West Nile virus (WNV) is a member of the
Flaviviridae family of positive-strand RNA viruses. Its viral RNA is translated to produce a polyprotein precursor that is further processed into three ...structural and seven non-structural proteins. The non-structural protein 3 (NS3) possess both protease and helicase activities. The C-terminal portion of the NS3 contains the ATPase/helicase domain presumably involved in viral replication. This domain has been expressed in
Escherichia coli, purified in soluble form and structurally characterized. As judged by analytical centrifugation and size exclusion chromatography, the purified enzyme behaves as a monomer in solution. It has ATPase activity that is stimulated by the presence of RNA and single-stranded DNA molecules (ssDNA). However, we were unable to detect helicase activity at protein concentrations up to 500
nM. It has been reported that longer constructions of NS3 helicase domains from other flavivirus, like those which include residues of the linker region between the protease and the helicase domains, have helicase activity. Since all the conformational features of the purified WNV NS3 domain are those of a native protein, it is tempting to assume that the linker region plays a critical role in determining the protein–protein interactions that leads to the formation of the active oligomer.
Abstract CD3ε chains are essential to the structure, expression and signaling of T cell receptors. Here, we extend to human CD3ε our previous data in mouse CD3ε showing that, in T cells, proteolytic ...processing of the acidic N-terminal sequence of CD3ε chains generate distinct polypeptide species that can be identified by two-dimension (IEF-SDS PAGE) electrophoresis and immunoblot. This was shown first by showing the processing of a fusion protein of GFP and the extracellular domain of mouse CD3ε (mCD3GFP) expressed in Jurkat cells. Secondly, pI heterogeneity was also found in human CD3ε chains immunoprecipitated from the surface of Jurkat cells or PHA blasts of human blood T lymphocytes. Comparison of CD3ε chains from 27 different species shows that their N-terminal sequences share a strong acidic nature, despite the large differences in terms of length and composition, even among closely related species. Our results suggest that generation of CD3ε chain isoforms with different N-terminal sequence and pI is a general phenomenon. Thus, as previously observed in the mouse, the relative abundance of CD3ε chain species might regulate TCR/CD3 structure and function, including the strength of the interactions between CD3 dimers and the TCR clonotypic receptors, as well as TCR/CD3 activation thresholds. Interestingly, CD3ε chains from 7 out of 27 species studied have putative N-glycosylation (NxS or NxT) motifs in their Ig extracellular domain. Their location, plus the conservation of residues involved in domain organization, the interactions with other CD3 chains, or the TCR, and signal triggering add new data useful to establish a permissive topology for the interaction between CD3 dimers and the TCR chains.
H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4+ Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti‐H4/ICOS ...antibodies strongly enhanced CD3‐mediated tyrosine phosphorylation of ZAP‐70, ζ, or Vav, as well as extracellular signal‐regulated kinase (ERK), Jun N‐terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3‐kinase (PI‐3K) to H4/ICOS was enhanced by H4/ICOS cross‐linking, and PI‐3K inhibitors inhibited ERK and JNK activation andIL‐4/IL‐10 secretion, but not p38 MAP kinase or ZAP‐70 activation. H4/ICOS‐mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP‐70, MAP kinase activation, or IL‐4/IL‐10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP‐70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL‐4, but not IL‐10 secretion. ERK or JNKinhibitors partially inhibited IL‐4 and IL‐10 secretion, while PKC or p38 inhibitors had no significant effects on IL‐4 or IL‐10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.
Background Gorlin-Goltz syndrome (GGS) is an autosomal-dominant disease characterized by the early onset of multiple basal cell carcinomas (BCCs), among other findings. Clinically, the BCCs may ...appear as soft pedunculated neoplasms that can be mistaken for true acrochordons. Objective We sought to describe the dermatoscopic characteristics of small acrochordon-like or polypoid BCCs in a child with GGS, and to perform histopathologic correlation. Methods Acrochordon-like growths from a child with GGS were studied. Clinical records and digital dermatoscopic images were collected, and excision and histopathologic examination of the most representative lesions were performed. Results Some acrochordon-like lesions showed specific dermatoscopic criteria for BCC, including multiple blue-gray globules and arborizing telangiectasia. Other polypoid lesions, especially the smaller ones, exhibited characteristics that suggested BCC, such as isolated blue-gray globules, small blue-gray ovoid nests, and fine elongated telangiectases. Limitations Conclusions are limited by the small sample size. Conclusion Dermatoscopy may be a useful diagnostic tool to analyze acrochordon-like lesions in children and to facilitate early diagnosis and treatment of BCCs in patients with GGS.
