150 research outputs found
Papillomavirus Capsid Binding and Uptake by Cells From Different Tissues and Species
The inability of papillomaviruses (PV) to replicate in tissue culture cells has hampered the study of the PV life cycle. We investigated virus-cell interactions by the following two methods: (i) using purified bovine PV virions or human PV type 11 (HPV type 11) virus-like particles (VLP) to test the binding to eukaryotic cells and (ii) using different VLP-reporter plasmid complexes of HPV6b, HPV11 L1 or HPV11 L1/L2, and HPV16 L1 or HPV16 L1/L2 to study uptake of particles into different cell lines. Our studies showed that PV capsids bind to a broad range of cells in culture in a dose-dependent manner. Binding of PV capsids to cells can be blocked by pretreating the cells with the protease trypsin. Penetration of PV into cells was monitored by using complexes in which the purified PV capsids were physically linked to DNA containing the gene for beta-galactosidase driven by the human cytomegalovirus promoter. Expression of beta-galactosidase occurred in < 1% of the cells, and the efficiency of PV receptor-mediated gene delivery was greatly enhanced (up to 10 to 20% positive cells) by the use of a replication-defective adenovirus which promotes endosomal lysis. The data generated by this approach further confirmed the results obtained from the binding assays, showing that PV enter a wide range of cells and that these cells have all functions required for the uptake of PV. Binding and uptake of PV particles can be blocked by PV-specific antisera, and different PV particles compete for particle uptake. Our results suggest that the PV receptor is a conserved cell surface molecule(s) used by different PV and that the tropism of infection by different PV is controlled by events downstream of the initial binding and uptake
CCR2 Acts as Scavenger for CCL2 during Monocyte Chemotaxis
<div><h3>Background</h3><p>Leukocyte migration is essential for effective host defense against invading pathogens and during immune homeostasis. A hallmark of the regulation of this process is the presentation of chemokines in gradients stimulating leukocyte chemotaxis via cognate chemokine receptors. For efficient migration, receptor responsiveness must be maintained whilst the cells crawl on cell surfaces or on matrices along the attracting gradient towards increasing concentrations of agonist. On the other hand agonist-induced desensitization and internalization is a general paradigm for chemokine receptors which is inconsistent with the prolonged migratory capacity.</p> <h3>Methodology/Principal Findings</h3><p>Chemotaxis of monocytes was monitored in response to fluorescent CCL2-mCherry by time-lapse video microscopy. Uptake of the fluorescent agonist was used as indirect measure to follow the endogenous receptor CCR2 expressed on primary human monocytes. During chemotaxis CCL2-mCherry becomes endocytosed as cargo of CCR2, however, the internalization of CCR2 is not accompanied by reduced responsiveness of the cells due to desensitization.</p> <h3>Conclusions/Significance</h3><p>During chemotaxis CCR2 expressed on monocytes internalizes with the bound chemoattractant, but cycles rapidly back to the plasma membrane to maintain high responsiveness. Moreover, following relocation of the source of attractant, monocytes can rapidly reverse their polarization axis organizing a new leading edge along the newly formed gradient, suggesting a uniform distribution of highly receptive CCR2 on the plasma membrane. The present observations further indicate that during chemotaxis CCR2 acts as scavenger consuming the chemokine forming the attracting cue.</p> </div
Operating a full tungsten actively cooled tokamak: overview of WEST first phase of operation
WEST is an MA class superconducting, actively cooled, full tungsten (W) tokamak, designed to operate in long pulses up to 1000 s. In support of ITER operation and DEMO conceptual activities, key missions of WEST are: (i) qualification of high heat flux plasma-facing components in integrating both technological and physics aspects in relevant heat and particle exhaust conditions, particularly for the tungsten monoblocks foreseen in ITER divertor; (ii) integrated steady-state operation at high confinement, with a focus on power exhaust issues. During the phase 1 of operation (2017â2020), a set of actively cooled ITER-grade plasma facing unit prototypes was integrated into the inertially cooled W coated startup lower divertor. Up to 8.8 MW of RF power has been coupled to the plasma and divertor heat flux of up to 6 MW mâ2 were reached. Long pulse operation was started, using the upper actively cooled divertor, with a discharge of about 1 min achieved. This paper gives an overview of the results achieved in phase 1. Perspectives for phase 2, operating with the full capability of the device with the complete ITER-grade actively cooled lower divertor, are also described
Migration of Th1 Lymphocytes Is Regulated by CD152 (CTLA-4)-Mediated Signaling via PI3 Kinase-Dependent Akt Activation
Efficient adaptive immune responses require the localization of T lymphocytes in secondary lymphoid organs and inflamed tissues. To achieve correct localization of T lymphocytes, the migration of these cells is initiated and directed by adhesion molecules and chemokines. It has recently been shown that the inhibitory surface molecule CD152 (CTLA-4) initiates Th cell migration, but the molecular mechanism underlying this effect remains to be elucidated. Using CD4 T lymphocytes derived from OVA-specific TCR transgenic CD152-deficient and CD152-competent mice, we demonstrate that chemokine-triggered signal transduction is differentially regulated by CD152 via phosphoinositide 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt). In the presence of CD152 signaling, the chemoattractant CCL4 selectively induces the full activation of Akt via phosphorylation at threonine 308 and serine 473 in pro-inflammatory Th lymphocytes expressing the cognate chemokine receptor CCR5. Akt signals lead to cytoskeleton rearrangements, which are indispensable for migration. Therefore, this novel Akt-modulating function of CD152 signals affecting T cell migration demonstrates that boosting CD152 or its down-stream signal transduction could aid therapies aimed at sensitizing T lymphocytes for optimal migration, thus contributing to a precise and effective immune response
