bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Physical Association of Protein Kinase Cθ with a Lipid Raft-Associated Inhibitor of κB Factor Kinase (IKK) Complex Plays a Role in the Activation of the NF-κB Cascade by TCR and CD28
Authors: Khoshnan Ali , Bae David , Tindell Charles A. , Nel Andre E. 
Published: April 2014

AbstractWe investigated the role of protein kinase C θ (PKCθ) in the activation of the NF-κB cascade in primary human CD4+ lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKCθ participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IκB kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca2+-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKCθ physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4+ T cells. The same set of stimuli also induced relocation of endogenous PKCθ and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IκBα sustrate. Confocal microscopy further demonstrated that exogenously expressed PKCθ and IKKβ colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKCθ activated IKKβ in Jurkat T cells. Expression of dominant-active PKCθ also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKCθ by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.
Peroxisome Proliferator Activator Receptor-γ Agonists and 15-Deoxy-Δ12,1412,14-PGJ2 Induce Apoptosis in Normal and Malignant B-Lineage Cells
Authors: Padilla Josué , Kaur Kuljeet , Cao H. James , Smith Terry J. , Phipps Richard P. 
Published: April 2014

AbstractThe research described herein evaluates the expression and functional significance of peroxisome proliferator activator receptor-γ (PPAR-γ) on B-lineage cells. Normal mouse B cells and a variety of B lymphoma cells reflective of stages of B cell differentiation (e.g., 70Z/3, CH31, WEHI-231, CH12, and J558) express PPAR-γ mRNA and, by Western blot analysis, the 67-kDa PPAR-γ protein. 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2), a PPAR-γ agonist, has a dose-dependent antiproliferative and cytotoxic effect on normal and malignant B cells as shown by [3H]thymidine and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays. Only PPAR-γ agonists (thiazolidinediones), and not PPAR-α agonists, mimicked the effect of 15d-PGJ2 on B-lineage cells, indicating that the mechanism by which 15d-PGJ2 negatively affects B-lineage cells involves in part PPAR-γ. The mechanism by which PPAR-γ agonists induce cytotoxicity is via apoptosis, as shown by annexin V staining and as confirmed by DNA fragmentation detected using the TUNEL assay. Interestingly, addition of PGF2α, which was not known to affect lymphocytes, dramatically attenuated the deleterious effects of PPAR-γ agonists on B lymphomas. Surprisingly, 15d-PGJ2 induced a massive increase in nuclear mitogen-activated protein kinase activation, and pretreatment with PGF2α blunted the mitogen-activated protein kinase activation. This is the first study evaluating PPAR-γ expression and its significance on B lymphocytes. PPAR-γ agonists may serve as a counterbalance to the stimulating effects of other PGs, namely PGE2, which promotes B cell differentiation. Finally, the use of PGs, such as 15d-PGJ2, and synthetic PPAR-γ agonists to induce apoptosis in B-lineage cells may lead to the development of novel therapies for fatal B lymphomas.
Crystal Structure of an Antibody Bound to an Immunodominant Peptide Epitope: Novel Features in Peptide-Antibody Recognition
Authors: Nair Deepak T. , Singh Kavita , Sahu Naresh , Rao Kanury V. S. , Salunke Dinakar M. 
Published: April 2014

Abstract
The crystal structure of Fab of an Ab PC283 complexed with its corresponding peptide Ag, PS1 (HQLDPAFGANSTNPD), derived from the hepatitis B virus surface Ag was determined. The PS1 stretch Gln2P to Phe7P is present in the Ag binding site of the Ab, while the next three residues of the peptide are raised above the binding groove. The residues Ser11P, Thr12P, and Asn13P then loop back onto the Ag-binding site of the Ab. The last two residues, Pro14P and Asp15P, extend outside the binding site without forming any contacts with the Ab. The PC283-PS1 complex is among the few examples where the light chain complementarity-determining regions show more interactions than the heavy chain complementarity-determining regions, and a distal framework residue is involved in Ag binding. As seen from the crystal structure, most of the contacts between peptide and Ab are through the five residues, Leu3-Asp4-Pro5-Ala6-Phe7, of PS1. The paratope is predominantly hydrophobic with aromatic residues lining the binding pocket, although a salt bridge also contributes to stabilizing the Ag-Ab interaction. The molecular surface area buried upon PS1 binding is 756 Å2 for the peptide and 625 Å2 for the Fab, which is higher than what has been seen to date for Ab-peptide complexes. A comparison between PC283 structure and a homology model of its germline ancestor suggests that paratope optimization for PS1 occurs by improving both charge and shape complementarity.
The Transcription FactorBrightAssociates with Bruton’s Tyrosine Kinase, the Defective Protein in Immunodeficiency Disease
Authors: Webb Carol F. , Yamashita Yoshio , Ayers Neil , Evetts Seth , Paulin Yolene , Conley Mary Ellen , Smith Elizabeth A. 
Published: April 2014