The complement regulatory protein CD46 (MCP, membrane cofactor protein) is used as a cell receptor by a number of bacterial and viral pathogens, including
Streptococcus pyogenes (Group A ...Streptococci). The highly variable M (Emm) proteins are virulence factors of
S. pyogenes, and Emm proteins of serotypes 5, 6 or 22 are able of binding to CD46, thus mediating the binding of Streptococci to human cells. In this work, using a soluble construction encompassing the extracellular domain of human CD46, we have analyzed its binding to clinical isolates of
S. pyogenes, including isolates of the M types 1, 3 and 18 that are frequently found in invasive infections or rheumatic fever. Our data show a strong binding of CD46 to bacteria of M types 1, 3, 8, 18, 24, 28, 29, 31 and 78; weak binding to M6 and M29 and no binding to M types 11, 12, M27 or M30. Surprisingly, CD46 bound to isogenic mutants of one clinical M18 isolate lacking the Emm protein or Emm and the Emm-related protein Enn, regardless of having capsule or not. In addition, these isogenic mutants bound to keratinocytes in a CD46-dependent manner, confirming the role of CD46 as one of the cell receptors for Group A Streptococci. Furthermore, CD46 did not bind to a recombinant Emm 18 construct, confirming that Emm is not involved in CD46 binding to M18 bacteria. Emm-dependent and -independent CD46 binding of clinical isolates of Streptococci confirms the importance of CD46 as a cell target that might confer pathogens some biological advantages over the host.
The T cell receptor-CD3 (TCR/CD3) complex is a multichain structure in charge of antigen recognition in T cells. Despite many genetic, structural, and functional data obtained in recent years, ...essential questions concerning the TCR/CD3 complex still remain open, including: 1) the precise number of polypeptides in each TCR/CD3 complex, their interactions and spatial arrangement, 2) the role(s) of each polypeptide in antigen recognition and/or in receptor signal transmission, and 3) the relationship between the TCR/CD3 complex and other membrane or cytoplasmic molecules involved in downstream signaling. In this work we shall review data concerning some of these issues, proposing a model of the overall structure of the TCR/CD3 complex to explain its known features.
The characterization of natural spaces by the precise observation of their material properties is highly demanded in remote sensing and computer vision. The production of novel sensors enables the ...collection of heterogeneous data to get a comprehensive knowledge of the living and non-living entities in the ecosystem. The high resolution of consumer-grade RGB cameras is frequently used for the geometric reconstruction of many types of environments. Nevertheless, the understanding of natural spaces is still challenging. The automatic segmentation of homogeneous materials in nature is a complex task because there are many overlapping structures and an indirect illumination, so the object recognition is difficult. In this paper, we propose a method based on fusing spatial and multispectral characteristics for the unsupervised classification of natural materials in a point cloud. A high-resolution camera and a multispectral sensor are mounted on a custom camera rig in order to simultaneously capture RGB and multispectral images. Our method is tested in a controlled scenario, where different natural objects coexist. Initially, the input RGB images are processed to generate a point cloud by applying the structure-from-motion (SfM) algorithm. Then, the multispectral images are mapped on the three-dimensional model to characterize the geometry with the reflectance captured from four narrow bands (green, red, red-edge and near-infrared). The reflectance, the visible colour and the spatial component are combined to extract key differences among all existing materials. For this purpose, a hierarchical cluster analysis is applied to pool the point cloud and identify the feature pattern for every material. As a result, the tree trunk, the leaves, different species of low plants, the ground and rocks can be clearly recognized in the scene. These results demonstrate the feasibility to perform a semantic segmentation by considering multispectral and spatial features with an unknown number of clusters to be detected on the point cloud. Moreover, our solution is compared to other method based on supervised learning in order to test the improvement of the proposed approach.
CD3ɛ chains are essential to the structure, expression and signaling of T cell receptors. Here, we extend to human CD3ɛ our previous data in mouse CD3ɛ showing that, in T cells, proteolytic ...processing of the acidic N-terminal sequence of CD3ɛ chains generate distinct polypeptide species that can be identified by two-dimension (IEF-SDS PAGE) electrophoresis and immunoblot. This was shown first by showing the processing of a fusion protein of GFP and the extracellular domain of mouse CD3ɛ (mCD3GFP) expressed in Jurkat cells. Secondly, pI heterogeneity was also found in human CD3ɛ chains immunoprecipitated from the surface of Jurkat cells or PHA blasts of human blood T lymphocytes.
