Protein crystals in adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489–492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors

Towards a manifestly supersymmetric action for 11-dimensional supergravity

We investigate the possibility of writing a manifestly supersymmetric action for 11-dimensional supergravity. The construction involves an explicit relation between the fields in the super-vielbein and the super-3-form, and uses non-minimal pure spinors. A simple cubic interaction term for a single scalar superfield is found.Comment: 22 pp., plain tex. v2: references adde

ISO spectroscopy of gas and dust: from molecular clouds to protoplanetary disks

Observations of interstellar gas-phase and solid-state species in the 2.4-200 micron range obtained with the spectrometers on board the Infrared Space Observatory are reviewed. Lines and bands due to ices, polycyclic aromatic hydrocarbons, silicates and gas-phase atoms and molecules (in particular H2, CO, H2O, OH and CO2) are summarized and their diagnostic capabilities illustrated. The results are discussed in the context of the physical and chemical evolution of star-forming regions, including photon-dominated regions, shocks, protostellar envelopes and disks around young stars.Comment: 56 pages, 17 figures. To appear in Ann. Rev. Astron. Astrophys. 2004. Higher resolution version posted at http://www.strw.leidenuniv.nl/~ewine/araa04.pd

Adenosine A2A receptor modulation of hippocampal CA3-CA1 synapse plasticity during associative learning in behaving mice

© 2009 Nature Publishing Group All rights reservedPrevious in vitro studies have characterized the electrophysiological and molecular signaling pathways of adenosine tonic modulation on long-lasting synaptic plasticity events, particularly for hippocampal long-term potentiation(LTP). However, it remains to be elucidated whether the long-term changes produced by endogenous adenosine in the efficiency of synapses are related to those required for learning and memory formation. Our goal was to understand how endogenous activation of adenosine excitatory A2A receptors modulates the associative learning evolution in conscious behaving mice. We have studied here the effects of the application of a highly selective A2A receptor antagonist, SCH58261, upon a well-known associative learning paradigm - classical eyeblink conditioning. We used a trace paradigm, with a tone as the conditioned stimulus (CS) and an electric shock presented to the supraorbital nerve as the unconditioned stimulus(US). A single electrical pulse was presented to the Schaffer collateral–commissural pathway to evoke field EPSPs (fEPSPs) in the pyramidal CA1 area during the CS–US interval. In vehicle-injected animals, there was a progressive increase in the percentage of conditioning responses (CRs) and in the slope of fEPSPs through conditioning sessions, an effect that was completely prevented (and lost) in SCH58261 (0.5 mg/kg, i.p.)-injected animals. Moreover, experimentally evoked LTP was impaired in SCH58261- injected mice. In conclusion, the endogenous activation of adenosine A2A receptors plays a pivotal effect on the associative learning process and its relevant hippocampal circuits, including activity-dependent changes at the CA3-CA1 synapse.This study was supported by grants from the Spanish Ministry of Education and Research (BFU2005-01024 and BFU2005-02512), Spanish Junta de Andalucía (BIO-122 and CVI-02487), and the Fundación Conocimiento y Cultura of the Pablo de Olavide University (Seville, Spain).B. Fontinha was in receipt of a studentship from a project grant (POCI/SAU-NEU/56332/2004) supported by Fundação para a Ciência e Tecnologia (FCT, Portugal), and of an STSM from Cost B30 concerted action of the EU

Mechanisms of confluence-dependent expression of CD26 in colon cancer cell lines

<p>Abstract</p> <p>Background</p> <p>CD26 (dipeptidyl peptidase IV, DPPIV) is a 110 kDa surface glycoprotein expressed in most normal tissues, and is a potential novel therapeutic target for selected cancers. Our work evaluates the mechanism involved in confluence-dependent CD26 expression in colon cancer.</p> <p>Methods</p> <p>Colon adenocarcinoma cells were grown to confluence, and expression of CD26 and transcription factors implicated in its regulation was confirmed by immunofluorescence and Western blotting. Real-time PCR was also performed to evaluate CD26 upregulation at the transcriptional level. The influence of c-Myc on CD26 expression during different growth conditions was further evaluated following transient transfection of a c-Myc-expressing plasmid and a c-Myc specific siRNA.</p> <p>Results</p> <p>We found that the colon cancer cell lines HCT-116 and HCT-15 exhibited a confluence-dependent increase in CD26 mRNA and protein, associated with decreased expression of c-Myc, increased USF-1 and Cdx 2 levels, and unchanged HNF-1α expression. Meanwhile, ectopic expression of c-Myc in both cell lines led to decreased CD26 expression. In contrast, transfection of a siRNA targeted to Cdx2 resulted in decreased CD26 level. Importantly, culturing of cells in serum-depleted media, but not acidic conditions, upregulated CD26. While HIF-1α level also increased when cells were cultured in serum-depleted media, its expression was required but not sufficient for CD26 upregulation.</p> <p>Conclusions</p> <p>CD26 mRNA and protein levels increase in a confluence-dependent manner in colon carcinoma cell lines, with c-Myc acting as a repressor and Cdx2 acting as an enhancer of CD26 expression. The enhanced expression of CD26 in serum-depleted media and a requirement for HIF-1α suggest a role for nutrients or growth factors in the regulation of CD26 protein expression.</p