AbstractBinding of the transcription factor Bright to Ig heavy chain loci after B cell activation is associated with increased heavy chain transcription. We now report that Bright coprecipitates with Bruton’s tyrosine kinase (Btk), the defective enzyme in X-linked immunodeficiency disease (xid). Furthermore, we observed Btk in the nucleus of activated murine B cells, and mobility shift assays suggest that it is a component of the Bright DNA-binding complex. While Bright protein was synthesized in activated spleen cells from xid mice, it did not bind DNA or associate stably with Btk. These data suggest that deficiencies in Bright DNA-binding activity may contribute to the defects in Ig production seen in xid mice.
Identification of Fetal Liver Tyrosine Kinase 3 (Flt3) Ligand Domain Required for Receptor Binding and Function Using Naturally Occurring Ligand Isoforms
Authors: Mwangi Waithaka , Brown Wendy C. , Palmer Guy H. 
Published: April 2014

AbstractWe used a comparative approach to identify the fetal liver tyrosine kinase 3 (flt3) ligand structure required for binding and function. Two conserved bovine flt3 ligand isoforms, which differ in a defined region within the extracellular domain, were identified and shown to be uniformly transcribed in individuals with diverse MHC haplotypes. Notably, at the amino acid level, the extracellular domain of the bovine flt3 ligand isoform 1 is 81 and 72% identical with the extracellular domains of the human and murine flt3 ligands, respectively, whereas isoform-2 has a deletion within this domain. Bovine flt3 ligand isoform 1, but not 2, bound the human flt3 receptor and stimulated murine pro B cells transfected with the murine flt3 receptor. This retention of binding and function allowed definition of key residues by identifying sequences conserved among species. We have shown that a highly conserved, 18 aa sequence within the flt3 ligand extracellular domain is required for flt3 receptor binding and function. However, a peptide representing this sequence is insufficient for receptor binding as demonstrated by its failure to inhibit the bovine flt3 ligand isoform 1 binding to the human flt3 receptor. The requirement for flanking structure was confirmed by testing bovine flt3 ligand isoform 1 constructs truncated at specific residues outside the 18 aa sequence. Overall, the flt3 ligand structure required for function is markedly similar to that of the related hemopoietic growth factors, CSF-1 and steel factor. This definition of the required flt3 ligand structure will facilitate development of agonists to enhance dendritic cell recruitment for vaccines and immunotherapy.
The MurineIL-2Promoter Contains Distal Regulatory Elements Responsive to the Ah Receptor, a Member of the Evolutionarily Conserved bHLH-PAS Transcription Factor Family
Authors: Jeon Myung-Shin , Esser Charlotte 
Published: April 2014

AbstractSignaling through the TCR and costimulatory signals primarily control transcription of the IL-2 gene in naive T cells. The minimal promoter necessary for this expression lies proximal, between −300 and the transcription start site. We had previously shown that activation of the arylhydrocarbon receptor (AHR), a member of the bHLH-PAS family of transcription factors, leads to increased mRNA expression of IL-2 in murine fetal thymocytes. The AHR is abundant in the thymus and may play a role for the development of the immune system. Moreover, its overactivation by chemicals such as dioxins leads to immunosuppression and thymic involution. Binding motifs for the liganded AHR can be identified in the distal region −1300 to −800 of the mouse IL-2 promoter. We show here that these DNA motifs, the so-called dioxin response elements, after binding to the liganded AHR are sufficient to transactivate luciferase expression in a reporter gene system. The IL-2 gene can be induced by the AHR also in thymocytes in vivo after injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent ligand of the AHR. The AHR mediates the IL-2 induction as shown with AHR-deficient mice. However, in spleen cells in vitro costimulation via the TCR is necessary for optimal IL-2 gene induction. Thus, the IL-2 promoter region contains novel distal regulatory elements that can be addressed by the AHR to induce IL-2 and can cooperate with the proximal promoter in this.
Conservation ofMhcClass III Region Synteny Between Zebrafish and Human as Determined by Radiation Hybrid Mapping
Authors: Sültmann Holger , Sato Akie , Murray Brent W. , Takezaki Naoko , Geisler Robert , Rauch Gerd-Jörg , Klein Jan 
Published: April 2014

