Fc gamma RII/III and CD2 expression mark distinct subpopulations of immature CD4-CD8- murine thymocytes: in vivo developmental kinetics and T cell receptor beta chain rearrangement status.

PubMed

Rodewald, H R; Awad, K; Moingeon, P; D'Adamio, L; Rabinowitz, D; Shinkai, Y; Alt, F W; Reinherz, E L

1993-04-01

We have recently identified a dominant wave of CD4-CD8- (double-negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III-CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double-positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision

PubMed Central

Hale, J. Scott; Nelson, Lisa T.; Simmons, Kalynn B.; Fink, Pamela J.

2010-01-01

Peripheral CD4+Vβ5+ T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of post-revision CD4+Vβ5− T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the post-revision population. Here, we investigate the role of Bim-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4+ T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR expressing cells are TCR revision intermediates, and that the population of RAG-expressing, revising CD4+ T cells is increased in Bim deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the post-revision repertoire. PMID:21148799

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision.

PubMed

Hale, J Scott; Nelson, Lisa T; Simmons, Kalynn B; Fink, Pamela J

2011-01-15

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.

Atomic structure of an alphabeta T cell receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody.

PubMed Central

Wang, J; Lim, K; Smolyar, A; Teng, M; Liu, J; Tse, A G; Liu, J; Hussey, R E; Chishti, Y; Thomson, C T; Sweet, R M; Nathenson, S G; Chang, H C; Sacchettini, J C; Reinherz, E L

1998-01-01

Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule via a clonotypic alphabeta heterodimeric structure (Ti) non-covalently associated with the monomorphic CD3 signaling components. A crystal structure of an alphabeta TCR-anti-TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb), interacting with the elongated FG loop of the Cbeta domain, situated beneath the Vbeta domain. This loop, along with the partially exposed ABED beta sheet of Cbeta, and glycans attached to both Cbeta and Calpha domains, forms a cavity of sufficient size to accommodate a single non-glycosylated Ig domain such as the CD3epsilon ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre-TCR complexes. Furthermore, quaternary structures of TCRs vary significantly even when they bind the same MHC molecule, as manifested by a unique twisting of the V module relative to the C module. PMID:9427737

Energetic, Structural, and Antimicrobial Analyses of [beta]-Lactam Side Chain Recognition by [beta]-Lactamases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caselli, E.; Powers, R.A.; Blaszczak, L.C.

2010-03-05

Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these {beta}-lactams, most often through bacterial expression of {beta}-lactamases, threatens public health. To understand how {beta}-lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because {beta}-lactams form covalent adducts with {beta}-lactamases. This has complicated functional analyses and inhibitor design. To investigate the contribution to interaction energy of the key amide (R1) side chain of {beta}-lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well asmore » four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine {beta}-lactamases. Therefore, binding energies can be calculated directly from K{sub i} values. The K{sub i} values measured span four orders of magnitude against the Group I {beta}-lactamase AmpC and three orders of magnitude against the Group II {beta}-lactamase TEM-1. The acylglycineboronic acids have K{sub i} values as low as 20 nM against AmpC and as low as 390 nM against TEM-1. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of {beta}-lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of {beta}-lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to {beta}-lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray crystallography to 1.90 {angstrom} and 1.75 {angstrom} resolution; these structures suggest interactions that are important to the affinity of the inhibitors. Acylglycineboronic acids allow us to begin to dissect interaction energies between {beta}-lactam side chains and {beta

Rag Deletion in Peripheral T Cells Blocks TCR Revision

PubMed Central

Hale, J. Scott; Ames, Kristina T.; Boursalian, Tamar E.; Fink, Pamela J.

2010-01-01

Mature CD4+Vβ5+ T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or T cell receptor (TCR) revision. In Vβ5 transgenic mice, this latter tolerance pathway results in the appearance of CD4+Vβ5−TCRβ+ T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of Vβ-DJβ recombination intermediates in peripheral CD4+ T cells. Because post-thymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We now show that Rag deletion in post-positive selection T cells in Vβ5 transgenic mice blocks TCR revision in vivo, and that mature peripheral T cells sorted to remove cells bearing endogenous TCRβ chains can express newly generated TCRβ molecules in adoptive hosts. These findings unambiguously demonstrate post-thymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4+ T cells. PMID:20435935

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri).

PubMed

Islam, A; Persson, B; Zaidi, Z H; Jörnvall, H

1989-08-01

The primary structure of Rose-ringed Parakeet hemoglobin beta-chain was established, completing the analysis of this hemoglobin. Comparison with other avian beta-chains show variations smaller than those for the corresponding alpha-chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform beta-chain, and a total of 35 positions are affected by differences among all avian beta-chains analyzed (versus 61 for the alpha-chains). At three positions, the Psittacula beta-chain has residues unique to this species. Three alpha 1 beta 1 contacts are modified, by substitutions at positions beta 51, beta 116, and beta 125.

Monoclonal TCR-redirected tumor cell killing.

PubMed

Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

2012-06-01

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

Dysfunctional C8 beta chain in patients with C8 deficiency.

PubMed

Tschopp, J; Penea, F; Schifferli, J; Späth, P

1986-12-01

Two sera from unrelated individuals, each lacking C8 activity, were examined by Western blot analysis. Using antisera raised against whole C8, the two sera are shown to lack the C8 beta chain, indicating a C8 beta deficiency, which is frequently observed in cases of dysfunctional C8. In contrast, by means of a specific anti-C8-beta antiserum, a C8 beta-like polypeptide chain of apparently identical molecular weight compared to normal C8 beta was detected. Digestion of normal and dysfunctional C8 beta with Staphylococcus aureus V8 protease revealed distinct differences in the enzymatic digestion pattern. We conclude that the dysfunction in the C8 protein in these two patients resides in the dysfunctional C8 beta chain, and that this form of C8 deficiency is distinct from C8 deficiencies previously reported, in which one or both C8 subunits are lacking.

The T-cell receptor beta chain CDR3 region of BV8S1/BJ1S5 transcripts in type 1 diabetes.

PubMed

Naserke, H E; Durinovic-Bellò, I; Seidel, D; Ziegler, A G

1996-01-01

We recently described the T-cell receptor (TCR) beta chain CDR3 motif S-SDRLG-NQPQH (BV8S1-BJ1S5) in an islet-specific T-cell clone (K2.12) from a type 1 diabetic patient (AS). A similar motif (RLGNQ) was also reported in a T-cell clone of non-obese diabetic (NOD) mice by others. In order to determine the frequency of our motif in selected and unselected T-cell populations, we cloned and sequenced the CDR3 region of BV8S1-BJ1S5 transcripts. These transcripts were derived from unstimulated peripheral blood T lymphocytes from two type 1 diabetic patients (AS and FS) and their non-diabetic sibling (WS), as well as from an islet-specific T-cell line of one of the patients. In addition, we compared the structure and composition of the CDR3 region in BV8S1-BJ1S5 transcripts from peripheral blood T cells between the patients and their non-diabetic sibling (>50 sequences each). We found that 30% of the islet-specific T-cell line cDNA clones expressed the entire sequence-motif, whereas it was absent in the clones of unstimulated peripheral blood T cells from both patients and their non-diabetic sibling. The average length of the CDR3 region was shorter in the patients (mean AS 9.9, FS 9.9, versus WS 10.7, p = 0.0037) and the number of inserted nucleotides in N nucleotide addition at the DJ-junction lower (mean AS 3.5, FS 3. 2, versus WS 5.2, P = <10(-4)) as compared with their non-diabetic sibling. Moreover, the pattern of amino acid usage in the CDR3 region was dissimilar at positions 5 and 6, where polar amino acids predominated in both diabetic siblings. In contrast, basic amino acids are preferentially used at position 5 in the clones of the non-diabetic sibling. These data provide information on the general structure of the TCR(BV8S1-BJ1S5) CDR3 region in type 1 diabetes and may indicate differences in the amino and nucleic acid composition of the TCR beta chain CDR3 region between two type 1 diabetic patients and their non-diabetic sibling.

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

PubMed Central

Friend, Samantha F.; Peterson, Lisa K.; Kedl, Ross M.; Dragone, Leonard L.

2014-01-01

How T cell receptor (TCR) avidity influences CD8+ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP−/− mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP−/− Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8+ T cell development and repertoire selection. In comparing SLAP−/− OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP−/− Vβ5 mice. We have found that SLAP−/− OT-1 mice have fewer CD8+ thymocytes but have increased CD5 expression. SLAP−/− OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8+ splenocytes upon tetramer staining. Our data demonstrate that SLAP−/− Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8+ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8+ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8+ T cell development influences repertoire selection. PMID:22956467

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells.

PubMed

Friend, Samantha F; Peterson, Lisa K; Kedl, Ross M; Dragone, Leonard L

2013-03-01

How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vβ5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCRzeta for degradation.

PubMed

Myers, Margaret D; Dragone, Leonard L; Weiss, Arthur

2005-07-18

Src-like adaptor protein (SLAP) down-regulates expression of the T cell receptor (TCR)-CD3 complex during a specific stage of thymocyte development when the TCR repertoire is selected. Consequently, SLAP-/- thymocytes display alterations in thymocyte development. Here, we have studied the mechanism of SLAP function. We demonstrate that SLAP-deficient thymocytes have increased TCRzeta chain expression as a result of a defect in TCRzeta degradation. Failure to degrade TCRzeta leads to an increased pool of fully assembled TCR-CD3 complexes that are capable of recycling back to the cell surface. We also provide evidence that SLAP functions in a pathway that requires the phosphorylated TCRzeta chain and the Src family kinase Lck, but not ZAP-70 (zeta-associated protein of 70 kD). These studies reveal a unique mechanism by which SLAP contributes to the regulation of TCR expression during a distinct stage of thymocyte development.

Topological side-chain classification of beta-turns: ideal motifs for peptidomimetic development.

PubMed

Tran, Tran Trung; McKie, Jim; Meutermans, Wim D F; Bourne, Gregory T; Andrews, Peter R; Smythe, Mark L

2005-08-01

Beta-turns are important topological motifs for biological recognition of proteins and peptides. Organic molecules that sample the side chain positions of beta-turns have shown broad binding capacity to multiple different receptors, for example benzodiazepines. Beta-turns have traditionally been classified into various types based on the backbone dihedral angles (phi2, psi2, phi3 and psi3). Indeed, 57-68% of beta-turns are currently classified into 8 different backbone families (Type I, Type II, Type I', Type II', Type VIII, Type VIa1, Type VIa2 and Type VIb and Type IV which represents unclassified beta-turns). Although this classification of beta-turns has been useful, the resulting beta-turn types are not ideal for the design of beta-turn mimetics as they do not reflect topological features of the recognition elements, the side chains. To overcome this, we have extracted beta-turns from a data set of non-homologous and high-resolution protein crystal structures. The side chain positions, as defined by C(alpha)-C(beta) vectors, of these turns have been clustered using the kth nearest neighbor clustering and filtered nearest centroid sorting algorithms. Nine clusters were obtained that cluster 90% of the data, and the average intra-cluster RMSD of the four C(alpha)-C(beta) vectors is 0.36. The nine clusters therefore represent the topology of the side chain scaffold architecture of the vast majority of beta-turns. The mean structures of the nine clusters are useful for the development of beta-turn mimetics and as biological descriptors for focusing combinatorial chemistry towards biologically relevant topological space.

Analysis of the CDR3 length repertoire and the diversity of TCR alpha chain in human peripheral blood T lymphocytes.

PubMed

Yao, Xin Sheng; Diao, Ying; Sun, Wan Bang; Luo, Jun Min; Qin, Ming; Tang, Xian Ying

2007-06-01

Analysis of complementarity determining region 3 (CDR3) length of T lymphocyte receptors (TCRs) by immunoscope spectratyping technique has been used successfully to investigate the diversity of TCR in autoimmune diseases and infection diseases. In this study, we investigated the patterns of CDR3 length distribution for all 32 TCR AV gene families in human peripheral blood lymphocytes of four normal volunteers by the immunoscope spectratyping technique. It was found that PCR products exhibited an obscure band on 1.5% agarose gel electrophoresis. Each TCR AV family exhibited more than 8 bands on 6% sequencing gel electrophoresis. The CDR3 spectratyping of all TCR AV families showed a standard Gaussian distribution with different CDR3 length, and the expression frequency of CDR3 was similar among the gene families. Most of CDR3 in TCR AV family recombine in frame. However, some of the CDR3 showed out-of frame gene rearrangement. Additionally, we found that in some of TCR AV families there were 18 amino acid discrepancies between the longest CDR3 and shortest CDR3. These results may be helpful to further study the recombination mechanism of human TCR genes, the TCR CDR3 gene repertoire, and the repertoire drift in health people and disease state.

Immune selection of tumor cells in TCR β-chain transgenic mice.

PubMed

Silaeva, Yulia Yu; Grinenko, Tatyana S; Vagida, Murad S; Kalinina, Anastasia A; Khromykh, Ludmila M; Kazansky, Dmitry B

2014-10-01

The concept of immunological surveillance implies that immunogenic variants of tumor cells arising in the organism can be recognized by the immune system. Tumor progression is provided by somatic evolution of tumor cells under the pressure of the immune system. The loss of MHC Class I molecules on the surface of tumor cells is one of the most known outcomes of immune selection. This study developed a model of immune selection based on the immune response of TCR 1d1 single β-chain transgenic B10.D2(R101) (K(d)I(d)D(b)) mice to allogeneic EL4 (H-2(b)) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2K(b) molecule and results in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in mixed-lymphocyte response assays and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, CD8(+) T-lymphocytes with endogenous β-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic β-chains became CD8(+)CD44(+)CD62L(-) effectors. Then, instead of a full elimination of tumor cells as in wild-type mice, a reproducible prolonged equilibrium phase and subsequent escape was observed in transgenic mice that resulted in death of 90% of the mice in 40-60 days after grafting. Prolonged exposure of tumor cells to the pressure of the immune system in transgenic mice in vivo resulted in a stable loss of H-2K(b) molecules on the EL4 cell surface. Genetic manipulation of the T-lymphocyte repertoire was sufficient to reproduce the classic pattern of interactions between tumor cells and the immune system, usually observed in reliable syngeneic models of anti-tumor immunity. This newly-developed model could be used in further studies of immunoregulatory circuits common for transplantational and anti-tumor immune responses.

Reduced TCR signaling potential impairs negative selection but does not result in autoimmune disease

PubMed Central

Hwang, SuJin; Song, Ki-Duk; Lesourne, Renaud; Lee, Jan; Pinkhasov, Julia; Li, LiQi; El-Khoury, Dalal

2012-01-01

Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability. PMID:22945921

Radiation leukemia virus-induced thymic lymphomas express a restricted repertoire of T-cell receptor V beta gene products.