The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins
The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins
The 2017 May 20 stellar occultation by the elongated centaur (95626) 2002 GZ
We predicted a stellar occultation of the bright star Gaia DR1
4332852996360346368 (UCAC4 385-75921) (m= 14.0 mag) by the centaur
2002 GZ for 2017 May 20. Our latest shadow path prediction
was favourable to a large region in Europe. Observations were arranged in a
broad region inside the nominal shadow path. Series of images were obtained
with 29 telescopes throughout Europe and from six of them (five in Spain and
one in Greece) we detected the occultation. This is the fourth centaur, besides
Chariklo, Chiron and Bienor, for which a multi-chord stellar occultation is
reported. By means of an elliptical fit to the occultation chords we obtained
the limb of 2002 GZ during the occultation, resulting in an ellipse with
axes of 305 17 km 146 8 km. From this limb, thanks to a
rotational light curve obtained shortly after the occultation, we derived the
geometric albedo of 2002 GZ ( = 0.043 0.007) and a 3-D
ellipsoidal shape with axes 366 km 306 km 120 km. This shape
is not fully consistent with a homogeneous body in hydrostatic equilibrium for
the known rotation period of 2002 GZ. The size (albedo) obtained from
the occultation is respectively smaller (greater) than that derived from the
radiometric technique but compatible within error bars. No rings or debris
around 2002 GZ were detected from the occultation, but narrow and thin
rings cannot be discarded.Comment: Accepted for publication in MNRAS (8-Dec.-2020), 15 pages, 9 figure
A large topographic feature on the surface of the trans-Neptunian object (307261) 2002 MS measured from stellar occultations
This work aims at constraining the size, shape, and geometric albedo of the
dwarf planet candidate 2002 MS4 through the analysis of nine stellar
occultation events. Using multichord detection, we also studied the object's
topography by analyzing the obtained limb and the residuals between observed
chords and the best-fitted ellipse. We predicted and organized the
observational campaigns of nine stellar occultations by 2002 MS4 between 2019
and 2022, resulting in two single-chord events, four double-chord detections,
and three events with three to up to sixty-one positive chords. Using 13
selected chords from the 8 August 2020 event, we determined the global
elliptical limb of 2002 MS4. The best-fitted ellipse, combined with the
object's rotational information from the literature, constrains the object's
size, shape, and albedo. Additionally, we developed a new method to
characterize topography features on the object's limb. The global limb has a
semi-major axis of 412 10 km, a semi-minor axis of 385 17 km, and
the position angle of the minor axis is 121 16. From
this instantaneous limb, we obtained 2002 MS4's geometric albedo and the
projected area-equivalent diameter. Significant deviations from the fitted
ellipse in the northernmost limb are detected from multiple sites highlighting
three distinct topographic features: one 11 km depth depression followed by a
25 km height elevation next to a crater-like depression with an
extension of 322 39 km and 45.1 1.5 km deep. Our results present an
object that is 138 km smaller in diameter than derived from thermal
data, possibly indicating the presence of a so-far unknown satellite. However,
within the error bars, the geometric albedo in the V-band agrees with the
results published in the literature, even with the radiometric-derived albedo
HGF-Induced PKCζ Activation Increases Functional CXCR4 Expression in Human Breast Cancer Cells
The chemokine receptor CXCR4 and its ligand CXCL12 have been shown to mediate the metastasis of many malignant tumors including breast carcinoma. Interaction between hepatocyte growth factor (HGF) and the Met receptor tyrosine kinase mediates development and progression of cancers. HGF is able to induce CXCR4 expression and contributes to tumor cell invasiveness in breast carcinoma. However, the mechanism of the CXCR4 expression modulated by c-Met-HGF axis to enhance the metastatic behavior of breast cancer cells is still unclear. In this study, we found that HGF induced functional CXCR4 receptor expression in breast cancer cells. The effect of HGF was specifically mediated by PKCζ activity. After transfection with PKCζ-siRNA, the phosphorylation of PKCζ and CXCR4 was abrogated in breast cancer cells. Interference with the activation of Rac1, a downstream target of HGF, prevented the HGF-induced increase in PKCζ activity and CXCR4 levels. The HGF-induced, LY294002-sensitive translocation of PKCζ from cytosol to plasma membrane indicated that HGF was capable of activating PKCζ, probably via phosphoinositide (PI) 3-kinases. HGF treatment also increased MT1-MMP secretion. Inhibition of PKCζ, Rac-1 and phosphatidylinositol 3-kinase may attenuate MT1-MMP expression in cells exposed to HGF. Functional manifestation of the effects of HGF revealed an increased ability for migration, chemotaxis and metastasis in MDA-MB-436 cells in vitro and in vivo. Our findings thus provided evidence that the process of HGF-induced functional CXCR4 expression may involve PI 3-kinase and atypical PKCζ. Moreover, HGF may promote the invasiveness and metastasis of breast tumor xenografts in BALB/c-nu mice via the PKCζ-mediated pathway, while suppression of PKCζ by RNA interference may abrogate cancer cell spreading
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