AbstractIn the HLA, H2, and other mammalian Mhc, the class I and II loci are separated by the so-called class III region comprised of ∼60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the Mhc 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-Mhc that existed before the class I/II genes arose. The pre-Mhc may not have contained the complement and other class III genes involved in immune response.
Distinct T Cell Interactions with HLA Class II Tetramers Characterize a Spectrum of TCR Affinities in the Human Antigen-Specific T Cell Response
Authors: Reichstetter Sandra , Ettinger Ruth A. , Liu Andrew W. , Gebe John A. , Nepom Gerald T. , Kwok William W. 
Published: April 2014

AbstractThe polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16369–379-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16369–379 peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16369–379 tetramer at either 23 or 37°C. Weak staining was also observed at 4°C. Clone 5 showed weaker staining compared with clone 48 at 37°C, and no staining was observed at 23°C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16369–379 complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.
Antibody Repertoire Development in Fetal and Neonatal Piglets. II. Characterization of Heavy Chain Complementarity-Determining Region 3 Diversity in the Developing Fetus
Authors: Butler J. E. , Weber P. , Sinkora M. , Sun J. , Ford S. J. , Christenson R. K. 
Published: April 2014

AbstractSince the actual combinatorial diversity in the VH repertoire in fetal piglets represents &lt;1% of the potential in mice and humans, we wondered whether 1) complementarity-determining region 3 (CDR3) diversity was also restricted; 2) CDR3 diversity changed with fetal age; and 3) to what extent CDR3 contributed to the preimmune VDJ repertoire. CDR3 spectratyping and sequence analyses of 213 CDR3s recovered from &gt;30 fetal animals of different ages showed that &gt;95% of VDJ diversity resulted from junctional diversity. Unlike sheep and cattle, somatic hypermutation does not contribute to the repertoire. These studies also revealed that 1) N region additions are as extensive in VDJ rearrangements recovered at 30 days as those in late term fetuses, suggesting that TdT is fully active at the onset of VDJ rearrangement; 2) nearly 90% of all rearrangement are in-frame until late gestation; 3) the oligoclonal CDR3 spectratype of 30-day fetal liver becomes polyclonal by 50 days, while this change occurs much later in spleen; 4) there is little evidence of individual variation in CDR3 spectratype or differences in spectratype among lymphoid tissues with the exception of the thymus; and 4) there is a tendency for usage of the most JH proximal DH segment (DHB) to decrease in older fetuses and for the longer DH segment to be trimmed to the same length as the shorter DH when used in CDR3. These findings suggest that in the fetal piglet, highly restricted combinatorial diversity and the lack of somatic mutation are compensated by early onset of TdT activity and other mechanisms that contribute to CDR3 junctional diversity.
Delineation of the Human Systemic Lupus Erythematosus Anti-Smith Antibody Response Using Phage-Display Combinatorial Libraries
Authors: del Rincon Inmaculada , Zeidel Maria , Rey Elena , Harley John B. , James Judith A. , Fischbach Michael , Sanz Iñaki 
Published: April 2014

AbstractThe anti-Smith (Sm) autoantibody response is highly specific for systemic lupus erythematosus and is predominantly targeted to the Sm-B/B′ and -D1 polypeptides. In all animal species thus far studied, anti-Sm Abs initially recognize proline-rich epitopes in the carboxyl terminus of the Sm-B/B′ protein and subsequently to multiple other epitopes in B/B′ and D. The absence of appropriate mAbs has limited our understanding of the genetic and structural basis of this autoimmune response. Using phage-display technology and lymphocytes from a systemic lupus erythematosus patient we have generated the first and only panel of human IgG anti-Sm mAbs thus far available. These Abs reproduced to a remarkable extent the serological reactivity of the patient. Epitope mapping and genetic studies revealed that the anti-Sm response is produced by distinct B cell clones with restricted epitope reactivity. All of the Abs in our study were exclusively encoded by different members of the VH4 gene family. On the aggregate, our results demonstrate that combinatorial libraries can recapitulate the immune repertoire of peripheral blood B memory cells and that epitope spreading appears to occur through the sequential recruitment of nonclonally related autoreactive B cell clones.
Activation of MHC Class I, II, and CD40 Gene Expression by Histone Deacetylase Inhibitors
Authors: Magner William J. , Kazim A. Latif , Stewart Carleton , Romano Michelle A. , Catalano Geoffrey , Grande Catherine , Keiser Nicholas , Santaniello Frank , Tomasi Thomas B. 
Published: April 2014