PubMed Central

Sen-Majumdar, A; Weissman, I L; Hansteen, G; Marian, J; Waller, E K; Lieberman, M

1994-01-01

We have investigated the phenotypic changes that take place during the process of neoplastic transformation in the thymocytes of C57BL/Ka mice infected by the radiation leukemia virus (RadLV). By the combined use of antibodies against the envelope glycoprotein gp70 of RadLV, the transformation-associated cell surface marker 1C11, and the CD3-T-cell receptor (TCR) complex, we found that in the RadLV-infected thymus, the earliest expression of viral gp70 is in 1C11hi cells; a small but significant percentage of these cells also express CD3. A first wave of viral replication, manifested by the expression of high levels of gp70 in thymocytes (over 70% positive), reaches a peak at 2 weeks; during this period, no significant changes are observed in the expression of 1C11 or CD3. The population of gp70+ cells is drastically reduced at 3 to 4 weeks after infection. However, a second cohort of gp70+ cells appears after 4 weeks, and these cells express high levels of 1C11 and TCR determinants as well. RadLV-induced lymphomas differ from normal thymocytes in their CD4 CD8 phenotype, with domination by one or more subsets. Characterization of TCR gene rearrangements in RadLV-induced lymphomas shows that most of these tumors are clonal or oligoclonal with respect to the J beta 2 TCR gene, while the J beta 1 TCR gene is rearranged in a minority (4 of 11) of lymphomas. TCR V beta repertoire analysis of 12 tumors reveals that 6 (50%) express exclusively the V beta 6 gene product, 2 (17%) are V beta 5+, and 1 (8%) each are V beta 8+ and V beta 9+. In normal C57BL/Ka mice, V beta 6 is expressed on 12%, V beta 5 is expressed on 9%, V beta 8 is expressed on 22%, and V beta 9 is expressed on 4% of TCRhi thymocytes. Thus, it appears that RadLV-induced thymic lymphomas are not randomly selected with respect to expressed TCR V beta type. Images PMID:8289345

[Application of the polymerase chain reaction (PCR) in the diagnosis of Hb S-beta(+)-thalassemia].

PubMed

Harano, K; Harano, T; Kushida, Y; Ueda, S

1991-08-01

Isoelectric focusing of the hemolysate prepared from a two-year-old American black boy with microcytic hypochromia showed the presence of a high percentage (63.3%) of such Hb variant as Hb S, while the levels of Hb A, Hb F and Hb A2 were 20.0%, 12.7%, and 4.0%, respectively. The ratio of the non-alpha-chain to the alpha-chain of the biosynthesized globin chains was 0.49. The variant was identified as Hb S by amino acid analysis of the abnormal peptide (beta T-1) and digestion of DNA amplified by the polymerase chain reaction with enzyme Eco 81 I. This was further confirmed by DNA sequencing. DNA sequencing of a beta-gene without the beta s-mutation revealed a nucleotide change of T to C in the polyadenylation signal sequence AATAAA 3' to the beta-gene, resulting in beta(+)-thalassemia. These results are consistent with the existence of a beta s-gene and a beta(+)-thalassemia gene in trans.

HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer.

PubMed

Foley, Kendra C; Spear, Timothy T; Murray, David C; Nagato, Kaoru; Garrett-Mayer, Elizabeth; Nishimura, Michael I

2017-06-16

T cell receptor (TCR)-gene-modified T cells for adoptive cell transfer can mediate objective clinical responses in melanoma and other malignancies. When introducing a second TCR, mispairing between the endogenous and introduced α and β TCR chains limits expression of the introduced TCR, which can result in impaired efficacy or off-target reactivity and autoimmunity. One approach to promote proper TCR chain pairing involves modifications of the introduced TCR genes: introducing a disulfide bridge, substituting murine for human constant regions, codon optimization, TCR chain leucine zipper fusions, and a single-chain TCR. We have introduced these modifications into our hepatitis C virus (HCV) reactive TCR and utilize a marker gene, CD34t, which allows us to directly compare transduction efficiency with TCR expression and T cell function. Our results reveal that of the TCRs tested, T cells expressing the murine Cβ2 TCR or leucine zipper TCR have the highest levels of expression and the highest percentage of lytic and interferon-γ (IFN-γ)-producing T cells. Our studies give us a better understanding of how TCR modifications impact TCR expression and T cell function that may allow for optimization of TCR-modified T cells for adoptive cell transfer to treat patients with malignancies.

Cutting edge: rescue of pre-TCR but not mature TCR signaling in mice expressing membrane-targeted SLP-76.

PubMed

Bezman, Natalie A; Baker, Rebecca G; Lenox, Laurie E; Jordan, Martha S; Koretzky, Gary A

2009-05-01

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) organizes signaling from immunoreceptors, including the platelet collagen receptor, the pre-TCR, and the TCR, and is required for T cell development. In this study we examine a mouse in which wild-type SLP-76 is replaced with a mutant constitutively targeted to the cell membrane. Membrane-targeted SLP-76 (MTS) supports ITAM signaling in platelets and from the pre-TCR. Signaling from the mature TCR, however, is defective in MTS thymocytes, resulting in failed T cell differentiation. Defective thymic selection by MTS is not rescued by a SLP-76 mutant whose localization is restricted to the cytosol. Thus, fixed localization of SLP-76 reveals differential requirements for the subcellular localization of signaling complexes downstream of the pre-TCR vs mature TCR.

Promyelocytic leukemia zinc finger turns on the effector T cell program without requirement for agonist TCR signaling.

PubMed

Savage, Adam K; Constantinides, Michael G; Bendelac, Albert

2011-05-15

Thymocytes expressing the NKT cell semi-invariant αβ TCR are thought to undergo agonist interactions with CD1d ligands prior to expressing promyelocytic leukemia zinc finger (PLZF), a broad complex, tramtrack, bric-a-brac, poxvirus, and zinc finger transcription factor that directs acquisition of the effector program of these innate-like T cells. Whether PLZF can mediate this effector conversion independently of agonist signaling has not been investigated. We demonstrated that transgenic (Tg) expression of PLZF under the CD4 promoter induced the innate effector program in two different MHC class II-restricted TCR-Tg Rag1(-/-) models examined. In CD4 thymocytes expressing a fixed Tg TCR β-chain, the associated TCRα sequences in wild-type and PLZF-Tg mice overlapped extensively, further demonstrating that PLZF could induce the effector program in most CD4 T cells that would normally be selected as naive cells. In contrast, PLZF altered the negative selection of thymocytes expressing TCR β-chains reactive against several retroviral superantigens. Thus, PLZF is remarkable in that it is a transcription factor capable of inducing an effector program in the absence of T cell agonist interactions or cell division. Its expression may also enhance the survival of agonist-signaled thymocytes.

PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

PubMed Central

Li, Qiongshu; Liu, Muyun; Wu, Man; Zhou, Xin; Wang, Shaobin; Hu, Yuan; Wang, Youfu; He, Yixin; Zeng, Xiaoping; Chen, Junhui; Liu, Qubo; Xiao, Dong; Hu, Xiang; Liu, Weibin

2018-01-01

Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and β-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/β-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRβ), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative

Differential regulation of peripheral CD4+ T cell tolerance induced by deletion and TCR revision.

PubMed

Ali, Mohamed; Weinreich, Michael; Balcaitis, Stephanie; Cooper, Cristine J; Fink, Pamela J

2003-12-01

In Vbeta5 transgenic mice, mature Vbeta5(+)CD4(+) T cells are tolerized upon recognition of a self Ag, encoded by a defective endogenous retrovirus, whose expression is confined to the lymphoid periphery. Cells are driven by the tolerogen to enter one of two tolerance pathways, deletion or TCR revision. CD4(+) T cells entering the former pathway are rendered anergic and then eliminated. In contrast, TCR revision drives gene rearrangement at the endogenous TCR beta locus and results in the appearance of Vbeta5(-), endogenous Vbeta(+), CD4(+) T cells that are both self-tolerant and functional. An analysis of the molecules that influence each of these pathways was conducted to understand better the nature of the interactions that control tolerance induction in the lymphoid periphery. These studies reveal that deletion is efficient in reconstituted radiation chimeras and is B cell, CD28, inducible costimulatory molecule, Fas, CD4, and CD8 independent. In contrast, TCR revision is radiosensitive, B cell, CD28, and inducible costimulatory molecule dependent, Fas and CD4 influenced, and CD8 independent. Our data demonstrate the differential regulation of these two divergent tolerance pathways, despite the fact that they are both driven by the same tolerogen and restricted to mature CD4(+) T cells.

Hb Molfetta [beta126(H4)Val-->Leu, GTG-->CTG]: a new, silent, neutral beta chain variant found in an Italian woman.

PubMed

Qualtieri, Antonio; Le, Pera Maria; Pedace, Vera; Magariello, Angela; Brancati, Carlo

2002-02-01

We have identified a new neutral hemoglobin variant in a pregnant Italian woman, that resulted from a GTG-->CTG replacement at codon 126 of the beta chain, corresponding to a Val-->Leu amino acid change at position beta126(H4). Thermal and isopropanol stability tests were normal and there were no abnormal clinical features. Routine electrophoretic and ion exchange chromatographic methods for hemoglobin separation failed to show this variant, but reversed phase high performance liquid chromatography revealed an abnormal peak eluting near the normal beta chain. No abnormal tryptic peptide was revealed on the high performance liquid chromatographic elution pattern of the total globin digest. The mutation was determined at the DNA level by amplification of the three beta exons by polymerase chain reaction and direct sequencing of one exon that showed an abnormal migration on single strand conformational polymorphism analysis.

TCR tuning of T cell subsets.

PubMed

Cho, Jae-Ho; Sprent, Jonathan

2018-05-01

After selection in the thymus, the post-thymic T cell compartments comprise heterogenous subsets of naive and memory T cells that make continuous T cell receptor (TCR) contact with self-ligands bound to major histocompatibility complex (MHC) molecules. T cell recognition of self-MHC ligands elicits covert TCR signaling and is particularly important for controlling survival of naive T cells. Such tonic TCR signaling is tightly controlled and maintains the cells in a quiescent state to avoid autoimmunity. Here, we review how naive and memory T cells are differentially tuned and wired for TCR sensitivity to self and foreign ligands. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

TCR repertoires of intratumoral T-cell subsets.

PubMed

Linnemann, Carsten; Mezzadra, Riccardo; Schumacher, Ton N M

2014-01-01

The infiltration of human tumors by T cells is a common phenomenon, and over the past decades, it has become increasingly clear that the nature of such intratumoral T-cell populations can predict disease course. Furthermore, intratumoral T cells have been utilized therapeutically in clinical studies of adoptive T-cell therapy. In this review, we describe how novel methods that are either based on T-cell receptor (TCR) sequencing or on cancer exome analysis allow the analysis of the tumor reactivity and antigen-specificity of the intratumoral TCR repertoire with unprecedented detail. Furthermore, we discuss studies that have started to utilize these techniques to probe the link between cancer exomes and the intratumoral TCR pool. Based on the observation that both the cancer epitope repertoire and intratumoral TCR repertoire appear highly individual, we outline strategies, such as 'autologous TCR gene therapy', that exploit the tumor-resident TCR repertoire for the development of personalized immunotherapy. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Increased myosin heavy chain-beta with atrial expression of ventricular light chain-2 in canine cardiomyopathy.

PubMed

Fuller, Geraldine A; Bicer, Sabahattin; Hamlin, Robert L; Yamaguchi, Mamoru; Reiser, Peter J

2007-10-01

Dilated cardiomyopathy is a naturally occurring disease in humans and dogs. Human studies have shown increased levels of myosin heavy chain (MHC)-beta in failing ventricles and the left atria (LA) and of ventricular light chain (VLC)-2 in the right atria in dilated cardiomyopathy. This study evaluates the levels of MHC-beta in all heart chambers in prolonged canine right ventricular pacing. In addition, we determined whether levels of VLC2 were altered in these hearts. Failing hearts demonstrated significantly increased levels of MHC-beta in the right atria, right atrial appendage, LA, left atrial appendage (LAA), and right ventricle compared with controls. Significant levels of VLC2 were detected in the right atria of paced hearts. Differences in MHC-beta expression were observed between the LA and the LAA of paced and control dogs. MHC-beta expression was significantly greater in the LA of paced and control dogs compared with their respective LAA. The cardiac myosin isoform shifts in this study were similar to those observed in end-stage human heart failure and more severe than those reported in less prolonged pacing models, supporting the use of this model for further study of end-stage human heart failure. The observation of consistent differences between sampling sites, especially LA versus LAA, indicates the need for rigorous sampling consistency in future studies.

Modulation of TCRβ surface expression during TCR revision.

PubMed

Simmons, Kalynn B; Wubeshet, Maramawit; Ames, Kristina T; McMahan, Catherine J; Hale, J Scott; Fink, Pamela J

2012-01-01

TCR revision is a tolerance mechanism by which self-reactive TCRs expressed by mature CD4(+) peripheral T cells are replaced by receptors encoded by genes generated by post-thymic DNA rearrangement. The downmodulation of surface TCR expression initiates TCR revision, and serves as a likely trigger for the induction of the recombinase machinery. We show here in a Vβ5 transgenic mouse model system that downregulation of the self-reactive transgene-encoded TCR is not maintained by transgene loss or diminished transcription or translation. The downregulation of surface TCR expression likely occurs in two stages, only one of which requires tolerogen expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex.

PubMed

Myers, Margaret D; Sosinowski, Tomasz; Dragone, Leonard L; White, Carmen; Band, Hamid; Gu, Hua; Weiss, Arthur

2006-01-01

The adaptor molecule SLAP and E3 ubiquitin ligase c-Cbl each regulate expression of T cell receptor (TCR)-CD3 on thymocytes. Here we provide genetic and biochemical evidence that both molecules function in the same pathway. TCR-CD3 expression was similar in the absence of SLAP and/or c-Cbl. SLAP and c-Cbl were found to interact, and their expression together downregulated CD3epsilon. This required multiple domains in SLAP and the ring finger of c-Cbl. Furthermore, expression of SLAP and c-Cbl together induced TCRzeta ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes. These studies indicate that SLAP links the E3 ligase activity of c-Cbl to the TCR, allowing for stage-specific regulation of TCR expression.

[Perissodactyla: the primary structure of hemoglobins from the lowland tapir (Tapirus terrestris): glutamic acid in position 2 of the beta chains].