Comparison of CD3ɛ chains from 27 different species shows that their N-terminal sequences share a strong acidic nature, despite the large differences in terms of length and composition, even among closely related species. Our results suggest that generation of CD3ɛ chain isoforms with different N-terminal sequence and pI is a general phenomenon. Thus, as previously observed in the mouse, the relative abundance of CD3ɛ chain species might regulate TCR/CD3 structure and function, including the strength of the interactions between CD3 dimers and the TCR clonotypic receptors, as well as TCR/CD3 activation thresholds.
Interestingly, CD3ɛ chains from 7 out of 27 species studied have putative N-glycosylation (NxS or NxT) motifs in their Ig extracellular domain. Their location, plus the conservation of residues involved in domain organization, the interactions with other CD3 chains, or the TCR, and signal triggering add new data useful to establish a permissive topology for the interaction between CD3 dimers and the TCR chains.
The antigen T cell receptor (TCR)-CD3 complexes present on the cell surface of CD4 + T lymphocytes and T cell lines express CD3ϵ chain isoforms with different isoelectric points (pI), with important ...structural
and functional consequences. The pI values of the isoforms fit the predicted pI values of CD3ϵ chains lacking one, two, and
three negatively charged amino acid residues present in the N-terminal region. Different T cells have different ratios of
CD3ϵ chain isoforms. At a high pI, degraded CD3ϵ isoforms can be better recognized by certain anti-CD3 monoclonal antibodies
such as YCD3-1, the ability of which to bind to the TCR-CD3 complex is directly correlated with the pI of CD3ϵ. The abundance
of CD3ϵ isoforms can be modified by treatment of T cells with the proteinase inhibitor phenanthroline. In addition, these
CD3ϵ isoforms have functional importance. This is shown, first, by the different structure of TCR-CD3 complexes in cells possessing
different amounts of isoforms (as observed in surface biotinylation experiments), by their different antigen responses, and
by the stronger interaction between low pI CD3ϵ isoforms and the TCR. Second, incubation of cells with phenanthroline diminished
the proportion of degraded high pI CD3ϵ isoforms, but also the ability of the cells to deliver early TCR activation signals.
Third, cells expressing mutant CD3ϵ chains lacking N-terminal acid residues showed facilitated recognition by antibody YCD3-1
and enhanced TCR-mediated activation. Furthermore, the binding avidity of antibody YCD3-1 was different in distinct thymus
populations. These results suggest that changes in CD3ϵ N-terminal chains might help to fine-tune the response of the TCR
to its ligands in distinct activation situations or in thymus selection.
The antigen T cell receptor (TCR)-CD3 complexes present on the cell surface of CD4+ T lymphocytes and T cell lines express CD3ɛ chain isoforms with different isoelectric points (pI), with important ...structural and functional consequences. The pI values of the isoforms fit the predicted pI values of CD3ɛ chains lacking one, two, and three negatively charged amino acid residues present in the N-terminal region. Different T cells have different ratios of CD3ɛ chain isoforms. At a high pI, degraded CD3ɛ isoforms can be better recognized by certain anti-CD3 monoclonal antibodies such as YCD3-1, the ability of which to bind to the TCR-CD3 complex is directly correlated with the pI of CD3ɛ. The abundance of CD3ɛ isoforms can be modified by treatment of T cells with the proteinase inhibitor phenanthroline. In addition, these CD3ɛ isoforms have functional importance. This is shown, first, by the different structure of TCR-CD3 complexes in cells possessing different amounts of isoforms (as observed in surface biotinylation experiments), by their different antigen responses, and by the stronger interaction between low pI CD3ɛ isoforms and the TCR. Second, incubation of cells with phenanthroline diminished the proportion of degraded high pI CD3ɛ isoforms, but also the ability of the cells to deliver early TCR activation signals. Third, cells expressing mutant CD3ɛ chains lacking N-terminal acid residues showed facilitated recognition by antibody YCD3-1 and enhanced TCR-mediated activation. Furthermore, the binding avidity of antibody YCD3-1 was different in distinct thymus populations. These results suggest that changes in CD3ɛ N-terminal chains might help to fine-tune the response of the TCR to its ligands in distinct activation situations or in thymus selection.
PDF