Leishmania major Survival in Selective Phlebotomus papatasi Sand Fly Vector Requires a Specific SCG-Encoded Lipophosphoglycan Galactosylation Pattern

Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In “selective” sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the “selective” fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both ‘poly-scGal’ and ‘null-scGal’ lines survived poorly relative to PpapJ-sympatric L. major FV1 and other ‘mono-scGal’ lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing ‘null-scGal’-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a ‘PpapJ-optimal’ scGal-LPG PAMP. Unexpectedly, these “L. major FV1-cloaked” L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific ‘mono-scGal’ pattern. However, failure of ‘mono-scGal’ L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is “selective” or “permissive”, with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania

Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

<p>Abstract</p> <p>Background</p> <p>Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D) co-culture assay.</p> <p>Methods</p> <p>Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy.</p> <p>Results</p> <p>In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors.</p> <p>Conclusion</p> <p>Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.</p

Oncostatin M Protects Rod and Cone Photoreceptors and Promotes Regeneration of Cone Outer Segment in a Rat Model of Retinal Degeneration

Retinitis pigmentosa (RP) is a group of photoreceptor degenerative disorders that lead to loss of vision. Typically, rod photoreceptors degenerate first, resulting in loss of night and peripheral vision. Secondary cone degeneration eventually affects central vision, leading to total blindness. Previous studies have shown that photoreceptors could be protected from degeneration by exogenous neurotrophic factors, including ciliary neurotrophic factor (CNTF), a member of the IL-6 family of cytokines. Using a transgenic rat model of retinal degeneration (the S334-ter rat), we investigated the effects of Oncostatin M (OSM), another member of the IL-6 family of cytokines, on photoreceptor protection. We found that exogenous OSM protects both rod and cone photoreceptors. In addition, OSM promotes regeneration of cone outer segments in early stages of cone degeneration. Further investigation showed that OSM treatment induces STAT3 phosphorylation in Müller cells but not in photoreceptors, suggesting that OSM not directly acts on photoreceptors and that the protective effects of OSM on photoreceptors are mediated by Müller cells. These findings support the therapeutic strategy using members of IL-6 family of cytokines for retinal degenerative disorders. They also provide evidence that activation of the STAT3 pathway in Müller cells promotes photoreceptor survival. Our work highlights the importance of Müller cell-photoreceptor interaction in the retina, which may serve as a model of glia-neuron interaction in general

Circulating CD62E+ Microparticles and Cardiovascular Outcomes

BACKGROUND: Activated endothelial cells release plasma membrane submicron vesicles expressing CD62E (E-selectin) into blood, known as endothelial microparticles (EMPs). We studied whether the levels of endothelial microparticles expressing CD62E(+), CD31(+)/Annexin-V(+), or CD31(+)/CD42(-) predict cardiovascular outcomes in patients with stroke history. METHODS/PRINCIPAL FINDINGS: Patients with stroke history at least 3 months prior to enrolment were recruited. Peripheral blood EMP levels were measured by flow cytometry. Major cardiovascular events and death were monitored for 36 months. Three hundred patients were enrolled, of which 298 completed the study according to protocol. Major cardiovascular events occurred in 29 patients (9.7%). Nine patients died, five from cardiovascular causes. Cumulative event-free survival rates were lower in patients with high levels of CD62E(+) microparticles. Multivariate Cox regression analysis adjusted for cardiovascular risk factors, medications and stroke etiologic groups showed an association between a high CD62E(+) microparticle level and a risk of major cardiovascular events and hospitalization. Levels of other kinds of EMPs expressing CD31(+)/Annexin-V(+) or CD31(+)/CD42(-) markers were not predictive of cardiovascular outcomes. CONCLUSION: A high level of CD62E(+) microparticles is associated with cardiovascular events in patients with stroke history, suggesting that the systemic endothelial activation increases the risk for cardiovascular morbidities

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 A2A receptors (A2ARs) and BDNF have been recently reported. In this article, we report some recent findings from our group showing that A2ARs 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 A2AR knock-out (KO) mice. Furthermore, enzyme immunoassay studies showed a significant reduction in hippocampal BDNF levels in A2AR KO vs. WT mice. Having found an even marked reduction in the striatum of A2AR KO mice, and as both BDNF and A2ARs have been implicated in the pathogenesis of Huntington’s disease (HD), an inherited striatal neurodegenerative disease, we then evaluated whether the pharmacological blockade of A2ARs 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 A2AR antagonist SCH58261 significantly reduced striatal BDNF levels. These results indicate that the presence and the tonic activation of A2ARs 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