PubMed

Mazur, G; Braunitzer, G

1984-09-01

The hemoglobins from a lowland tapir (Tapirus terrestris) were analysed and the complete primary structure is described. The globin chains were separated on CM cellulose column in 8M urea and the amino-acid sequences were determined in the liquid phase sequenator. The results show that globin consists of two alpha chains (alpha I and alpha II) and beta major and beta minor components. The alpha chains differ only at one position: alpha I contains aspartic acid and alpha II glycine. The beta chains are heterogeneous: aspartic and glutamic acid were found at position beta 21 and beta 73 of the beta major components and asparagine and serine at position beta 139. In the beta minor components four positions were found with more than one amino acid, namely beta 2, beta 4, beta 6 and beta 56. The sequences are compared with those of man, horse and rhinoceros. Four residues of horse methemoglobin, which are involved in the alpha 1 beta 1 contacts are substituted in tapir hemoglobins. In the alpha chains: alpha 107(G14)Ser----Val, alpha 111-(G18) Val----Leu, alpha 115(GH3) Asn----Asp or Gly; in the beta chains: beta 116(G18) Arg----Gln. The amino acid at beta 2 of the major components is glutamic acid while glutamine and histidine are found in the minor components. Although glutamic acid, a binding site for ATP, does not interact with 2,3-bisphosphoglycerate, glutamine and histidine in the minor components are responsible for the slight effect of 2,3-bisphosphoglycerate on tapir hemoglobin.

A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

PubMed Central

Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

2014-01-01

Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals.

PubMed

Zarin, Payam; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Zúñiga-Pflücker, Juan Carlos

2014-04-15

Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.

Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants.

PubMed

Xie, Gary; Forst, Christian; Bonner, Carol; Jensen, Roy A

2002-01-01

Tryptophan synthase consists of two subunits, alpha and beta. Two distinct subgroups of beta chain exist. The major group (TrpEb_1) includes the well-studied beta chain of Salmonella typhimurium. The minor group of beta chain (TrpEb_2) is most frequently found in the Archaea. Most of the amino-acid residues important for catalysis are highly conserved between both TrpE subfamilies. Conserved amino-acid residues of TrpEb_1 that make allosteric contact with the TrpEa subunit (the alpha chain) are absent in TrpEb_2. Representatives of Archaea, Bacteria and higher plants all exist that possess both TrpEb_1 and TrpEb_2. In those prokaryotes where two trpEb genes coexist, one is usually trpEb_1 and is adjacent to trpEa, whereas the second is trpEb_2 and is usually unlinked with other tryptophan-pathway genes. TrpEb_1 is nearly always partnered with TrpEa in the tryptophan synthase reaction. However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the functional beta chain, as TrpEb_1 is absent. The six lineages show a distinctive divergence within the overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in TrpEa that are otherwise conserved for interfacing with TrpEb_1. We suggest that the standalone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established catalytic capability of tryptophan synthase beta chains. A coincident finding of interest is that the Archaea seem to use the citramalate pathway, rather than threonine deaminase (IlvA), to initiate the pathway of isoleucine biosynthesis.

CD25 and CD69 induction by α4β1 outside-in signalling requires TCR early signalling complex proteins

PubMed Central

Cimo, Ann-Marie; Ahmed, Zamal; McIntyre, Bradley W.; Lewis, Dorothy E.; Ladbury, John E.

2013-01-01

Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4β1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4β1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation. PMID:23758320

CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells.

PubMed

Legut, Mateusz; Dolton, Garry; Mian, Afsar Ali; Ottmann, Oliver G; Sewell, Andrew K

2018-01-18

Adoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR-modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer-reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4 + and CD8 + T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies. © 2018 by The American Society of Hematology.

Selective amplification of T-cell receptor variable region species is demonstrable but not essential in early lesions of psoriasis vulgaris: analysis by anchored polymerase chain reaction and hypervariable region size spectratyping.

PubMed

Vekony, M A; Holder, J E; Lee, A J; Horrocks, C; Eperon, I C; Camp, R D

1997-07-01

Several groups have investigated the role of T cells in the pathogenesis of psoriasis by determination of T-cell receptor (TCR) B-chain variable (V) region usage, both in chronic plaque (psoriasis vulgaris) and guttate forms, with various results. Because there are no data on TCR expression in early psoriasis vulgaris, when specific cellular immune events may be expected to be most pronounced, we have analyzed early lesions (less than 3 wk old) of ten patients, with highly reproducible results. We have developed a highly controlled anchored polymerase chain reaction (PCR) method in which TCR beta chain species are all amplified with the same primer pair and products are quantified by dot blot hybridization with BV family-specific oligonucleotide probes. Overexpression of certain TCR BV genes was observed in the majority of lesional biopsies, but in samples in which the expanded BV family formed more than 10% of total lesional BV (half of the samples analyzed), BV2 and BV6 predominated. The consistency of overexpression of these BV species between patients was much less than in previous studies of TCRBV usage in established chronic plaque psoriasis lesions. Complementarity-determining region 3 (CDR3) size spectratyping demonstrated evidence for selective clonal T cell accumulation in less than half of the lesional samples showing BV expansion. These results indicate that selective amplification of TCRBV species occurs in early psoriasis vulgaris but is not essential to the pathogenic process and may be more important in the maintenance or expansion of chronic lesions.

Differential TCR signals for T helper cell programming.

PubMed

Morel, Penelope A

2018-05-02

Upon encounter with their cognate antigen naïve CD4 T cells become activated and are induced to differentiate into several possible T helper (Th) cell subsets. This differentiation depends on a number of factors including antigen presenting cells, cytokines and costimulatory molecules. The strength of the T cell receptor (TCR) signal, related to the affinity of TCR for antigen and antigen dose, has emerged as a dominant factor in determining Th cell fate. Recent studies have revealed that TCR signals of high or low strength do not simply induce quantitatively different signals in the T cells, but rather qualitatively distinct pathways can be induced based on TCR signal strength. This review examines the recent literature in this area and highlights important new developments in our understanding of Th cell differentiation and TCR signal strength. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Organization of the resting TCR in nanoscale oligomers.

PubMed

Schamel, Wolfgang W A; Alarcón, Balbino

2013-01-01

Despite the low affinity of the T-cell antigen receptor (TCR) for its peptide/major histocompatibility complex (pMHC) ligand, T cells are very sensitive to their antigens. This paradox can be resolved if we consider that the TCR may be organized into pre-existing oligomers or nanoclusters. Such structures could improve antigen recognition by increasing the functional affinity (avidity) of the TCR-pMHC interaction and by allowing cooperativity between individual TCRs. Up to approximately 20 TCRs become tightly apposed in these nanoclusters, often in a linear manner, and such structures could reflect a relatively generalized phenomenon: the non-random concentration of membrane receptors in specific areas of the plasma membrane known as protein islands. The association of TCRs into nanoclusters can explain the enhanced kinetics of the pMHC-TCR interaction in two dimensional versus three dimensional systems, but also their existence calls for a revision of the TCR triggering models based on pMHC-induced TCR clustering. Interestingly, the B-cell receptor and the FcεRI have also been shown to form nanoclusters, suggesting that the formation of pre-existing receptor oligomers could be widely used in the immune system. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paessler, Slobodan; Yun, Nadezhda E.; Judy, Barbara M.

2007-10-25

We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta ({alpha}{beta}) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta ({gamma}{delta}) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chainmore » and a minority of vaccinated immunoglobulin heavy chain-deficient ({mu}MT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3{sup +} T cells are required for protection.« less

Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg.

PubMed

Stockis, Julie; Colau, Didier; Coulie, Pierre G; Lucas, Sophie

2009-12-01

Human Treg and Th clones secrete the latent form of TGF-beta, in which the mature TGF-beta protein is bound to the latency-associated peptide (LAP), and is thereby prevented from binding to the TGF-beta receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF-beta and bear LAP on their surface. Here, we show that latent TGF-beta, i.e. both LAP and mature TGF-beta, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF-beta mediated by binding to GARP may be necessary for the ability of Treg to activate TGF-beta upon TCR stimulation. However, it is not sufficient as lentiviral-mediated expression of GARP in human Th cells induces binding of latent TGF-beta to the cell surface, but does not result in the production of active TGF-beta upon stimulation of these Th cells.

Alternative splicing produces transcripts coding for alpha and beta chains of a hetero-dimeric phosphagen kinase.

PubMed

Ellington, W Ross; Yamashita, Daisuke; Suzuki, Tomohiko

2004-06-09

Glycocyamine kinase (GK) catalyzes the reversible phosphorylation of glycocyamine (guanidinoacetate), a reaction central to cellular energy homeostasis in certain animals. GK is a member of the phosphagen kinase enzyme family and appears to have evolved from creatine kinase (CK) early in the evolution of multi-cellular animals. Prior work has shown that GK from the polychaete Neanthes (Nereis) diversicolor exits as a hetero-dimer in vivo and that the two polypeptide chains (termed alpha and beta) are coded for by unique transcripts. In the present study, we demonstrate that the GK from a congener Nereis virens is also hetero-dimeric and is coded for by alpha and beta transcripts, which are virtually identical to the corresponding forms in N. diversicolor. The GK gene from N. diversicolor was amplified by PCR. Sequencing of the PCR products showed that the alpha and beta chains are the result of alternative splicing of the GK primary mRNA transcript. These results also strongly suggest that this gene underwent an early tandem exon duplication event. Full-length cDNAs for N. virens GKalpha and GKbeta were individually ligated into expression vectors and the resulting constructs used to transform Escherichia coli expression hosts. Regardless of expression conditions, minimal GK activity was observed in both GKalpha and GKbeta constructs. Inclusion bodies for both were harvested, unfolded in urea and alpha chains, beta chains and mixtures of alpha and beta chains were refolded by sequential dialysis. Only modest amounts of GK activity were observed when alpha and beta were refolded individually. In contrast, when refolded the alpha and beta mixture yielded highly active hetero-dimers, as validated by size exclusion chromatography, electrophoresis and mass spectrometry, with a specific activity comparable to that of natural GK. The above evidence suggests that there is a preference for hetero-dimer formation in the GKs from these two polychaetes. The evolution of the

Mushroom acidic glycosphingolipid induction of cytokine secretion from murine T cells and proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nozaki, Hirofumi; Itonori, Saki; Sugita, Mutsumi

2008-08-29

Interferon (IFN)-{gamma} and interleukin (IL)-4 regulate many types of immune responses. Here we report that acidic glycosphingolipids (AGLs) of Hypsizigus marmoreus and Pleurotus eryngii induced secretion of IFN- {gamma} and IL-4 from T cells in a CD11c-positive cell-dependent manner similar to that of {alpha}-galactosylceramide ({alpha}-GalCer) and isoglobotriaosylceramide (iGb3), although activated T cells by AGLs showed less secretion of cytokine than those activated by {alpha}-GalCer. In addition, stimulation of these mushroom AGLs induced proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in splenocytes. Administration of a mixture of {alpha}-GalCer and AGLs affected the stimulation of {alpha}-GalCer and generally induced a subtle Th1more » bias for splenocytes but induced an extreme Th2 bias for thymocytes. These results suggested that edible mushroom AGLs contribute to immunomodulation.« less

Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection

PubMed Central

Paessler, Slobodan; Yun, Nadezhda E.; Judy, Barbara M.; Dziuba, Natallia; Zacks, Michele A.; Grund, Anna H.; Frolov, Ilya; Campbell, Gerald A.; Weaver, Scott C.; Estes, D. Mark

2007-01-01

We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta (αβ) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta ( γδ) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chain and a minority of vaccinated immunoglobulin heavy chain-deficient (μMT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3+ T cells are required for protection. PMID:17610927

Proline substitution independently enhances H-2D(b) complex stabilization and TCR recognition of melanoma-associated peptides.

PubMed

Uchtenhagen, Hannes; Abualrous, Esam T; Stahl, Evi; Allerbring, Eva B; Sluijter, Marjolein; Zacharias, Martin; Sandalova, Tatyana; van Hall, Thorbald; Springer, Sebastian; Nygren, Per-Åke; Achour, Adnane

2013-11-01

The immunogenicity of H-2D(b) (D(b)) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D(b) in complex with an optimized version of the melanoma-associated epitope gp10025-33 . Furthermore, the p3P modification directly increased the affinity of the D(b)/gp10025-33 -specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D(b)/gp10025-33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

PubMed Central

Xia, Zhen; Chen, Huabiao; Kang, Seung-gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

2014-01-01

Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function. PMID:24522437

The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

NASA Astrophysics Data System (ADS)

Xia, Zhen; Chen, Huabiao; Kang, Seung-Gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

2014-02-01

Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.

T-cell receptor accessory and co-receptor molecules in channel catfish

USDA-ARS?s Scientific Manuscript database

T cell receptor (TCR) associated invariant chains CD3gamma/delta,epsilon, and zeta as well as TCR co-receptors CD8alpha and CD8beta were isolated from the channel catfish, Ictalurus punctatus, at both the gene and cDNA levels. All of catfish CD3 sequences encode for proteins that resemble their resp...

High-throughput identification of antigen-specific TCRs by TCR gene capture.

PubMed

Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia; Kluin, Roelof J C; Bolotin, Dmitriy A; Chen, Xiaojing; Bresser, Kaspar; Nieuwland, Marja; Schotte, Remko; Michels, Samira; Gomez-Eerland, Raquel; Jahn, Lorenz; Hombrink, Pleun; Legrand, Nicolas; Shu, Chengyi Jenny; Mamedov, Ilgar Z; Velds, Arno; Blank, Christian U; Haanen, John B A G; Turchaninova, Maria A; Kerkhoven, Ron M; Spits, Hergen; Hadrup, Sine Reker; Heemskerk, Mirjam H M; Blankenstein, Thomas; Chudakov, Dmitriy M; Bendle, Gavin M; Schumacher, Ton N M

2013-11-01

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

PubMed Central

Williams, Chad M.; Schonnesen, Alexandra A.; Zhang, Shu-Qi; Ma, Ke-Yue; He, Chenfeng; Yamamoto, Tori; Eckhardt, S. Gail; Klebanoff, Christopher A.; Jiang, Ning

2017-01-01

The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously

Inverted repeats in the promoter as an autoregulatory sequence for TcrX in Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Monolekha; Das, Amit Kumar, E-mail: amitk@hijli.iitkgp.ernet.in

Highlights: Black-Right-Pointing-Pointer The regulatory sequences recognized by TcrX have been identified. Black-Right-Pointing-Pointer The regulatory region comprises of inverted repeats segregated by 30 bp region. Black-Right-Pointing-Pointer The mode of binding of TcrX with regulatory sequence is unique. Black-Right-Pointing-Pointer In silico TcrX-DNA docked model binds one of the inverted repeats. Black-Right-Pointing-Pointer Both phosphorylated and unphosphorylated TcrX binds regulatory sequence in vitro. -- Abstract: TcrY, a histidine kinase, and TcrX, a response regulator, constitute a two-component system in Mycobacterium tuberculosis. tcrX, which is expressed during iron scarcity, is instrumental in the survival of iron-dependent M. tuberculosis. However, the regulator of tcrX/Y has notmore » been fully characterized. Crosslinking studies of TcrX reveal that it can form oligomers in vitro. Electrophoretic mobility shift assays (EMSAs) show that TcrX recognizes two regions in the promoter that are comprised of inverted repeats separated by {approx}30 bp. The dimeric in silico model of TcrX predicts binding to one of these inverted repeat regions. Site-directed mutagenesis and radioactive phosphorylation indicate that D54 of TcrX is phosphorylated by H256 of TcrY. However, phosphorylated and unphosphorylated TcrX bind the regulatory sequence with equal efficiency, which was shown with an EMSA using the D54A TcrX mutant.« less

Identification and characterization of TCRgamma and TCRdelta chains in channel catfish, Ictalurus punctatus

USDA-ARS?s Scientific Manuscript database

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) gamma and delta were identified by mining of expressed sequence tag databases and full length sequences were obtained by 5'-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), (diversity; D), joining ...

Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

PubMed Central

Hemmers, Saskia; Schizas, Michail; Faire, Mehlika B.; Konopacki, Catherine; Schmidt-Supprian, Marc; Germain, Ronald N.

2017-01-01

The regulatory T cell (T reg cell) T cell receptor (TCR) repertoire is highly diverse and skewed toward recognition of self-antigens. TCR expression by T reg cells is continuously required for maintenance of immune tolerance and for a major part of their characteristic gene expression signature; however, it remains unknown to what degree diverse TCR-mediated interactions with cognate self-antigens are required for these processes. In this study, by experimentally switching the T reg cell TCR repertoire to a single T reg cell TCR, we demonstrate that T reg cell function and gene expression can be partially uncoupled from TCR diversity. An induced switch of the T reg cell TCR repertoire to a random repertoire also preserved, albeit to a limited degree, the ability to suppress lymphadenopathy and T helper cell type 2 activation. At the same time, these perturbations of the T reg cell TCR repertoire led to marked immune cell activation, tissue inflammation, and an ultimately severe autoimmunity, indicating the importance of diversity and specificity for optimal T reg cell function. PMID:28130403

PU.1 regulates TCR expression by modulating GATA-3 activity

PubMed Central

Chang, Hua-Chen; Han, Ling; Jabeen, Rukhsana; Carotta, Sebastian; Nutt, Stephen L.; Kaplan, Mark H.

2009-01-01

The Ets transcription factor PU.1 is a master regulator for the development of multiple lineages during hematopoiesis. The expression pattern of PU.1 is dynamically regulated during early T lineage development in the thymus. We previously revealed that PU.1 delineates heterogeneity of effector Th2 populations. In this study, we further define the function of PU.1 on the Th2 phenotype using mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1lck-/-). While deletion of PU.1 by the lck-Cre transgene does not affect T cell development, Sfpi1lck-/- T cells have a lower activation threshold than wild type T cells. When TCR engagement is limiting, Sfpi1lck-/- T cells cultured in Th2 polarizing conditions secrete higher levels of Th2 cytokines and have greater cytokine homogeneity than wild type cells. We show that PU.1 modulates the levels of TCR expression in CD4+ T cells by regulating the DNA-binding activity of GATA-3 and limiting GATA-3 regulation of TCR gene expression. GATA-3 dependent regulation of TCR expression is also observed in Th1 and Th2 cells. In CD4+ T cells, PU.1 expression segregates into subpopulations of cells that have lower levels of surface TCR, suggesting that PU.1 contributes to the heterogeneity of TCR expression. Thus, we have identified a mechanism whereby increased GATA-3 function in the absence of the antagonizing activity of PU.1 leads to increased TCR expression, a reduced activation threshold and increased homogeneity in Th2 populations. PMID:19801513

Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

NASA Technical Reports Server (NTRS)

Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.;

Characterization and membrane organization of beta 1----3- and beta 1----4-galactosyltransferases from human colonic adenocarcinoma cell lines Colo 205 and SW403: basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures.

PubMed

Holmes, E H

1989-05-01

Evidence indicates that activation of a beta 1----3N-acetylglucosaminyltransferase is responsible for accumulation of large quantities of lacto-series tumor-associated antigens in human colonic adenocarcinomas. Expression of type 1 and 2 core chain derivatives characterize human colonic adenocarcinomas, whereas normal adult colonic epithelial cells express detectable quantities of only type 1 chain derivatives. The basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures characteristic of normal colonic mucosa and human colonic adenocarcinoma Colo 205 cells has been studied. The beta 1----3- and beta 1----4galactosyltransferase enzymes associated with synthesis of type 1 and 2 core chain structures, respectively, have been separated from a Triton X-100 solubilized membrane fraction of Colo 205 cells by chromatography on an alpha-lactalbumin-Sepharose column and their properties studied. Optimal transfer of beta 1----3-linked galactose to acceptor Lc3 occurred in the presence of 0.1% Triton CF-54 with Triton X-100 providing 75% of maximal activity. The enzyme was active over a broad pH range from 6.5 to 7.5 and had a near absolute requirement for Mn2+. The Km values for donor UDPgalactose and acceptor Lc3 were determined to be 48 and 13 microM, respectively. In contrast, the beta 1----4galactosyltransferase required taurodeoxycholate for maximal activity and the Km for Lc3 was found to be 20-fold higher than that for the beta 1----3-specific enzyme under the same assay conditions. Studies with membrane-bound beta 1----3- and beta 1----4galactosyltransferases as found in Golgi-rich membrane fractions of SW403 and Colo 205 adenocarcinoma cells showed that preferential synthesis of type 1 chain structures occurs under conditions similar to those in vivo for biosynthesis of lacto-series core chains. The results suggest that both the higher affinity of the beta 1----3galactosyltransferase for acceptor Lc3 and the membrane organizational

Evolution of the antigen-specific CD8+ TCR repertoire across the life span: evidence for clonal homogenization of the old TCR repertoire.

PubMed

Rudd, Brian D; Venturi, Vanessa; Davenport, Miles P; Nikolich-Zugich, Janko

2011-02-15

Defects in T cell responses against pathogens and reduced diversity of TCRs have been described at both extremes of the life span. Yet, we still lack information on how Ag-specific T cell populations are maintained and/or altered from birth to old age. In this study, for the first time to our knowledge, we provide insight into Ag-specific TCR repertoire changes over the life span at the single-cell level. We have examined the TCR diversity of the primary CD8(+) T cell response to the immunodominant HSV-1 epitope HSV glycoprotein B 495-502 (HSV gB(498-505); SSIEFARL) (gB-8p) in neonatal, adult, and old C57BL/6 mice. The global distinctive features of the gB-8p-specific TCR repertoire were preserved in mice of different ages. However, both old and especially neonatal mice exhibited significant decreases in TCR diversity compared with that of adult mice. Still, although the neonatal Ag-specific repertoire comprised expectedly shorter germline-biased CDR3β lengths, the repertoire was surprisingly complex, and only a minority of responding cells lacked random nucleotide additions. Changes with aging included increased use of the already dominant TCRVβ10 family, a trend for lower content of the TCR containing the germline WG motif in the CDR3, and a remarkable sharing of one dominant clonotype between individual old mice, implying operation of selective mechanisms. Implications for the rational design of vaccines for neonates and the elderly are discussed.

BCR CDR3 length distributions differ between blood and spleen and between old and young patients, and TCR distributions can be used to detect myelodysplastic syndrome

NASA Astrophysics Data System (ADS)

Pickman, Yishai; Dunn-Walters, Deborah; Mehr, Ramit

2013-10-01

Complementarity-determining region 3 (CDR3) is the most hyper-variable region in B cell receptor (BCR) and T cell receptor (TCR) genes, and the most critical structure in antigen recognition and thereby in determining the fates of developing and responding lymphocytes. There are millions of different TCR Vβ chain or BCR heavy chain CDR3 sequences in human blood. Even now, when high-throughput sequencing becomes widely used, CDR3 length distributions (also called spectratypes) are still a much quicker and cheaper method of assessing repertoire diversity. However, distribution complexity and the large amount of information per sample (e.g. 32 distributions of the TCRα chain, and 24 of TCRβ) calls for the use of machine learning tools for full exploration. We have examined the ability of supervised machine learning, which uses computational models to find hidden patterns in predefined biological groups, to analyze CDR3 length distributions from various sources, and distinguish between experimental groups. We found that (a) splenic BCR CDR3 length distributions are characterized by low standard deviations and few local maxima, compared to peripheral blood distributions; (b) healthy elderly people's BCR CDR3 length distributions can be distinguished from those of the young; and (c) a machine learning model based on TCR CDR3 distribution features can detect myelodysplastic syndrome with approximately 93% accuracy. Overall, we demonstrate that using supervised machine learning methods can contribute to our understanding of lymphocyte repertoire diversity.

TCR revision generates functional CD4+ T cells.

PubMed

Hale, J Scott; Wubeshet, Maramawit; Fink, Pamela J

2010-12-01

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes, driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ, and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, postrevision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host.

Expression and function of CD8 alpha/beta chains on rat and human mast cells.

PubMed

Kim, Mi-Sun; Kim, Sung-Hoon; Lee, Hye-Jung; Kim, Hyung-Min

2004-03-01

The expression and functional role of CD8 glycoprotein, a marker of cytotoxic/suppressor T lymphocytes and NK cells, were not studied on freshly isolated connective tissue type rat peritoneal mast cells, a rat mucosal type mast cell line (RBL 2H3), or human mast cell line (HMC-1). We used the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, immunohistochemistry and enzyme-linked immunosorbent assay. RT-PCR and Western blot analysis identified the presence of CD8 alpha/beta chains on the mast cells, and immunohistochemistry confirmed CD8alpha expression on rat or human mast cells. Functional studies demonstrated that stimulation of CD8 alpha/beta chains on rat mast cells induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), which are regarded as important mediators during infection. However, co-stimulation with stem cell factor had no effect on CD8-induced mediator secretion. Our findings demonstrate novel biological roles of CD8 molecules in mast cells.

Ikaros promotes rearrangement of TCR alpha genes in an Ikaros null thymoma cell line

PubMed Central

Collins, Bernard; Clambey, Eric T.; Scott-Browne, James; White, Janice; Marrack, Philippa; Hagman, James; Kappler, John W.

2014-01-01

Summary Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4+CD8+ thymocyte using an Ikaros null CD4−CD8− mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR β gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry non-functional rearrangements on both alleles of their TCR α loci. Retroviral re-introduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αβTCR+ cells. The process is RAG dependent, requires SWI/SNF chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/NuRD complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αβTCR+ CD4+CD8+ thymocyte transition. PMID:23172374

Ikaros promotes rearrangement of TCR α genes in an Ikaros null thymoma cell line.

PubMed

Collins, Bernard; Clambey, Eric T; Scott-Browne, James; White, Janice; Marrack, Philippa; Hagman, James; Kappler, John W

2013-02-01

Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4(+) CD8(+) thymocyte using an Ikaros null CD4(-) CD8(-) mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR β gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry nonfunctional rearrangements on both alleles of their TCR α loci. Retroviral reintroduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αβTCR(+) cells. The process is RAG dependent, requires switch/sucrose nonfermentable chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/nucleosome remodeling and deacetylase complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αβTCR(+) CD4(+) CD8(+) thymocyte transition. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteolytic processing of the pro beta chain of beta-hexosaminidase occurs at basic residues contained within an exposed disulfide loop structure.

PubMed

Sagherian, C; Poroszlay, S; Vavougios, G; Mahuran, D

1993-01-01

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes, Hex A (alpha beta) and Hex B (beta beta). The alpha and beta subunits are encoded by the HEXA and HEXB genes, respectively. Extensive homology in both the gene structures and deduced primary sequences demonstrate their common evolutionary origin. While undergoing similar proteolytic modifications in the lysosome, the pro beta polypeptide is additionally cleaved internally to produce the mature 24-30 kilodalton beta b and beta a chains. Previous data have suggested that this processing event occurs somewhere between residues Ser311 and Lys315. In this report we demonstrate that this area is located in a hydrophilic disulfide-loop structure (between Cys309 and Cys360). The cleavage event is prevented by the deletion through in vitro mutagenesis of the Arg312-Gln-Asn-Lys tetrapeptide or by its substitution with the aligned alpha residues (Gly-Ser-Glu-Pro). Reintroduction of either Arg312 or Lys315 reinstates the processing. Furthermore, we show that this area is not involved in lysosomal targeting of pro-Hex B, or in the increased stability or the variation in substrate specificity of the beta as compared with the alpha subunit. Our data suggest the presence of a novel lysosomal endoprotease. Like other endoproteases it is specific for basic amino acids; however, it cleaves on the amino-terminal side rather than the conventional carboxy-terminal side of such residues and then only if they are fully exposed to the lysosomal environment.(ABSTRACT TRUNCATED AT 250 WORDS)

TCR-independent CD28-mediated gene expression in peripheral blood lymphocytes from donors chronically infected with HIV-1.

PubMed Central

Wong, J G; Smithgall, M D; Haffar, O K

1997-01-01

Complete activation of peripheral blood T cells requires both T-cell receptor (TCR) stimulation and CD28 costimulation. Signalling pathways associated specifically with CD28 are not well understood, however, because ligation of CD28 in the absence of TCR stimulation does not give rise to cellular responses in normal cells. In peripheral blood lymphocytes (PBL) from donors chronically infected with human immunodeficiency virus-1 (HIV-1), CD28 can induce viral replication through an alternative pathway that does not require TCR ligation. We have exploited this observation to study CD28-mediated signal transduction using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) to amplify viral RNA. Independent ligation of CD28 on donor PBL induced expression of the HIV-1 tat gene but not the interleukin-2 (IL-2) gene. Viral induction did not occur following pretreatment of cells with actinomycin D, suggesting it was mediated through transcriptional activation of the viral long terminal repeat (LTR). tat was induced in the presence of the protein kinase C inhibitor H-7, but was inhibited by cyclosporin A. Our results demonstrate that CD28 is linked directly to specific signalling pathways leading to de novo induction of genes in PBL. Images Figure 1 Figure 2 Figure 3 PMID:9135558

Purification and characterization of an amidohydrolase for N4-long-chain fatty acyl derivatives of 1-beta-D-arabinofuranosylcytosine from mouse liver microsomes.

