³C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and γ-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83–90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.     

VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.