Expression analysis and protein localization of the human HPC-1/syntaxin 1A, a gene deleted in Williams syndrome

The HPC-1/syntaxin 1A (STX1A) gene maps to the Williams syndrome (WS) commonly deleted region on chromosome 7q11.23 and encodes a protein implicated in the docking of synaptic vesicles with the presynaptic plasma membrane. To assess the potential role of STX1A in the WS phenotype, we carried out expression studies at the RNA and protein levels, in fetal and adult human tissues. RNA in situ hybridization on human embryo sections showed strong STX1A expression in spinal cord and ganglia. However, in adulthood, this gene was preferentially expressed in brain, as shown by Northern blot and RT-PCR experiments. Marked expression levels were observed in cerebellum and cerebral cortex. The STX1A protein was prevalently distributed in the molecular layer of the cerebellar cortex. A qualitative and quantitative analysis using a specific anti-STX1A antibody did not disclose any significant difference among frontal, temporal, and occipital poles of the human adult cortex in the two hemispheres. This is the first study focused on STX1A expression in humans. Our results indicate that this gene is strongly expressed in cerebral areas involved in cognitive process, supporting a likely role in the neurological symptoms of WS.     

Cloning and Molecular Characterization of the First Innexin of the Phylum Annelida—Expression of the Gene During Development

A novel member of the innexin family (cv-inx) has been isolated from the annelid polychaete worm Chaetopterus variopedatus using a PCR approach on genomic DNA and sequence analysis on genomic DNA clones. The gene is present in a HindIII-HindIII segment of 2250 bp containing an uninterrupted open reading frame of 1196 bp encoding a protein of 399 amino acids. The predicted protein shows the typical structural features of innexins and consensus sites for phosphorylation. Analyses on genomic DNA demonstrate that cv-inx is a single copy gene with no introns in the coding region, exactly corresponding to the cDNA sequence. The gene expression is regulated during development as shown by Northern blots analyses of the RNA and by immunoreaction with antibodies against the protein at several embryonic stages. The finding of an innexin in the phylum Annelida, outside of the Ecdysozoa clade, and its peculiar gene structure suggest the necessity to reconsider the current hypothesis on the origin and evolution of gap junctional proteins. Received: 15 December 2000 / Accepted: 27 August 2001     

Identification of differentially expressed cDNA clones in Tilia platyphyllos-Tuber borchii ectomycorrhizae using a differential screening approach

No information is presently available on the molecular mechanisms that control the morphogenesis of the truffle, an ectomycorrhizal ascomycetous fungus of great economic interest not only for forestry and agronomy but also for the organoleptic properties of its hypogeous fruitbodies. A Tilia platyphyllos- Tuber borchii model system was used in order to identify genes induced or up-regulated during symbiosis, since their isolation is a prerequisite for the understanding of the molecular bases of mycorrhizal development and regulation. The strategy applied involved the construction of an ectomycorrhizal cDNA library and random selection of clones, followed by a differential screening procedure to analyse cDNA expression in uninfected roots, ectomycorrhizae and free-living mycelia. The results revealed that many genes - and more plant genes than fungal genes - are expressed at higher levels during the symbiotic phase. Several clones were also investigated in order to understand their biological function. This study represents the first attempt to extend our knowledge of the molecular mechanisms underlying the establishment of ectomycorrhiza in Tuber species.     

Adenosine A2A receptors modulate BDNF both in normal conditions and in experimental models of Huntington’s disease