PubMed

Hori, K; Tsuruo, T; Tsukagoshi, S; Sakurai, Y

1984-03-01

N4-Long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase, a metabolizing enzyme for N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with long-chain fatty acids, was purified from mouse liver microsomes. The purification was accomplished by solubilization of liver microsomes with Triton X-100, diethylaminoethyl cellulose chromatography, gel filtrations, hydroxyapatite chromatography, and concanavalin A:Sepharose chromatography. On sodium dodecyl sulfate:polyacrylamide gel electrophoresis, the purified enzyme preparation produced a single protein band with a molecular weight of 54,000. The enzyme had an optimal pH of 9.0, and the Michaelis constant for N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was 67 microM. The thiols such as dithiothreitol or 2-mercaptoethanol stabilized the enzyme and stimulated its activity. p-Chloromercuribenzoate, N-ethylmaleimide, diisopropylfluorophosphate, and phenylmethylsulfonyl fluoride strongly inhibited the reaction. Bovine serum albumin markedly stimulated the enzyme activity, whereas detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate had little effect. The enzyme did not require monovalent or divalent cations. Among the series of N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with different chain lengths of acyl residues, the purified enzyme preferentially hydrolyzed the derivatives with long-chain fatty acids (C12 to C18), and N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was the most susceptible. The purified enzyme was inactive on various N-acylamino acids, amides, oligopeptides, proteins, N-acylsphingosines (ceramides), triglyceride, lecithin, and lysolecithin. These results suggest that N4-long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase may be a new type of linear amidase.

A Co-Receptor Independent Transgenic Human TCR Mediates Anti-Tumor and Anti-Self Immunity in Mice

PubMed Central

Mehrotra, Shikhar; Al-Khami, Amir A.; Klarquist, Jared; Husain, Shahid; Naga, Osama; Eby, Jonathan M.; Murali, Anuradha K.; Lyons, Gretchen E.; Li, Mingli; Spivey, Natali D.; Norell, Håkan; Martins da Palma, Telma; Onicescu, Georgiana; Diaz-Montero, C. Marcela; Garrett-Mayer, Elizabeth; Cole, David J.; Le Poole, I. Caroline; Nishimura, Michael I.

2013-01-01

Recent advancements in T cell immunotherapy suggest that T cells engineered with high affinity T cell receptors (TCR) can offer better tumor regression. However, whether a high affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope reactive, CD8 independent, high affinity TCR isolated from MHC class-I restricted CD4+ T cells obtained from tumor infiltrating lymphocytes of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2 restricted TCR was positively selected on both CD4+ and CD8+ single-positive (SP) cells. However, when the TCR transgenic mouse was developed with an HLA-A2 background, the transgenic TCR was primarily expressed by CD3+CD4-CD8- double-negative (DN) T cells. TIL 1383I TCR transgenic CD4+, CD8+ and CD4-CD8- T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2+/human tyrosinase TCR double transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high affinity TIL 1383I TCR alone in CD3+ T cells is sufficient to control the growth of murine and human melanoma and the presence or absence of CD4 and CD8 co-receptors had little effect on its functional capacity. PMID:22798675

Thy-1+ dendritic epidermal cells express T3 antigen and the T-cell receptor gamma chain.

PubMed Central

Stingl, G; Koning, F; Yamada, H; Yokoyama, W M; Tschachler, E; Bluestone, J A; Steiner, G; Samelson, L E; Lew, A M; Coligan, J E

1987-01-01

The murine epidermis is a heterogeneous epithelium composed of keratinocytes, melanocytes, Langerhans cells, and a recently described subpopulation (2-3%) of bone-marrow-derived leukocytes with a dendritic morphology and the cell surface phenotype Thy-1+, L3T4-, Lyt-2-. Previous studies have demonstrated that cell lines derived from freshly explanted Thy-1+ dendritic epidermal cells (DEC) have abundant mRNA for rearranged T-cell receptor (TCR) gamma-chain genes. Analysis of Thy-1+ DEC in situ, freshly isolated cell suspensions of Thy-1+ DEC, and long-term Thy-1+ DEC lines demonstrated that 100% of the Thy-1+ DEC reacted with a monoclonal antibody to the epsilon chain of the murine T3 complex and that 40-60% of resident Thy-1+ DEC were also reactive with an antiserum to the TCR gamma chain. Two Thy-1+ DEC lines expressed a disulfide-linked 70-kDa molecule that could be precipitated with an anti-gamma-chain antiserum and could be coprecipitated with an antiserum to the T3 delta chain; the molecule appeared as a single 34-kDa band under reducing conditions. The phenotype of Thy-1+ DEC (T3+, L3T4-, Lyt-2-, TCR gamma chain+) thus resembles that of the recently described subpopulation of murine and human lymphocytes that have been identified in the thymus, peripheral blood, and fetal blood. Images PMID:2885839

Pontin is required for pre-TCR signaling at the β-selection checkpoint in T cell development.

PubMed

Boo, Kyungjin; Baek, Sung Hee; Lee, Ho

2014-04-25

Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Based on high expression in lymphoid tissues, we examined whether Pontin has a T cell-specific function. We generated Pontin(f/f);Lck-Cre mice, in which Pontin can be conditionally deleted in T cells and then explored T cell-specific function of Pontin in vivo. Here, we show that specific abrogation of Pontin expression in T cells almost completely blocked development of αβ T cells at the β-selection checkpoint by inducing cell apoptosis indicating that Pontin is essential for early T cell development. Pontin-deficient thymocytes show a comparable expression level of T cell receptor (TCR)β chain, but have enhanced activation of p53 and Notch signaling compared to wild-type thymocytes. Intriguingly, the developmental block of αβ T cells can be partially rescued by loss of p53. Together, our data demonstrate a novel role of Pontin as a crucial regulator in pre-TCR signaling during T cell development. Copyright © 2014 Elsevier Inc. All rights reserved.

TCR revision generates functional CD4+ T cells1

PubMed Central

Hale, J. Scott; Wubeshet, Maramawit; Fink, Pamela J.

2010-01-01

CD4+Vβ5+ peripheral T cells in B6 mice respond to encounter with a peripherally-expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes, driving surface expression of novel TCRs. While post-revision CD4+Vβ5−TCRβ+ T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli, and thus may benefit the host. We now demonstrate that post-revision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, post-revision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of post-revision cells is not driven by the transgene-encoded receptor. Post-revision cells proliferate extensively to commensal bacterial Ags and can generate I-Ab-restricted responses to Ag by producing IFNγ following Listeria monocytogenes challenge. These data show that rescued post-revision T cells are responsive to homeostatic signals and recognize self and foreign peptides in the context of self MHC, and are thus useful to the host. PMID:20971922

A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer

PubMed Central

Jahn, Lorenz; Hagedoorn, Renate S.; van der Steen, Dirk M.; Hombrink, Pleun; Kester, Michel G.D.; Schoonakker, Marjolein P.; de Ridder, Daniëlle; van Veelen, Peter A.; Falkenburg, J.H. Frederik; Heemskerk, Mirjam H.M.

2016-01-01

CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy. PMID:27689397

A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer.

PubMed

Jahn, Lorenz; Hagedoorn, Renate S; van der Steen, Dirk M; Hombrink, Pleun; Kester, Michel G D; Schoonakker, Marjolein P; de Ridder, Daniëlle; van Veelen, Peter A; Falkenburg, J H Frederik; Heemskerk, Mirjam H M

2016-11-01

CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy.

Identification of functional domains within the alpha and beta subunits of beta-hexosaminidase A through the expression of alpha-beta fusion proteins.

PubMed

Tse, R; Wu, Y J; Vavougios, G; Hou, Y; Hinek, A; Mahuran, D J

1996-08-20

There are three human beta-hexosaminidase isozymes which are composed of all possible dimeric combinations of an alpha and/or a beta subunit; A (alpha beta), and B (beta beta), and S (alpha alpha). The amino acid sequences of the two subunits are 60% identical. The homology between the two chains varies with the middle > the carboxy-terminal > > the amino-terminal portions. Although dimerization is required for activity, each subunit contains its own active site and differs in its substrate specificity and thermal stability. The presence of the beta subunit in hexosaminidase A also influences the substrate specificity of the alpha subunit; e.g., in vivo only the A heterodimer can hydrolyze GM2 ganglioside. In this report, we localize functional regions in the two subunits by cellular expression of alpha/beta fusion proteins joined at adjacently aligned residues. First, a chimeric alpha/beta chain was made by replacing the least well-conserved amino-terminal section of the beta chain with the corresponding alpha section. The biochemical characteristics of this protein were nearly identical to hexosaminidase B. Therefore, the most dissimilar regions in the subunits are not responsible for their dissimilar biochemical properties. A second fusion protein was made that also included the more homologous middle section of the alpha chain. This protein expressed the substrate specificity unique to isozymes containing an alpha subunit (A and S). We conclude that the region responsible for the ability of the alpha subunit to bind negatively charged substrates is located within residues alpha 132-283. Interestingly, the remaining carboxy-terminal section from the beta chain, beta 316-556, was sufficient to allow this chimera to hydrolyze GM2 ganglioside with 10% the specific activity of heterodimeric hexosaminidase A. Thus, the carboxy-terminal section of each subunit is likely involved in subunit-subunit interactions.

Allele related mutation specific-polymerase chain reaction for rapid diagnosis of Hb New York (beta 113 (G15) Val-->Glu, beta(CD113 GTG-->GAG)).

PubMed

Viprakasit, Vip; Tachavanich, Kalaya; Suwantol, Lerlugsn; Pung-Amritt, Parichat; Chinchang, Worawut; Tanphaichitr, Voravarn S

2002-08-01

Hemoglobin New York (beta 113 (G15) Val-->Glu), a beta-globin variant, was first reported in a Chinese family living in New York. Subsequently, this abnormal hemoglobin was reported in many Chinese descendants from several groups and it was also known as Hb Kaohsiung. The subtle change in alpha1beta1 contact region apart from the heme group connecting area by Val-->Glu substitution has minor changes in both the electrophoretic mobility and stability making this hemoglobin variant difficult to distinguish from Hb A using routine hemoglobin analysis. The authors described a case of heterozygosity of Hb New York diagnosed by a molecular technique and revealed a mutation in beta(CD113 GTG-->GAG). A novel Allele Related Mutation Specific-Polymerase Chain Reaction (ARMS-PCR) for rapid diagnosis of this mutation has been proposed.

BetaSCPWeb: side-chain prediction for protein structures using Voronoi diagrams and geometry prioritization

PubMed Central

Ryu, Joonghyun; Lee, Mokwon; Cha, Jehyun; Laskowski, Roman A.; Ryu, Seong Eon; Kim, Deok-Soo

2016-01-01

Many applications, such as protein design, homology modeling, flexible docking, etc. require the prediction of a protein's optimal side-chain conformations from just its amino acid sequence and backbone structure. Side-chain prediction (SCP) is an NP-hard energy minimization problem. Here, we present BetaSCPWeb which efficiently computes a conformation close to optimal using a geometry-prioritization method based on the Voronoi diagram of spherical atoms. Its outputs are visual, textual and PDB file format. The web server is free and open to all users at http://voronoi.hanyang.ac.kr/betascpweb with no login requirement. PMID:27151195

CD8+ TCR repertoire formation is guided primarily by the peptide component of the antigenic complex.

PubMed

Koning, Dan; Costa, Ana I; Hoof, Ilka; Miles, John J; Nanlohy, Nening M; Ladell, Kristin; Matthews, Katherine K; Venturi, Vanessa; Schellens, Ingrid M M; Borghans, Jose A M; Kesmir, Can; Price, David A; van Baarle, Debbie

2013-02-01

CD8(+) T cells recognize infected or dysregulated cells via the clonotypically expressed αβ TCR, which engages Ag in the form of peptide bound to MHC class I (MHC I) on the target cell surface. Previous studies have indicated that a diverse Ag-specific TCR repertoire can be beneficial to the host, yet the determinants of clonotypic diversity are poorly defined. To better understand the factors that govern TCR repertoire formation, we conducted a comprehensive clonotypic analysis of CD8(+) T cell populations directed against epitopes derived from EBV and CMV. Neither pathogen source nor the restricting MHC I molecule were linked with TCR diversity; indeed, both HLA-A and HLA-B molecules were observed to interact with an overlapping repertoire of expressed TRBV genes. Peptide specificity, however, markedly impacted TCR diversity. In addition, distinct peptides sharing HLA restriction and viral origin mobilized TCR repertoires with distinct patterns of TRBV gene usage. Notably, no relationship was observed between immunodominance and TCR diversity. These findings provide new insights into the forces that shape the Ag-specific TCR repertoire in vivo and highlight a determinative role for the peptide component of the peptide-MHC I complex on the molecular frontline of CD8(+) T cell-mediated immune surveillance.

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response.

PubMed

Mukhopadhyay, Himadri; de Wet, Ben; Clemens, Lara; Maini, Philip K; Allard, Jun; van der Merwe, P Anton; Dushek, Omer

2016-04-26

Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors.

PubMed Central

Blom, B; Heemskerk, M H; Verschuren, M C; van Dongen, J J; Stegmann, A P; Bakker, A Q; Couwenberg, F; Res, P C; Spits, H

1999-01-01

Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint. PMID:10329625

T follicular helper and T follicular regulatory cells have different TCR specificity

PubMed Central

Maceiras, Ana Raquel; Almeida, Silvia Cristina Paiva; Mariotti-Ferrandiz, Encarnita; Chaara, Wahiba; Jebbawi, Fadi; Six, Adrien; Hori, Shohei; Klatzmann, David; Faro, Jose; Graca, Luis

2017-01-01

Immunization leads to the formation of germinal centres (GCs) that contain both T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Whether T-cell receptor (TCR) specificity defines the differential functions of Tfh and Tfr cells is unclear. Here we show that antigen-specific T cells after immunization are preferentially recruited to the GC to become Tfh cells, but not Tfr cells. Tfh cells, but not Tfr cells, also proliferate efficiently on restimulation with the same immunizing antigen in vitro. Ex vivo TCR repertoire analysis shows that immunization induces oligoclonal expansion of Tfh cells. By contrast, the Tfr pool has a TCR repertoire that more closely resembles that of regulatory T (Treg) cells. Our data thus indicate that the GC Tfh and Tfr pools are generated from distinct TCR repertoires, with Tfh cells expressing antigen-responsive TCRs to promote antibody responses, and Tfr cells expressing potentially autoreactive TCRs to suppress autoimmunity. PMID:28429709

Cutting Edge: TCR Revision Affects Predominantly Foxp3− Cells and Skews Them toward the Th17 Lineage1

PubMed Central

Zehn, Dietmar; Bevan, Michael J.; Fink, Pamela J.

