Localization and functional relevance of system a neutral amino acid transporters in cultured hippocampal neurons.

Glutamine and alanine are important precursors for the synthesis of glutamate. Provided to neurons by neighboring astrocytes, these amino acids are internalized by classical system A amino acid carriers. In particular, System A transporter (SAT1) is a highly efficient glutamine transporter, whereas SAT2 exhibits broad specificity for neutral amino acids with a preference for alanine. We investigated the localization and the functional relevance of SAT1 and SAT2 in primary cultures of hippocampal neurons. Both carriers have been expressed since early developmental stages and are uniformly distributed throughout all neuronal processes. However, whereas SAT1 is present in axonal growth cones and can be detected at later developmental stages at the sites of synaptic contacts, SAT2 does not appear to be significantly expressed in these compartments. The non-metabolizable amino acid analogue alpha-(methylamino)-isobutyric acid, a competitive inhibitor of system A carriers, significantly reduced miniature excitatory postsynaptic current amplitude in neurons growing on top of astrocytes, being ineffective in pure neuronal cultures. alpha-(Methylamino)-isobutyric acid did not alter neuronal responsitivity to glutamate, thus excluding a postsynaptic effect. These data indicate that system A carriers are expressed with a different subcellular distribution in hippocampal neurons and play a crucial role in controlling the astrocyte-mediated supply of glutamatergic neurons with neurotransmitter precursors.     

Functional Gene Hybridization Patterns of Terrestrial Ammonia-Oxidizing Bacteria

Abstract The biochemical pathway and genetics of autotrophic ammonia oxidation have been studied almost exclusively in Nitrosomonas europaea. Terrestrial autotrophic ammonia-oxidizing bacteria (AAOs), however, comprise two distinct phylogenetic groups in the beta-Proteobacteria, the Nitrosomonas and Nitrosospira groups. Hybridization patterns were used to assess the potential of functional probes in non-PCR-based molecular analysis of natural AAO populations and their activity. The objective of this study was to obtain an overview of functional gene homologies by hybridizing probes derived from N. europaea gene sequences ranging in size from 0.45 to 4.5 kb, and labeled with 32P to Southern blots containing genomic DNA from four Nitrosospira representatives. Probes were specific for genes encoding ammonia monooxygenase (amoA and amoB), hydroxylamine oxidoreductase (hao), and cytochrome c-554 (hcy). These probes produced hybridization signals, at low stringency (30 degreesC), with DNA from each of the four representatives; signals at higher stringency (42 degreesC) were greatly reduced or absent. The hybridization signals at low stringency ranged from 20 to 76% of the total signal obtained with N. europaea DNA. These results indicate that all four functional genes in the ammonia oxidation pathway have diverged between the Nitrosomonas and Nitrosospira groups. The hao probe produced the most consistent hybridization intensities among the Nitrosospira representatives, suggesting that hao sequences would provide the best probes for non-PCR-based molecular analysis of terrestrial AAOs. Since N. europaea can also denitrify, an additional objective was to hybridize genomic DNA from AAOs with probes for Pseudomonas genes involved in denitrification. These probes were specific for genes encoding heme-type dissimilatory nitrite reductase (dNir), Cu-type dNir, and nitrous oxide reductase (nosz). No hybridization signals were observed from probes for the heme-type dNir or nosz, but Nitrosospira sp. NpAV and Nitrosolobus sp. 24-C hybridized, under low-stringency conditions, with the Cu-type dNir probe. These results indicate that AAOs may also differ in their mechanisms and capacities for denitrification.     

Acid sphingomyelinase activity triggers microparticle release from glial cells

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1β, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X₇, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1β release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1β release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1β release, thus, opening new strategies for the treatment of neuroinflammatory diseases.     

Centrosomal localization of cyclins E and A: Structural similarities and functional differences

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G₁/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.Key words: cyclin A, cyclin E, Cdk2, centrosome, CLS, PSTAIRE, DNA synthesis     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

FCI: an R-based algorithm for evaluating uncertainty of absolute real-time PCR quantification

Background  

FCI is an R code for analyzing data from real-time PCR experiments. This algorithm estimates standard curve features as well as nucleic acid concentrations and confidence intervals according to Fieller's theorem.     

Calcium-dependent cleavage of endogenous wild-type huntingtin in primary cortical neurons

Huntington's disease (HD) is caused by a polyglutamine expansion in the amino-terminal region of huntingtin. Mutant huntingtin is proteolytically cleaved by caspases, generating amino-terminal aggregates that are toxic for cells. The addition of calpains to total brain homogenates also leads to cleavage of wild-type huntingtin, indicating that proteolysis of mutant and wild-type huntingtin may play a role in HD. Here we report that endogenous wild-type huntingtin is promptly cleaved by calpains in primary neurons. Exposure of primary neurons to glutamate or 3-nitropropionic acid increases intracellular calcium concentration, leading to loss of intact full-length wild-type huntingtin. This cleavage could be prevented by calcium chelators and calpain inhibitors. Degradation of wild-type huntingtin by calcium-dependent proteases thus occurs in HD neurons, leading to loss of wild-type huntingtin neuroprotective activity.     

Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: The SPIDIA experience

In this note, we present an ad hoc procedure that combines qualitative (visual evaluation) and quantitative (ImageJ software) evaluations of Pulsed-Field Gel Electrophoresis (PFGE) images to assess the genomic DNA (gDNA) integrity of analyzed samples. This procedure could be suitable for the analysis of a large number of images by taking into consideration both the expertise of researchers and the objectiveness of the software. We applied this procedure on the first SPIDIA DNA External Quality Assessment (EQA) samples. Results show that the classification obtained by this ad hoc procedure allows a more accurate evaluation of gDNA integrity with respect to a single approach.