PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

In Bacillus subtilis W23, the duet sigmaXsigmaM, two sigma factors of the extracytoplasmic function subfamily, are required for septum and wall synthesis under batch culture conditions.

The synthesis of poly(RboP), the main Bacillus subtilis W23 teichoic acid, is encoded by tarDF-tarABIJKL operons, the latter being controlled by two promoters designated PtarA-int and PtarA-ext. Analysis by lacZ fusions reveals that PtarA-int activity exhibits sharp increases at the beginning and end of the transition between exponential and stationary growth phase. As confirmed by mRNA quantification, these increases are mediated by ECF sigma factors sigmaX and sigmaM respectively. In liquid media, strain W23 sigX sigM double mutants experience serious difficulties in the transition and stationary growth phases. Inactivation of sigmaX- and sigmaM-controlled regulons, which precludes transcription from PtarA-int, leads to (i) delays in chromosome segregation and septation and (ii) a transient loss of up to 30% of the culture OD or lysis. However, specific inactivation of PtarA-int, leading mainly to a shortage of poly(RboP), does not affect growth while, nevertheless, interfering with normal septation, as revealed by electron microscopy. The different sigM transcription in strains W23 and 168 is discussed. In W23, expression of tarA and sigM, which is shown to control divIC, is inversely correlated with growth rate, suggesting that the sigM regulon is involved in the control of cell division.

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.

MP2RAGE, a self bias-field corrected sequence for improved segmentation and T-1-mapping at high field

The large spatial inhomogeneity in transmit B, field (B-1(+)) observable in human MR images at hi h static magnetic fields (B-0) severely impairs image quality. To overcome this effect in brain T-1-weighted images the, MPRAGE sequence was modified to generate two different images at different inversion times MP2RAGE By combining the two images in a novel fashion, it was possible to create T-1-weigthed images where the result image was free of proton density contrast, T-2* contrast, reception bias field, and, to first order transmit field inhomogeneity. MP2RAGE sequence parameters were optimized using Bloch equations to maximize contrast-to-noise ratio per unit of time between brain tissues and minimize the effect of B-1(+) variations through space. Images of high anatomical quality and excellent brain tissue differentiation suitable for applications such as segmentation and voxel-based morphometry were obtained at 3 and 7 T. From such T-1-weighted images, acquired within 12 min, high-resolution 3D T-1 maps were routinely calculated at 7 T with sub-millimeter voxel resolution (0.65-0.85 mm isotropic). T-1 maps were validated in phantom experiments. In humans, the T, values obtained at 7 T were 1.15 +/- 0.06 s for white matter (WM) and 1.92 +/- 0.16 s for grey matter (GM), in good agreement with literature values obtained at lower spatial resolution. At 3 T, where whole-brain acquisitions with 1 mm isotropic voxels were acquired in 8 min the T-1 values obtained (0.81 +/- 0.03 S for WM and 1.35 +/- 0.05 for GM) were once again found to be in very good agreement with values in the literature. (C) 2009 Elsevier Inc. All rights reserved.

Lesion Recognition in Multiple Sclerosis: A Sequence Comparison and Quantification Study at 3T