2009-01-01

CD4+ T cells respond to peripheral endogenous superantigen stimulation by undergoing deletion or TCR revision. The latter involves RAG re-expression, TCR gene rearrangement, and expression of a novel TCR. TCR-revised T cells are functional and express a diverse TCR repertoire. Because TCR revision harbors the potential to create self-reactivity, it is important to explore whether T cells known to be self-reactive (regulatory T cells) or those involved in autoimmunity (Th17 cells) arise from TCR revision. Interestingly, we observed that Foxp3+ cells are excluded from revising their TCR and that only a small fraction of postrevision cells expresses Foxp3. In contrast, Th17 cells are 20 times more frequent among revised than among C57BL/6 CD4+ T cells, indicating that postrevision cells are biased toward the Th17 lineage. The link between Th17 differentiation and TCR revision might be highly relevant to the role of Th17 cells in promoting autoimmunity. PMID:17947636

Intrathymic selection of NK1.1+α/β T cell antigen receptor (TCR)+ cells in transgenic mice bearing TCR specific for chicken ovalbumin and restricted to I-Ad

PubMed Central

Iwabuchi, Chikako; Iwabuchi, Kazuya; Nakagawa, Ken-ichi; Takayanagi, Toshiaki; Nishihori, Hiroki; Tone, Saori; Ogasawara, Kazumasa; Good, Robert A.; Onoé, Kazunori

1998-01-01

Generation and negative selection of NK1.1+α/β T cell receptor (TCR)+ thymocytes were analyzed using TCR-transgenic (B10.D2 × DO10)F1 and (C57BL/6 × DO10)F1 mice and Rag-1−/−/DO10 mice, which had been established by breeding and backcrossing between Rag-1−/− and DO10 mice. Almost all T cells from these mice were shown to bear Vα13/Vβ8.2 that is specific for chicken ovalbumin (cOVA) and restricted to I-Ad. A normal proportion of the NK1.1+ Vα13/Vβ8.2+ thymocytes was generated in these mice. However, the actual cell number of both NK1.1+ and NK1.1− thymocytes in I-Ad/d mice (positive selecting background) was larger than that in I-Ab/d mice (negative selecting background). Markedly low but significant proportions of NK1.1+ Vα13/Vβ8.2+ cells were detected in the spleens from I-Ad/d and I-Ab/d mice. It was shown that the splenic NK1.1+ T cells of the I-Ab/d mice were anergized against stimulation through TCR. When (B10.D2 × DO10)F1 and (C57BL/6 × DO10)F1 mice were given cOVA, extensive or intermediate elimination of NK1.1+α/βTCR+ thymocytes was induced in I-Ad/d or I-Ab/d mice, respectively. However, the clonal elimination was not as complete as that seen in the major NK1.1− thymocyte population. The present findings indicate that normal generation of NK1.1+α/βTCR+ thymocytes occurs in the absence of Vα14-Jα281 and that substantial negative selection operates on the NK1.1+α/βTCR+ cells. PMID:9653164

Conformation of methyl beta-lactoside bound to the ricin B-chain: Interpretation of transferred nuclear Overhauser effects facilitated by spin simulation and selective deuteration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bevilacqua, V.L.; Thomson, D.S.; Prestegard, J.H.

1990-06-12

Spin simulation and selective deuteration have been used to aid in the interpretation of 1D transferred nuclear Overhauser effect (TRNOE) NMR experiments on ricin B-chain/ligand systems. Application of these methods has revealed a change in the conformation of deuterated methyl beta-lactoside upon binding to the ricin B-chain which results in a slight change in glycosidic torsional angels which appear to dominate in the solution conformation. The combination of simulation and experiment also shows an important sensitivity of TRNOE magnitudes to dissociation rate constants and available spin-diffusion pathways for the ricin B-chain/ligand systems under study. The sensitivity to dissociation rates allowsmore » determination of rate constants for methyl beta-lactoside and methyl beta-galactoside of 50 and 300 s-1, respectively.« less

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

PubMed Central

Ghosh, D; Weeks, C M; Grochulski, P; Duax, W L; Erman, M; Rimsay, R L; Orr, J C

1991-01-01

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. Images PMID:1946424

Motion of spin label side chains in cellular retinol-binding protein: correlation with structure and nearest-neighbor interactions in an antiparallel beta-sheet.

PubMed

Lietzow, Michael A; Hubbell, Wayne L

2004-03-23

A goal in the development of site-directed spin labeling in proteins is to correlate the motion of a nitroxide side chain with local structure, interactions, and dynamics. Significant progress toward this goal has been made using alpha-helical proteins of known structure, and the present study is the first step in a similar exploration of a beta-sheet protein, cellular retinol-binding protein (CRBP). Nitroxide side chains were introduced along both interior and edge strands. At sites in interior strands, the side-chain motion is strongly influenced by interactions with side chains of neighboring strands, giving rise to a rich variety of dynamic modes (weakly ordered, strongly ordered, immobilized) and complex electron paramagnetic resonance spectra that are modulated by strand twist. The interactions giving rise to the dynamic modes are explored using mutagenesis, and the results demonstrate the particular importance of the non-hydrogen-bonded neighbor residue in giving rise to highly ordered states. Along edge strands of the beta-sheet, the motion of the side chain is simple and weakly ordered, resembling that at solvent-exposed surfaces of an alpha-helix. A simple working model is proposed that can account for the wide variety of dynamic modes encountered. Collectively, the results suggest that the nitroxide side chain is an effective probe of side-chain interactions, and that site-directed spin labeling should be a powerful means of monitoring conformational changes that involve changes in beta-sheet topology.

T-cell receptor variable genes and genetic susceptibility to celiac disease: an association and linkage study.

PubMed

Roschmann, E; Wienker, T F; Gerok, W; Volk, B A

1993-12-01

Genetic susceptibility of celiac disease is primarily associated with a particular combination of and HLA-DQA1/DQB1 gene; however, this does not fully account for the genetic predisposition. Therefore, the aim of this study was to examine whether T-cell receptor (TCR) genes may be susceptibility genes in celiac disease. HLA class II typing was performed by polymerase chain reaction amplification in combination with sequence-specific oligonucleotide hybridization. TCR alpha (TCRA), TCR gamma (TCRG), and TCR beta (TCRB) loci were investigated by restriction fragment length polymorphism analysis. Allelic frequencies of TCRA, TCRG, and TCRB variable genes were compared between patients with celiac disease (n = 53) and control patients (n = 67), and relative risk (RR) estimates were calculated. The RR was 1.67 for allele C1 at TCRA1, 3.35 for allele D2 at TCRA2, 1.66 for allele B2 at TCRG, and 1.35 for allele B at TCRB, showing no significant association. Additionally, linkage analysis was performed in 23 families. The logarithm of odd scores for celiac disease vs. the TCR variable genes at TCRA, TCRG, and TCRB showed no significant linkage. These data suggest that the analyzed TCR variable gene segments V alpha 1.2, V gamma 11, and V beta 8 do not play a major role in susceptibility to celiac disease.

BetaSCPWeb: side-chain prediction for protein structures using Voronoi diagrams and geometry prioritization.

PubMed

Ryu, Joonghyun; Lee, Mokwon; Cha, Jehyun; Laskowski, Roman A; Ryu, Seong Eon; Kim, Deok-Soo

2016-07-08

Many applications, such as protein design, homology modeling, flexible docking, etc. require the prediction of a protein's optimal side-chain conformations from just its amino acid sequence and backbone structure. Side-chain prediction (SCP) is an NP-hard energy minimization problem. Here, we present BetaSCPWeb which efficiently computes a conformation close to optimal using a geometry-prioritization method based on the Voronoi diagram of spherical atoms. Its outputs are visual, textual and PDB file format. The web server is free and open to all users at http://voronoi.hanyang.ac.kr/betascpweb with no login requirement. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Energetic and flexibility properties captured by long molecular dynamics simulations of a membrane-embedded pMHCII-TCR complex.

PubMed

Bello, Martiniano; Correa-Basurto, José

2016-04-01

Although crystallographic data have provided important molecular insight into the interactions in the pMHC-TCR complex, the inherent features of this structural approach cause it to only provide a static picture of the interactions. While unbiased molecular dynamics simulations (UMDSs) have provided important information about the dynamic structural behavior of the pMHC-TCR complex, most of them have modeled the pMHC-TCR complex as soluble, when in physiological conditions, this complex is membrane bound; therefore, following this latter UMDS protocol might hamper important dynamic results. In this contribution, we performed three independent 300 ns-long UMDSs of the pMHCII-TCR complex anchored in two opposing membranes to explore the structural and energetic properties of the recognition of pMHCII by the TCR. The conformational ensemble generated through UMDSs was subjected to clustering and Cartesian principal component analyses (cPCA) to explore the dynamical behavior of the pMHCII-TCR association. Furthermore, based on the conformational population sampled through UMDSs, the effective binding free energy, per-residue free energy decomposition, and alanine scanning mutations were explored for the native pMHCII-TCR complex, as well as for 12 mutations (p1-p12MHCII-TCR) introduced in the native peptide. Clustering analyses and cPCA provide insight into the rocking motion of the TCR onto pMHCII, together with the presence of new electrostatic interactions not observed through crystallographic methods. Energetic results provide evidence of the main contributors to the pMHC-TCR complex formation as well as the key residues involved in this molecular recognition process.

Hepatic beta-oxidation and carnitine palmitoyltransferase I in neonatal pigs after dietary treatments of clofibric acid, isoproterenol, and medium-chain triglycerides.

PubMed

Peffer, Pasha Lyvers; Lin, Xi; Odle, Jack

2005-06-01

A suckling piglet model was used to study nutritional and pharmacologic means of stimulating hepatic fatty acid beta-oxidation. Newborn pigs were fed milk diets containing either long- or medium-chain triglycerides (LCT or MCT). The long-chain control diet was supplemented further with clofibric acid (0.5%) or isoproterenol (40 ppm), and growth was monitored for 10-12 days. Clofibrate increased rates of hepatic peroxisomal and mitochondrial beta-oxidation of [1-(14)C]-palmitate by 60 and 186%, respectively. Furthermore, malonyl-CoA sensitive carnitine palmitoyltransferase (CPT I) activity increased 64% (P < 0.05) in pigs receiving clofibrate. Increased CPT I activity was not congruent with changes in message, as elevated abundance of CPT I mRNA was not detected (P = 0.16) when assessed by qRT-PCR. Neither rates of beta-oxidation nor CPT activities were affected by dietary MCT or by isoproterenol treatment (P > 0.1). Collectively, these findings indicate that clofibrate effectively induced hepatic CPT activity concomitant with increased fatty acid beta-oxidation.

Absence of Fc epsilonRI alpha chain results in upregulation of Fc gammaRIII-dependent mast cell degranulation and anaphylaxis. Evidence of competition between Fc epsilonRI and Fc gammaRIII for limiting amounts of FcR beta and gamma chains.

PubMed Central

Dombrowicz, D; Flamand, V; Miyajima, I; Ravetch, J V; Galli, S J; Kinet, J P

1997-01-01

In mouse mast cells, both Fc epsilonRI and Fc gammaRIII are alpha beta gamma2 tetrameric complexes in which different alpha chains confer IgE or IgG ligand recognition while the signaling FcR beta and gamma chains are identical. We used primarily noninvasive techniques (changes in body temperature, dye extravasation) to assess systemic anaphylactic responses in nonanesthetized wild-type, Fc epsilonRI alpha chain -/- and FcR gamma chain -/- mice. We confirm that systemic anaphylaxis in mice can be mediated largely through IgG1 and Fc gammaRIII and we provide direct evidence that these responses reflect activation of Fc gammaRIII rather than Fc gammaRI. Furthermore, we show that Fc gammaRIII-dependent responses are more intense in normal than in congenic mast cell-deficient KitW/KitW-v mice, indicating that Fc gammaRIII responses have mast cell-dependent and -independent components. Finally, we demonstrate that the upregulation of cell surface expression of Fc gammaRIII seen in Fc epsilonRI alpha chain -/- mice corresponds to an increased association of Fc gammaRIII alpha chains with FcR beta and gamma chains and is associated with enhanced Fc gammaRIII-dependent mast cell degranulation and systemic anaphylactic responses. Therefore, the phenotype of the Fc epsilonRI alpha chain -/- mice suggests that expression of Fc epsilonRI and Fc gammaRIII is limited by availability of the FcR beta and gamma chains and that, in normal mice, changes in the expression of one receptor (Fc epsilonRI) may influence the expression of functional responses dependent on the other (Fc gammaRIII). PMID:9062349

Cutting edge: spontaneous development of IL-17-producing gamma delta T cells in the thymus occurs via a TGF-beta 1-dependent mechanism.

PubMed

Do, Jeong-su; Fink, Pamela J; Li, Lily; Spolski, Rosanne; Robinson, Janet; Leonard, Warren J; Letterio, John J; Min, Booki

2010-02-15

In naive animals, gammadelta T cells are innate sources of IL-17, a potent proinflammatory cytokine mediating bacterial clearance as well as autoimmunity. However, mechanisms underlying the generation of these cells in vivo remain unclear. In this study, we show that TGF-beta1 plays a key role in the generation of IL-17(+) gammadelta T cells and that it mainly occurs in the thymus particularly during the postnatal period. Interestingly, IL-17(+) gammadelta TCR(+) thymocytes were mainly CD44(high)CD25(low) cells, which seem to derive from double-negative 4 gammadelta TCR(+) cells that acquired CD44 and IL-17 expression. Our findings identify a novel developmental pathway during which IL-17-competent gammadelta T cells arise in the thymus by a TGF-beta1-dependent mechanism.

Partial deletion of beta9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates.

PubMed

Dridi, Kaouthar; Amara, Sawsan; Bezzine, Sofiane; Rodriguez, Jorge A; Carrière, Frédéric; Gaussier, Hélène

2013-07-01

Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and beta loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of beta9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the beta9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of beta9 loop (GPLRP2-deltabeta9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-deltabeta9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within beta9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-deltabeta9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions.

Hamiltonian Markov Chain Monte Carlo Methods for the CUORE Neutrinoless Double Beta Decay Sensitivity

NASA Astrophysics Data System (ADS)

Graham, Eleanor; Cuore Collaboration

2017-09-01

The CUORE experiment is a large-scale bolometric detector seeking to observe the never-before-seen process of neutrinoless double beta decay. Predictions for CUORE's sensitivity to neutrinoless double beta decay allow for an understanding of the half-life ranges that the detector can probe, and also to evaluate the relative importance of different detector parameters. Currently, CUORE uses a Bayesian analysis based in BAT, which uses Metropolis-Hastings Markov Chain Monte Carlo, for its sensitivity studies. My work evaluates the viability and potential improvements of switching the Bayesian analysis to Hamiltonian Monte Carlo, realized through the program Stan and its Morpho interface. I demonstrate that the BAT study can be successfully recreated in Stan, and perform a detailed comparison between the results and computation times of the two methods.