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Functional interactions between adenosine A_2A receptors (A_2ARs) and BDNF have been recently reported. In this article, we report some recent findings from our group showing that A_2ARs regulate both BDNF functions and levels in the brain. Whereas BDNF (10 ng/ml) increased the slope of excitatory postsynaptic field potentials (fEPSPs) in hippocampal slices from wild-type (WT) mice, it was completely ineffective in slices taken from A_2AR knock-out (KO) mice. Furthermore, enzyme immunoassay studies showed a significant reduction in hippocampal BDNF levels in A_2AR KO vs. WT mice. Having found an even marked reduction in the striatum of A_2AR KO mice, and as both BDNF and A_2ARs have been implicated in the pathogenesis of Huntington’s disease (HD), an inherited striatal neurodegenerative disease, we then evaluated whether the pharmacological blockade of A_2ARs could influence striatal levels of BDNF in an experimental model of HD-like striatal degeneration (quinolinic acid-lesioned rats) and in a transgenic mice model of HD (R6/2 mice). In both QA-lesioned rats and early symptomatic R6/2 mice (8 weeks), the systemic administration of the A_2AR antagonist SCH58261 significantly reduced striatal BDNF levels. These results indicate that the presence and the tonic activation of A_2ARs are necessary to allow BDNF-induced potentiation of synaptic transmission and to sustain a normal BDNF tone. The possible functional consequences of reducing striatal BDNF levels in HD models need further investigation.     

Phylogenetic Characterization and In Situ Detection of a Cytophaga-Flexibacter-Bacteroides Phylogroup Bacterium in Tuber borchii Vittad. Ectomycorrhizal Mycelium

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.     

Influence of interferon on the functional expression of natural killer target structures of murine lymphoma cells

Murine lymphoma cells (YAC-1), induced by Moloney leukemia virus, nontreated (YAC) or pretreated in vitro with interferon (YAC-IF), were tested for their susceptibility to natural killer (NK)-mediated cytolysis. In line with previous reports YAC-IF were less susceptible to NK lysis than YAC cells. In cold competition assay, YAC-IF inhibited cytotoxicity to a lesser extent than YAC lymphoma when labeled target YAC cells were used. However, when radioactive YAC-IF cells were used as targets, cold competition attained with both YAC and YAC-IF was essentially the same. Furthermore, effector splenocytes, depleted of NK effector cells through immunoabsorption on YAC monolayer, were inactive against both YAC and YAC-IF targets. On the other hand, effector lymphocytes, absorbed on YAC-IF monolayer, retained NK activity against YAC cells but not against YAC-IF targets. These results are compatible with the hypothesis that interferon (IF) modulates negatively a subset of "interferon-susceptible" (IFS) NK target structure(s) (TS) of YAC cells, which would then express membrane determinants not functionally present on YAC-IF cells. On the other hand YAC and YAC-IF cells share "interferon-resistant" (IFR) TS not affected by pretreatment with IF. In order to test whether IFS X TS and IFR X TS are present on the same cell or clonally distributed, YAC cells were cloned and tested for NK susceptibility following IF pretreatment. The results did not support the hypothesis of a clonal distribution of both IFS X TS and IFR X TS since IF pretreatment of all clones, obtained by limiting dilution, resulted in a net impairment of target susceptibility to NK effector cells.     

Microsatellite primed-PCR to select molecular markers for Tuber species

The direct microsatellite-primed PCR and the RAMPO techniques were applied to detect inter-specific polymorphisms in Tuber species and to select species specific fragments. A T. borchii marker was identified and specific primers were selected.     

New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum

Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5-7.5μmolC(2)H(4)h(-1)g(-1), comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.     

Differential effect of creatine on oxidatively-injured mitochondrial and nuclear DNA

Creatine is a naturally occurring compound obtained in humans from endogenous production and consumption through the diet. It is used as an ergogenic aid to improve exercise performance and increase fat-free mass. Lately, creatine’s positive therapeutic benefits in various oxidative stress-associated diseases have been reported in literature and, more recently, creatine has also been shown to exert direct antioxidant effects. Oxidatively-challenged DNA was analysed to show possible protective effects of creatine. Acellular and cellular studies were carried out. Acellular assays, performed using molecular approaches, showed that creatine protects circular and linear DNA from oxidative attacks.     

Use of signal quality measurements to gain efficiency in the analysis of cDNA microarray data

This research provides a new way to measure error in microarray data in order to improve gene expression analysis.Microarray data contains many sources of error.In order to glean information about mRNA expression levels,the true signal must first be segregated from noise.This research focuses on the variation that can be captured at the spot level in cDNA microarray images.Variation at other levels,due to differences at the array,dye,and block levels,can be corrected for by a variety of existing normalizati...