Introduction Lesion detection in multiple sclerosis (MS) is an essential part of its clinical diagnosis. In addition, radiological characterisation of MS lesions is an important research field that aims at distinguishing different MS types, monitoring drug response and prognosis. To date, various MR protocols have been proposed to obtain optimal lesion contrast for early and comprehensive diagnosis of the MS disease. In this study, we compare the sensitivity of five different MR contrasts for lesion detection: (i) the DIR sequence (Double Inversion Recovery, [4]), (ii) the Dark-fluid SPACE acquisition schemes, a 3D variant of a 2D FLAIR sequence [1], (iii) the MP2RAGE [2], an MP-RAGE variant that provides homogeneous T1 contrast and quantitative T1-values, and the sequences currently used for clinical MS diagnosis (2D FLAIR, MP-RAGE). Furthermore, we investigate the T1 relaxation times of cortical and sub-cortical regions in the brain hemispheres and the cerebellum at 3T. Methods 10 early-stage female MS patients (age: 31.64.7y; disease duration: 3.81.9y; disability score, EDSS: 1.80.4) and 10 healthy controls (age and gender-matched: 31.25.8y) were included in the study after obtaining informed written consent according to the local ethic protocol. All experiments were performed at 3T (Magnetom Trio a Tim System, Siemens, Germany) using a 32-channel head coil [5]. The imaging protocol included the following sequences, (all except for axial FLAIR 2D with 1x1x1.2 mm3 voxel and 256x256x160 matrix): DIR (TI1/TI2/TR XX/3652/10000 ms, iPAT=2, TA 12:02 min), MP-RAGE (TI/TR 900/2300 ms, iPAT=3, TA 3:47 min); MP2RAGE (TI1/TI2/TR 700/2500/5000 ms, iPAT=3, TA 8:22 min, cf. [2]); 3D FLAIR SPACE (only for patient 4-6, TI/TR 1800/5000 ms, iPAT=2, TA=5;52 min, cf. [1]); Axial FLAIR (0.9x0.9x2.5 mm3, 256x256x44 matrix, TI/TR 2500/9000 ms, iPAT=2, TA 4:05 min). Lesions were identified by two experienced neurologist and radiologist, manually contoured and assigned to regional locations (s. table 1). Regional lesion masks (RLM) from each contrast were compared for number and volumes of lesions. In addition, RLM were merged in a single "master" mask, which represented the sum of the lesions of all contrasts. T1 values were derived for each location from this mask for patients 5-10 (3D FLAIR contrast was missing for patient 1-4). Results & Discussion The DIR sequence appears the most sensitive for total lesions count, followed by the MP2RAGE (table 1). The 3D FLAIR SPACE sequence turns out to be more sensitive than the 2D FLAIR, presumably due to reduced partial volume effects. Looking for sub-cortical hemispheric lesions, the DIR contrast appears to be equally sensitive to the MP2RAGE and SPACE, but most sensitive for cerebellar MS plaques. The DIR sequence is also the one that reveals cortical hemispheric lesions best. T1 relaxation times at 3T in the WM and GM of the hemispheres and the cerebellum, as obtained with the MP2RAGE sequence, are shown in table 2. Extending previous studies, we confirm overall longer T1-values in lesion tissue and higher standard deviations compared to the non-lesion tissue and control tissue in healthy controls. We hypothesize a biological (different degree of axonal loss and demyelination) rather than technical origin. Conclusion In this study, we applied 5 MR contrasts including two novel sequences to investigate the contrast of highest sensitivity for early MS diagnosis. In addition, we characterized for the first time the T1 relaxation time in cortical and sub-cortical regions of the hemispheres and the cerebellum. Results are in agreement with previous publications and meaningful biological interpretation of the data.

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites.

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.