TCR backscattering characterization for microwave remote sensing

NASA Astrophysics Data System (ADS)

Riccio, Giovanni; Gennarelli, Claudio

2014-05-01

A Trihedral Corner Reflector (TCR) is formed by three mutually orthogonal metal plates of various shapes and is a very important scattering structure since it exhibits a high monostatic Radar Cross Section (RCS) over a wide angular range. Moreover it is a handy passive device with low manufacturing costs and robust geometric construction, the maintenance of its efficiency is not difficult and expensive, and it can be used in all weather conditions (i.e., fog, rain, smoke, and dusty environment). These characteristics make it suitable as reference target and radar enhancement device for satellite- and ground-based microwave remote sensing techniques. For instance, TCRs have been recently employed to improve the signal-to-noise ratio of the backscattered signal in the case of urban ground deformation monitoring [1] and dynamic survey of civil infrastructures without natural corners as the Musmeci bridge in Basilicata, Italy [2]. The region of interest for the calculation of TCR's monostatic RCS is here confined to the first quadrant containing the boresight direction. The backscattering term is presented in closed form by evaluating the far-field scattering integral involving the contributions related to the direct illumination and the internal bouncing mechanisms. The Geometrical Optics (GO) laws allow one to determine the field incident on each TCR plate and the patch (integration domain) illuminated by it, thus enabling the use of a Physical Optics (PO) approximation for the corresponding surface current densities to consider for integration on each patch. Accordingly, five contributions are associated to each TCR plate: one contribution is due to the direct illumination of the whole internal surface; two contributions originate by the impinging rays that are simply reflected by the other two internal surfaces; and two contributions are related to the impinging rays that undergo two internal reflections. It is useful to note that the six contributions due to the

Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane.

PubMed

James, Scott E; Greenberg, Philip D; Jensen, Michael C; Lin, Yukang; Wang, Jinjuan; Till, Brian G; Raubitschek, Andrew A; Forman, Stephen J; Press, Oliver W

2008-05-15

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR(+) CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR(+) CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR(+) CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.

The Promiscuity of [beta]-Strand Pairing Allows for Rational Design of [beta]-Sheet Face Inversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makabe, Koki; Koide, Shohei

2009-06-17

Recent studies suggest the dominant role of main-chain H-bond formation in specifying {beta}-sheet topology. Its essentially sequence-independent nature implies a large degree of freedom in designing {beta}-sheet-based nanomaterials. Here we show rational design of {beta}-sheet face inversions by incremental deletions of {beta}-strands from the single-layer {beta}-sheet of Borrelia outer surface protein A. We show that a {beta}-sheet structure can be maintained when a large number of native contacts are removed and that one can design large-scale conformational transitions of a {beta}-sheet such as face inversion by exploiting the promiscuity of strand-strand interactions. High-resolution X-ray crystal structures confirmed the success ofmore » the design and supported the importance of main-chain H-bonds in determining {beta}-sheet topology. This work suggests a simple but effective strategy for designing and controlling nanomaterials based on {beta}-rich peptide self-assemblies.« less

Inhibition of Gαs/cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4(+) T Helper Cells.

PubMed

Hynes, Thomas R; Yost, Evan A; Yost, Stacy M; Hartle, Cassandra M; Ott, Braden J; Berlot, Catherine H

2015-07-06

The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4(+) T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4(+) T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gβγ. In contrast, Gαs siRNA and 2',5'-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.

Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation.

PubMed

Wanten, G J; Geijtenbeek, T B; Raymakers, R A; van Kooyk, Y; Roos, D; Jansen, J B; Naber, A H

2000-01-01

To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. Neutrophils, isolated from the blood of 10 healthy volunteers, were incubated in medium or physiologic (2.5 mmol/L) emulsions containing long-chain (LCT), medium-chain (MCT), mixed LCT/MCT, or structured (SL) triglycerides. Expression of adhesion molecules and degranulation markers was evaluated by flow cytometry. Also, functional adhesion was investigated by means of a flow cytometric assay using fluorescent beads coated with the integrin ligand intercellular adhesion molecule (ICAM)-1. Although LCT and SL had no effect, LCT/MCT significantly increased expression of the beta2 integrins lymphocyte-function-associated antigen 1 (+18%), macrophage antigen 1 (+387%), p150,95 (+82%), and (alphaDbeta2 (+230%). Degranulation marker expression for azurophilic (CD63, +210%) and specific granules (CD66b, +370%) also significantly increased, whereas L-selectin (CD62L, -70%) decreased. The effects of LCT/MCT were mimicked by the MCT emulsion. ICAM-1 adhesion (% beads bound) was increased by LCT/MCT (34% +/- 4%), whereas LCT (19% +/-3%) and SL (20% +/- 2%) had no effect compared with medium (17% +/- 3%). LCT/MCT and MCT, contrary to LCT and SL emulsions, increased neutrophil beta2 integrin expression, adhesion, and degranulation. Apart from other emulsion constituents, triglyceride chain length might therefore be a key feature in the interaction of lipid emulsions and the phagocyte immune system.

Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors.

PubMed

Haynes, Nicole M; Trapani, Joseph A; Teng, Michèle W L; Jackson, Jacob T; Cerruti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K

2002-11-01

Tumor cells are usually weakly immunogenic as they largely express self-antigens and can down-regulate major histocompatability complex/peptide molecules and critical costimulatory ligands. The challenge for immunotherapies has been to provide vigorous immune effector cells that circumvent these tumor escape mechanisms and eradicate established tumors. One promising approach is to engineer T cells with single-chain antibody receptors, and since T cells require 2 distinct signals for optimal activation, we have compared the therapeutic efficacy of erbB2-reactive chimeric receptors that contain either T-cell receptor zeta (TCR-zeta) or CD28/TCR-zeta signaling domains. We have demonstrated that primary mouse CD8(+) T lymphocytes expressing the single-chain Fv (scFv)-CD28-zeta receptor have a greater capacity to secrete Tc1 cytokines, induce T-cell proliferation, and inhibit established tumor growth and metastases in vivo. The suppression of established tumor burden by cytotoxic T cells expressing the CD28/TCR-zeta chimera was critically dependent upon their interferon gamma (IFN-gamma) secretion. Our study has illustrated the practical advantage of engineering a T-cell signaling complex that codelivers CD28 activation, dependent only upon the tumor's expression of the appropriate tumor associated antigen.

Preferential V beta gene usage and lack of junctional sequence conservation among human T cell receptors specific for a tetanus toxin- derived peptide: evidence for a dominant role of a germline-encoded V region in antigen/major histocompatibility complex recognition

PubMed Central

1992-01-01

To investigate the structural and genetic basis of the T cell response to defined peptide/major histocompatibility (MHC) class II complexes in humans, we established a large panel of T cell clones (61) from donors of different HLA-DR haplotypes and reactive with a tetanus toxin- derived peptide (tt830-844) recognized in association with most DR molecules (universal peptide). By using a bacterial enterotoxin-based proliferation assay and cDNA sequencing, we found preferential use of a particular V beta region gene segment, V beta 2.1, in three of the individuals studied (64%, n = 58), irrespective of whether the peptide was presented by the DR6wcI, DR4w4, or DRw11.1 and DRw11.2 alleles, demonstrating that shared MHC class II antigens are not required for shared V beta gene use by T cell receptors (TCRs) specific for this peptide. V alpha gene use was more heterogeneous, with at least seven different V alpha segments derived from five distinct families encoding alpha chains able to pair with V beta 2.1 chains to form a tt830-844/DR- specific binding site. Several cases were found of clones restricted to different DR alleles that expressed identical V beta and (or very closely related) V alpha gene segments and that differed only in their junctional sequences. Thus, changes in the putative complementary determining region 3 (CDR3) of the TCR may, in certain cases, alter MHC specificity and maintain peptide reactivity. Finally, in contrast to what has been observed in other defined peptide/MHC systems, a striking heterogeneity was found in the junctional regions of both alpha and beta chains, even for TCRs with identical V alpha and/or V beta gene segments and the same restriction. Among 14 anti-tt830-844 clones using the V beta 2.1 gene segment, 14 unique V beta-D-J beta junctions were found, with no evident conservation in length and/or amino acid composition. One interpretation for this apparent lack of coselection of specific junctional sequences in the context of

In-depth Characterization of a TCR-specific Tracer for Sensitive Detection of Tumor-directed Transgenic T Cells by Immuno-PET.

PubMed

Yusufi, Nahid; Mall, Sabine; Bianchi, Henrique de Oliveira; Steiger, Katja; Reder, Sybille; Klar, Richard; Audehm, Stefan; Mustafa, Mona; Nekolla, Stephan; Peschel, Christian; Schwaiger, Markus; Krackhardt, Angela M; D'Alessandria, Calogero

2017-01-01

A number of different technologies have been developed to monitor in vivo the distribution of gene-modified T cells used in immunotherapy. Nevertheless, in-depth characterization of novel approaches with respect to sensitivity and clinical applicability are so far missing. We have previously described a novel method to track engineered human T cells in tumors using 89 Zr-Df-aTCRmu-F(ab') 2 targeting the murinized part of the TCR beta domain (TCRmu) of a transgenic TCR. Here, we performed an in-depth in vitro characterization of the tracer in terms of antigen affinity, immunoreactivity, influence on T-cell functionality and stability in vitro and in vivo . Of particular interest, we have developed diverse experimental settings to quantify TCR-transgenic T cells in vivo . Local application of 89 Zr-Df-aTCRmu-F(ab') 2 -labeled T cells in a spot-assay revealed signal detection down to approximately 1.8x10 4 cells. In a more clinically relevant model, NSG mice were intravenously injected with different numbers of transgenic T cells, followed by injection of the 89 Zr-Df-aTCRmu-F(ab') 2 tracer, PET/CT imaging and subsequent ex vivo T-cell quantification in the tumor. Using this setting, we defined a comparable detection limit of 1.0x10 4 T cells. PET signals correlated well to total numbers of transgenic T cells detected ex vivo independently of the engraftment rates observed in different individual experiments. Thus, these findings confirm the high sensitivity of our novel PET/CT T-cell tracking method and provide critical information about the quantity of transgenic T cells in the tumor environment suggesting our technology being highly suitable for further clinical translation.

High-throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV-reactive CD8+ T cells.

PubMed

Lossius, Andreas; Johansen, Jorunn N; Vartdal, Frode; Robins, Harlan; Jūratė Šaltytė, Benth; Holmøy, Trygve; Olweus, Johanna

2014-11-01

Epstein-Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS). Here, we used high-throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV-reactivity of the T-cell receptor (TCR) repertoires in MS. TCR-β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T-cell clones, represented by TCR-β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR-β libraries generated from peripheral blood T cells reactive against autologous EBV-transformed B cells were highly enriched for public EBV-specific sequences and were used to quantify EBV-reactive TCR-β sequences in CSF. TCR-β sequences of EBV-reactive CD8+ T cells, including several public EBV-specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV-reactive CD4+ T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T-cell response in MS. The presented strategy links TCR sequence to intrathecal T-cell specificity, demonstrating enrichment of EBV-reactive CD8+ T cells in MS. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increased Peptide Contacts Govern High Affinity Binding of a Modified TCR Whilst Maintaining a Native pMHC Docking Mode

PubMed Central

Cole, David K.; Sami, Malkit; Scott, Daniel R.; Rizkallah, Pierre J.; Borbulevych, Oleg Y.; Todorov, Penio T.; Moysey, Ruth K.; Jakobsen, Bent K.; Boulter, Jonathan M.; Baker, Brian M.; Yi Li

2013-01-01

Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A∗0201) complexed with human T cell lymphotropic virus type 111–19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3β loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3β loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies. PMID:23805144

The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

PubMed Central

Awerkiew, Sabine; Schmidt, Annette; Hombach, Andreas A.; Pfister, Herbert; Abken, Hinrich

2012-01-01

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1+ CD57+ CD7− phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter. PMID:22292024

Selection of Fecal Enterococci Exhibiting tcrB-Mediated Copper Resistance in Pigs Fed Diets Supplemented with Copper † ▿

PubMed Central

Amachawadi, R. G.; Shelton, N. W.; Shi, X.; Vinasco, J.; Dritz, S. S.; Tokach, M. D.; Nelssen, J. L.; Scott, H. M.; Nagaraja, T. G.

2011-01-01

Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene, has been reported in Enterococcus faecium and E. faecalis. A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB-positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M-Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB-positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MICs of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ∼194-kbp size to which both tcrB and the erm(B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm(B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance. PMID:21705534

Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis

PubMed Central

1996-01-01

The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8+ EBV-transformed B lymphoblastoid cells, the primary virus- specific CTL response was shown to include specificities for two HLA-B8- restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TCR-beta sequence analysis of CTL clones specific for each epitope showed polyclonal TCR- beta repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed long-term preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TCR-beta clonotypes and antigen-specific TCR-V beta family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TCR-beta expansions were present at the clonotype level, but not at the TCR-V beta family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TCR stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease. PMID:8920869

Intrinsic folding of small peptide chains: spectroscopic evidence for the formation of beta-turns in the gas phase.

PubMed

Chin, Wutharath; Dognon, Jean-Pierre; Piuzzi, François; Tardivel, Benjamin; Dimicoli, Iliana; Mons, Michel

2005-01-19

Laser desorption of model peptides coupled to laser spectroscopic techniques enables the gas-phase observation of genuine secondary structures of biology. Spectroscopic evidence for the formation of beta-turns in gas-phase peptide chains containing glycine and phenylalanine residues establishes the intrinsic stability of these forms and their ability to compete with other stable structures. The precise characterization of local minima on the potential energy surface from IR spectroscopy constitutes an acute assessment for the state-of-the-art quantum mechanical calculations also presented. The observation of different types of beta-turns depending upon the residue order within the sequence is found to be consistent with the residue propensities in beta-turns of proteins, which suggests that the prevalence of glycine in type II and II' turns stems essentially from an energetic origin, already at play under isolated conditions.

Definition of APC presentation of phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate to Vgamma2Vdelta 2 TCR.

PubMed

Wei, Huiyong; Huang, Dan; Lai, Xiaomin; Chen, Meiling; Zhong, Weihua; Wang, Richard; Chen, Zheng W

2008-10-01

Although microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) can activate primate Vgamma2Vdelta2 T cells, molecular mechanisms by which HMBPP interacts with Vgamma2Vdelta2 T cells remain poorly characterized. Here, we developed soluble, tetrameric Vgamma2Vdelta2 TCR of rhesus macaques to define HMBPP/APC interaction with Vgamma2Vdelta2 TCR. While exogenous HMBPP was associated with APC membrane in an appreciable affinity, the membrane-associated HMBPP readily bound to the Vgamma2Vdelta2 TCR tetramer. The Vgamma2Vdelta2 TCR tetramer was shown to bind stably to HMBPP presented on membrane by various APC cell lines from humans and nonhuman primates but not those from mouse, rat, or pig. The Vgamma2Vdelta2 TCR tetramer also bound to the membrane-associated HMBPP on primary monocytes, B cells and T cells. Consistently, endogenous phosphoantigen produced in Mycobacterium-infected dendritic cells was transported and presented on membrane, and bound stably to the Vgamma2Vdelta2 TCR tetramer. The capability of APC to present HMBPP for recognition by Vgamma2Vdelta2 TCR was diminished after protease treatment of APC. Thus, our studies elucidated an affinity HMBPP-APC association conferring stable binding to the Vgamma2Vdelta2 TCR tetramer and the protease-sensitive nature of phosphoantigen presentation. The findings defined APC presentation of phosphoantigen HMBPP to Vgamma2Vdelta2 TCR.