AbstractEpigenetic mechanisms are involved in regulating chromatin structure and gene expression through repression. In this study, we show that histone deacetylase inhibitors (DAIs) that alter the acetylation of histones in chromatin enhance the expression of several genes on tumor cells including: MHC class I, II, and the costimulatory molecule CD40. Enhanced transcription results in a significant increase in protein expression on the tumor cell surface, and expression can be elicited on some tumors that are unresponsive to IFN-γ. The magnitude of induction of these genes cannot be explained by the effect of DAIs on the cell cycle or enhanced apoptosis. Induction of class II genes by DAIs was accompanied by activation of a repressed class II transactivator gene in a plasma cell tumor but, in several other tumor cell lines, class II was induced in the apparent absence of class II transactivator transcripts. These findings also suggest that the abnormalities observed in some tumors in the expression of genes critical to tumor immunity may result from epigenetic alterations in chromatin and gene regulation in addition to well-established mutational mechanisms.
Factors Controlling the Trafficking and Processing of a Leader-Derived Peptide Presented by Qa-1
Authors: Bai Ailin , Aldrich Carla J. , Forman James 
Published: April 2014

AbstractMany leader-derived peptides require TAP for presentation by class I molecules. This TAP dependence can either be ascribed to the inability of proteases resident in the endoplasmic reticulum (ER) to trim leader peptide precursors into the appropriate epitope or the failure of a portion of the leader segment to gain access to the lumen of the ER. Using the Qa-1 binding epitope, Qdm derived from a class Ia leader as a model, we show that many cell types lack ER protease activity to trim this peptide at its C terminus. However, both T1 and T2 cells contain appropriate protease activity to process the full length Dd leader (DL) when introduced into the ER lumen. Nevertheless, both T1 cells treated with the TAP inhibitor ICP47 and TAP− T2 cells fail to present this epitope from either the intact Dd molecule or a minigene encoding the DL. This indicates that the portion of the leader containing Qdm does not gain access to the ER. However, changing the Arg at P7 of the DL to a Cys can alter its trafficking and allows for TAP-independent presentation of the Qdm epitope.
HIV-1 Tat Represses Transcription from the Mannose Receptor Promoter
Authors: Caldwell Robert L. , Egan Brian S. , Shepherd Virginia L. 
Published: April 2014

AbstractThe mannose receptor is expressed on mature macrophages and immature dendritic cells, and functions to mediate phagocytosis of pathogens and capture of Ags for delivery to MHC class II-containing intracellular compartments. It has been previously reported that HIV-1-infected macrophages have reduced functions associated with the mannose receptor, including impaired Pneumocystis carinii phagocytosis and mannosylated albumin uptake. Several HIV-1-derived proteins including the Tat protein have been shown to transcriptionally repress host cell genes. The present study was undertaken to define the role of the HIV-1-derived protein Tat in HIV-mediated mannose receptor down-regulation. Cotransfection of the human macrophage cell line U937 with a Tat expression vector and a mannose receptor promoter-luciferase reporter construct resulted in down-regulation of mannose receptor promoter activity. This repression was targeted to the basal promoter. Expression of either one- or two-exon Tat resulted in decreased promoter activity. The addition of the transactivation response element (TAR) sequence enhanced the Tat-mediated repression. Down-regulation was also seen when transfected cells were treated with exogenously added Tat protein. These results are consistent with a mechanism whereby Tat reduces mannose receptor promoter activity by interfering with the host transcriptional initiation machinery, potentially resulting in decreased levels of surface mannose receptor available for Ag or pathogen capture.
Two Different Epitopes of the Signal Transducer gp130 Sequentially Cooperate on IL-6-Induced Receptor Activation
Authors: Pflanz Stefan , Kurth Ingo , Grötzinger Joachim , Heinrich Peter C. , Müller-Newen Gerhard 
Published: April 2014

Abstract
Cytokines are key mediators for the regulation of hemopoiesis and the coordination of immune responses. They exert their various functions through activation of specific cell surface receptors, thereby initiating intracellular signal transduction cascades which lead to defined cellular responses. As the common signal-transducing receptor subunit of at least seven different cytokines, gp130 is an important member of the family of hemopoietic cytokine receptors which are characterized by the presence of at least one cytokine-binding module. Mutants of gp130 that either lack the Ig-like domain D1 (ΔD1) or contain a distinct mutation (F191E) within the cytokine-binding module have been shown to be severely impaired with respect to IL-6 induced signal transduction. After cotransfection of COS-7 cells with a combination of both inactive gp130 mutants, signal transduction in response to IL-6 is restored. Whereas cells transfected with ΔD1 do not bind IL-6/sIL-6R complexes, cells transfected with the F191E mutant bind IL-6/sIL-6R with low affinity. Combination of ΔD1 and F191E, however, leads to high-affinity ligand binding. These data suggest that two different gp130 epitopes, one on each receptor chain, sequentially cooperate in asymmetrical binding of IL-6/IL-6R in a tetrameric signaling complex. On the basis of our data, a model for the mechanism of IL-6-induced gp130 activation is proposed.