ARCHITECTING USER-CENTRIC PRIVACY-AS-A-SET-OF-SERVICES: DIGITAL IDENTITY RELATED PRIVACY FRAMEWORK

AbstractDigitalization gives to the Internet the power by allowing several virtual representations of reality, including that of identity. We leave an increasingly digital footprint in cyberspace and this situation puts our identity at high risks. Privacy is a right and fundamental social value that could play a key role as a medium to secure digital identities. Identity functionality is increasingly delivered as sets of services, rather than monolithic applications. So, an identity layer in which identity and privacy management services are loosely coupled, publicly hosted and available to on-demand calls could be more realistic and an acceptable situation. Identity and privacy should be interoperable and distributed through the adoption of service-orientation and implementation based on open standards (technical interoperability). Ihe objective of this project is to provide a way to implement interoperable user-centric digital identity-related privacy to respond to the need of distributed nature of federated identity systems. It is recognized that technical initiatives, emerging standards and protocols are not enough to guarantee resolution for the concerns surrounding a multi-facets and complex issue of identity and privacy. For this reason they should be apprehended within a global perspective through an integrated and a multidisciplinary approach. The approach dictates that privacy law, policies, regulations and technologies are to be crafted together from the start, rather than attaching it to digital identity after the fact. Thus, we draw Digital Identity-Related Privacy (DigldeRP) requirements from global, domestic and business-specific privacy policies. The requirements take shape of business interoperability. We suggest a layered implementation framework (DigldeRP framework) in accordance to model-driven architecture (MDA) approach that would help organizations' security team to turn business interoperability into technical interoperability in the form of a set of services that could accommodate Service-Oriented Architecture (SOA): Privacy-as-a-set-of- services (PaaSS) system. DigldeRP Framework will serve as a basis for vital understanding between business management and technical managers on digital identity related privacy initiatives. The layered DigldeRP framework presents five practical layers as an ordered sequence as a basis of DigldeRP project roadmap, however, in practice, there is an iterative process to assure that each layer supports effectively and enforces requirements of the adjacent ones. Each layer is composed by a set of blocks, which determine a roadmap that security team could follow to successfully implement PaaSS. Several blocks' descriptions are based on OMG SoaML modeling language and BPMN processes description. We identified, designed and implemented seven services that form PaaSS and described their consumption. PaaSS Java QEE project), WSDL, and XSD codes are given and explained.

Production of recombinant TRAIL and TRAIL receptor: Fc chimeric proteins.

The tumor necrosis factor (TNF)/TNF receptor (TNFR) families of ligands and receptors are implicated in a variety of physiological and pathological processes and regulate cellular functions as diverse as proliferation, differentiation, and death. Recombinant forms of these ligands and receptors can act to agonize or antagonize these functions and are therefore useful for laboratory studies and may have clinical applications. A protocol is presented for the expression and purification of dimeric soluble receptors fused to the Fc portion of human IgG1 and of soluble, N-terminally Flag-tagged ligands. Soluble recombinant proteins are easier to handle than membrane-bound proteins and the use of tags greatly facilitates their detection and purification. In addition, some tags may provide enhanced biological activity to the recombinant proteins (mainly by oligomerization and stabilization effects) and facilitate their functional characterization. Expression in bacterial (for selected ligands) and eukaryotic expression systems (for ligands and receptors) was performed using M15 pREP4 bacteria and human embryonic kidney 293 cells, respectively. The yield of purified protein is about 1 mg/liter for the mammalian expression system and several milligrams per liter for the bacterial expression system. Protocols are given for a specific ligand-receptor pair, namely TRAIL (Apo-2L) and TRAIL receptor 2 (DR5), but can be applied to other ligands and receptors of the TNF family.

Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase.

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

Constitutively active alpha-1b adrenergic receptor mutants display different phosphorylation and internalization features.

We compared the phosphorylation and internalization properties of constitutively active alpha-1b adrenergic receptor (AR) mutants carrying mutations in two distant receptor domains, i.e., at A293 in the distal part of the third intracellular loop and at D142 of the DRY motif lying at the end of the third transmembrane domain. For the A293E and A293I mutants the levels of agonist-independent phosphorylation were 150% and 50% higher than those of the wild-type alpha-1b AR, respectively. On the other hand, for the constitutively active D142A and D142T mutants, the basal levels of phosphorylation were similar to those of the wild-type alpha-1b AR and did not appear to be further stimulated by epinephrine. Overexpression of the guanyl nucleotide binding regulatory protein-coupled receptor kinase GRK2 further increases the basal phosphorylation of the A293E mutant, but not that of D142A mutant. Both the wild-type alpha-1b AR and the A293E mutant could undergo beta-arrestin-mediated internalization. The epinephrine-induced internalization of the constitutively active A293E mutant was significantly higher than that of the wild-type alpha-1b AR. In contrast, the D142A mutant was impaired in its ability to interact with beta-arrestin and to undergo agonist-induced internalization. Interestingly, a double mutant A293E/D142A retained very high constitutive activity and regulatory properties of both the A293E and D142A receptors. These findings demonstrate that two constitutively activating mutations occurring in distant receptor domains of the alpha-1b AR have divergent effects on the regulatory properties of the receptor.

Large-scale gene-centric analysis identifies novel variants for coronary artery disease.

Coronary artery disease (CAD) has a significant genetic contribution that is incompletely characterized. To complement genome-wide association (GWA) studies, we conducted a large and systematic candidate gene study of CAD susceptibility, including analysis of many uncommon and functional variants. We examined 49,094 genetic variants in ∼2,100 genes of cardiovascular relevance, using a customised gene array in 15,596 CAD cases and 34,992 controls (11,202 cases and 30,733 controls of European descent; 4,394 cases and 4,259 controls of South Asian origin). We attempted to replicate putative novel associations in an additional 17,121 CAD cases and 40,473 controls. Potential mechanisms through which the novel variants could affect CAD risk were explored through association tests with vascular risk factors and gene expression. We confirmed associations of several previously known CAD susceptibility loci (eg, 9p21.3:p<10(-33); LPA:p<10(-19); 1p13.3:p<10(-17)) as well as three recently discovered loci (COL4A1/COL4A2, ZC3HC1, CYP17A1:p<5×10(-7)). However, we found essentially null results for most previously suggested CAD candidate genes. In our replication study of 24 promising common variants, we identified novel associations of variants in or near LIPA, IL5, TRIB1, and ABCG5/ABCG8, with per-allele odds ratios for CAD risk with each of the novel variants ranging from 1.06-1.09. Associations with variants at LIPA, TRIB1, and ABCG5/ABCG8 were supported by gene expression data or effects on lipid levels. Apart from the previously reported variants in LPA, none of the other ∼4,500 low frequency and functional variants showed a strong effect. Associations in South Asians did not differ appreciably from those in Europeans, except for 9p21.3 (per-allele odds ratio: 1.14 versus 1.27 respectively; P for heterogeneity = 0.003). This large-scale gene-centric analysis has identified several novel genes for CAD that relate to diverse biochemical and cellular functions and clarified the literature with regard to many previously suggested genes.

Identification and functional characterization of a monofunctional peroxisomal enoyl-CoA hydratase 2 that participates in the degradation of even cis-unsaturated fatty acids in Arabidopsis thaliana.

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high beta-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the beta-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core beta-oxidation cycle.

SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis.

Several evidences suggest that astrocytes release small transmitter molecules, peptides, and protein factors via regulated exocytosis, implying that they function as specialized neurosecretory cells. However, very little is known about the molecular and functional properties of regulated secretion in astrocytes in the adult brain. Establishing these properties is central to the understanding of the communication mode(s) of these cells and their role(s) in the control of synaptic functions and of cerebral blood flow. In this study, we have set-up a high-resolution confocal microscopy approach to distinguish protein expression in astrocytic structures and neighboring synaptic terminals in adult brain tissue. This approach was applied to investigate the expression pattern of core SNARE proteins for vesicle fusion in the dentate gyrus and CA1 regions of the mouse hippocampus. Our comparative analysis shows that astrocytes abundantly express, in their cell body and main processes, all three protein partners necessary to form an operational SNARE complex but not in the same isoforms expressed in neighbouring synaptic terminals. Thus, SNAP25 and VAMP2 are absent from astrocytic processes and typically concentrated in terminals, while SNAP23 and VAMP3 have the opposite expression pattern. Syntaxin 1 is present in both synaptic terminals and astrocytes. These data support the view that astrocytes in the adult hippocampus can communicate via regulated exocytosis and also indicates that astrocytic exocytosis may differ in its properties from action potential-dependent exocytosis at neuronal synapses, as it relies on a distinctive set of SNARE proteins.