Tubulin Beta-3 Chain as a New Candidate Protein Biomarker of Human Skin Aging: A Preliminary Study

PubMed Central

2017-01-01

Skin aging is a complex process, and a lot of efforts have been made to identify new and specific targets that could help to diagnose, prevent, and treat skin aging. Several studies concerning skin aging have analyzed the changes in gene expression, and very few investigations have been performed at the protein level. Moreover, none of these proteomic studies has used a global quantitative labeled proteomic offgel approach that allows a more accurate description of aging phenotype. We applied such an approach on human primary keratinocytes obtained from sun-nonexposed skin biopsies of young and elderly women. A total of 517 unique proteins were identified, and 58 proteins were significantly differentially expressed with 40 that were downregulated and 18 upregulated with aging. Gene ontology and pathway analysis performed on these 58 putative biomarkers of skin aging evidenced that these dysregulated proteins were mostly involved in metabolism and cellular processes such as cell cycle and signaling pathways. Change of expression of tubulin beta-3 chain was confirmed by western blot on samples originated from several donors. Thus, this study suggested the tubulin beta-3 chain has a promising biomarker in skin aging. PMID:28626498

Tubulin Beta-3 Chain as a New Candidate Protein Biomarker of Human Skin Aging: A Preliminary Study.

PubMed

Lehmann, Sylvia G; Bourgoin-Voillard, Sandrine; Seve, Michel; Rachidi, Walid

2017-01-01

Skin aging is a complex process, and a lot of efforts have been made to identify new and specific targets that could help to diagnose, prevent, and treat skin aging. Several studies concerning skin aging have analyzed the changes in gene expression, and very few investigations have been performed at the protein level. Moreover, none of these proteomic studies has used a global quantitative labeled proteomic offgel approach that allows a more accurate description of aging phenotype. We applied such an approach on human primary keratinocytes obtained from sun-nonexposed skin biopsies of young and elderly women. A total of 517 unique proteins were identified, and 58 proteins were significantly differentially expressed with 40 that were downregulated and 18 upregulated with aging. Gene ontology and pathway analysis performed on these 58 putative biomarkers of skin aging evidenced that these dysregulated proteins were mostly involved in metabolism and cellular processes such as cell cycle and signaling pathways. Change of expression of tubulin beta-3 chain was confirmed by western blot on samples originated from several donors. Thus, this study suggested the tubulin beta-3 chain has a promising biomarker in skin aging.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji

Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a verymore » low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V{alpha}/V{beta

Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

PubMed

Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Vázquez-Chávez, Elena; Lasserre, Rémi; Agüera-González, Sonia; Cuche, Céline; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

2017-04-01

The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production. Copyright © 2017 by The American Association of Immunologists, Inc.

Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines: a case report.

PubMed

Sonntag, Katja; Hashimoto, Hisayoshi; Eyrich, Matthias; Menzel, Moritz; Schubach, Max; Döcker, Dennis; Battke, Florian; Courage, Carolina; Lambertz, Helmut; Handgretinger, Rupert; Biskup, Saskia; Schilbach, Karin

2018-02-06

Cancer vaccines can effectively establish clinically relevant tumor immunity. Novel sequencing approaches rapidly identify the mutational fingerprint of tumors, thus allowing to generate personalized tumor vaccines within a few weeks from diagnosis. Here, we report the case of a 62-year-old patient receiving a four-peptide-vaccine targeting the two sole mutations of his pancreatic tumor, identified via exome sequencing. Vaccination started during chemotherapy in second complete remission and continued monthly thereafter. We tracked IFN-γ + T cell responses against vaccine peptides in peripheral blood after 12, 17 and 34 vaccinations by analyzing T-cell receptor (TCR) repertoire diversity and epitope-binding regions of peptide-reactive T-cell lines and clones. By restricting analysis to sorted IFN-γ-producing T cells we could assure epitope-specificity, functionality, and T H 1 polarization. A peptide-specific T-cell response against three of the four vaccine peptides could be detected sequentially. Molecular TCR analysis revealed a broad vaccine-reactive TCR repertoire with clones of discernible specificity. Four identical or convergent TCR sequences could be identified at more than one time-point, indicating timely persistence of vaccine-reactive T cells. One dominant TCR expressing a dual TCRVα chain could be found in three T-cell clones. The observed T-cell responses possibly contributed to clinical outcome: The patient is alive 6 years after initial diagnosis and in complete remission for 4 years now. Therapeutic vaccination with a neoantigen-derived four-peptide vaccine resulted in a diverse and long-lasting immune response against these targets which was associated with prolonged clinical remission. These data warrant confirmation in a larger proof-of concept clinical trial.

Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volarevic, S.; Burns, C.M.; Sussman, J.J.

1990-09-01

Immunoprecipitation of Thy-1 from Triton X-100 detergent lysates of surface-iodinated and chemically cross-linked T cells precipitated at least first major and discrete bands. Four of these bands were identified as Thy-1, CD45 (a trasmembrane tyrosine phosphatase), a major histocompatibility complex-encoded class I molecule, and {beta}{sub 2}-microglobulin. Similar analyses revealed that CD45 was coprecipitated from lysates of cross-linker-treated cells by antibodies to the T-cell antigen receptor (TCR). The same pattern of coprecipitated bands was observed when digitonin was used to lyse untreated cells. Immunoprecipitation of Thy-1 or the TCR from lysates of cross-linked T cells precipitated CD45 tyrosine phosphatase activity. Calculationsmore » based upon the amounts of coprecipitated enzymatic activity or TCR {zeta} chain indicate that a substantial fraction of Thy-1 and TCR complexes can be cross-linked to CD45. These data support a model in which the dependence of Thy-1 signaling on TCR coexpression is due to their common interaction with a tyrosine phosphatase and provide a possible structural basis for the influence of CD45 on TCR-mediated signaling.« less

In vivo regulation of the beta-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats

NASA Technical Reports Server (NTRS)

Giger, J. M.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

2000-01-01

In the weight-bearing hindlimb soleus muscle of the rat, approximately 90% of muscle fibers express the beta-myosin heavy chain (beta-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from beta toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the beta-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length beta-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest beta-promoter fragment (-3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the -3500-, -914-, and -408-bp promoter constructs were significantly reduced ( approximately 40%), compared with the control muscles. However, using the -215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the -408 and -215 bp of the promoter.

Interpreting T-Cell Cross-reactivity through Structure: Implications for TCR-Based Cancer Immunotherapy.

PubMed

Antunes, Dinler A; Rigo, Maurício M; Freitas, Martiela V; Mendes, Marcus F A; Sinigaglia, Marialva; Lizée, Gregory; Kavraki, Lydia E; Selin, Liisa K; Cornberg, Markus; Vieira, Gustavo F

2017-01-01

Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient's own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide-ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide-MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC "hot-spots" for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus-derived peptides. Our

Interpreting T-Cell Cross-reactivity through Structure: Implications for TCR-Based Cancer Immunotherapy

PubMed Central

Antunes, Dinler A.; Rigo, Maurício M.; Freitas, Martiela V.; Mendes, Marcus F. A.; Sinigaglia, Marialva; Lizée, Gregory; Kavraki, Lydia E.; Selin, Liisa K.; Cornberg, Markus; Vieira, Gustavo F.

2017-01-01

Immunotherapy has become one of the most promising avenues for cancer treatment, making use of the patient’s own immune system to eliminate cancer cells. Clinical trials with T-cell-based immunotherapies have shown dramatic tumor regressions, being effective in multiple cancer types and for many different patients. Unfortunately, this progress was tempered by reports of serious (even fatal) side effects. Such therapies rely on the use of cytotoxic T-cell lymphocytes, an essential part of the adaptive immune system. Cytotoxic T-cells are regularly involved in surveillance and are capable of both eliminating diseased cells and generating protective immunological memory. The specificity of a given T-cell is determined through the structural interaction between the T-cell receptor (TCR) and a peptide-loaded major histocompatibility complex (MHC); i.e., an intracellular peptide–ligand displayed at the cell surface by an MHC molecule. However, a given TCR can recognize different peptide–MHC (pMHC) complexes, which can sometimes trigger an unwanted response that is referred to as T-cell cross-reactivity. This has become a major safety issue in TCR-based immunotherapies, following reports of melanoma-specific T-cells causing cytotoxic damage to healthy tissues (e.g., heart and nervous system). T-cell cross-reactivity has been extensively studied in the context of viral immunology and tissue transplantation. Growing evidence suggests that it is largely driven by structural similarities of seemingly unrelated pMHC complexes. Here, we review recent reports about the existence of pMHC “hot-spots” for cross-reactivity and propose the existence of a TCR interaction profile (i.e., a refinement of a more general TCR footprint in which some amino acid residues are more important than others in triggering T-cell cross-reactivity). We also make use of available structural data and pMHC models to interpret previously reported cross-reactivity patterns among virus

Light-induced yellowing of selectively 13C-enriched dehydrogenation polymers (DHPs). Part 1, Side-chain 13C-enriched DHP ([alpha], [beta], and [gamma]-13C)

Treesearch

Jim Parkas; Magnus Paulsson; Terashima Noritsugu; Ulla Westermark; Sally Ralph

2004-01-01

Light-induced yellowing has been studied using side-chain ([alpha], [beta], and [gamma]) 13C-enriched DHP (dehydrogenation polymer) and quantitative solution state 13C NMR spectroscopy. The DHP was formed from 13C-enriched coniferin using an enzymatic system consisting of [beta]-glucosidase, glucose oxidase, and peroxidase in a pH 6 buffer solution. The DHP was applied...

Long-chain aliphatic beta-diketones from epicuticular wax of Vanilla bean species. Synthesis of nervonoylacetone.

PubMed

Ramaroson-Raonizafinimanana, B; Gaydou, E M; Bombarda, I

2000-10-01

Analysis of the neutral lipids from Vanilla fragrans and Vanilla tahitensis (Orchidaceae) without saponification resulted in the isolation and identification of a new product family in this plant: beta-dicarbonyl compounds. The compound structures are composed of a long aliphatic chain with 2,4-dicarbonyl carbons and a cis double bond at the n-9 position. They represent approximately 28% of the neutral lipids, that is, 1.5%, in immature beans, and approximately 10% of the neutral lipids, that is, 0.9%, in mature beans. Using retention indices, gas chromatography-mass spectrometry, derivatization reactions, and chemical degradation, five beta-dicarbonyl compounds have been identified including 16-pentacosene-2,4-dione, 18-heptacosene-2,4-dione, 20-nonacosene-2, 4-dione, 22-hentriacontene-2,4-dione, and 24-tritriacontene-2, 4-dione. Among them (Z)-18-heptacosene-2,4-dione, or nervonoylacetone, has been synthesized in two steps starting from nervonic acid. The major constituent, nervonoylacetone, represented 74.5% of the beta-dicarbonyl fraction. The range of these compounds has been studied in relation with bean maturity for V. fragrans and V. tahitensis species. This compound family has not been found in the leaves or stems of any of the three vanilla species studied and is markedly absent in the beans of V. madagascariensis.

Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

PubMed

Bueno, Clara; Lemke, Caitlin D; Criado, Gabriel; Baroja, Miren L; Ferguson, Stephen S G; Rahman, A K M Nur-Ur; Tsoukas, Constantine D; McCormick, John K; Madrenas, Joaquin

2006-07-01

The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.

Unbiased analysis of TCRα/β chains at the single-cell level in human CD8+ T-cell subsets.

PubMed

Sun, Xiaoming; Saito, Masumichi; Sato, Yoshinori; Chikata, Takayuki; Naruto, Takuya; Ozawa, Tatsuhiko; Kobayashi, Eiji; Kishi, Hiroyuki; Muraguchi, Atsushi; Takiguchi, Masafumi

2012-01-01

T-cell receptor (TCR) α/β chains are expressed on the surface of CD8(+) T-cells and have been implicated in antigen recognition, activation, and proliferation. However, the methods for characterization of human TCRα/β chains have not been well established largely because of the complexity of their structures owing to the extensive genetic rearrangements that they undergo. Here we report the development of an integrated 5'-RACE and multiplex PCR method to amplify the full-length transcripts of TCRα/β at the single-cell level in human CD8(+) subsets, including naive, central memory, early effector memory, late effector memory, and effector phenotypic cells. Using this method, with an approximately 47% and 62% of PCR success rate for TCRα and for TCRβ chains, respectively, we were able to analyze more than 1,000 reads of transcripts of each TCR chain. Our comprehensive analysis revealed the following: (1) chimeric rearrangements of TCRδ-α, (2) control of TCRα/β transcription with multiple transcriptional initiation sites, (3) altered utilization of TCRα/β chains in CD8(+) subsets, and (4) strong association between the clonal size of TCRα/β chains and the effector phenotype of CD8(+) T-cells. Based on these findings, we conclude that our method is a useful tool to identify the dynamics of the TCRα/β repertoire, and provides new insights into the study of human TCRα/β chains.

The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

PubMed Central

Ophir, Michael J.; Liu, Beiyun C.

2013-01-01

The T cell receptor (TCR) triggers the assembly of “SLP-76 microclusters,” which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase–associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A “tandem dimer” containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP–interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and “inside-out” signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins. PMID:24368808

Nanostructured vanadium oxide thin film with high TCR at room temperature for microbolometer

NASA Astrophysics Data System (ADS)

Wang, Bin; Lai, Jianjun; Li, Hui; Hu, Haoming; Chen, Sihai

2013-03-01

In order to obtain high quality of thermal sensitive material, VOx thin film of high temperature coefficient of resistance (TCR) of 6.5%/K at room temperature has been deposited by reactive ion beam sputtering and post annealing method. AFM and XRD measurements indicate that the VOx thin film with nanostructured crystalline is composed of VO2 and V2O3. The nanostructured VOx microbolometer has been designed and fabricated. The measurement of the film system with TiN absorbing layer indicates that it has about 92% infrared absorption in the range of 8-14 μm. The performance of this bolometer, comparing with that of bolometer with common VOx, has a better result. At 20 Hz frequency and 10 μA bias current, the bolometer with high TCR has reached detectivity of 1.0 × 109 cm Hz1/2/W. It also indicates that this nanostructured VOx thin film has not only a higher TCR but also a lower noise than common VOx thin film without annealing.

Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity

PubMed Central

Saitakis, Michael; Dogniaux, Stéphanie; Goudot, Christel; Bufi, Nathalie; Asnacios, Sophie; Maurin, Mathieu; Randriamampita, Clotilde; Asnacios, Atef; Hivroz, Claire

2017-01-01

T cells are mechanosensitive but the effect of stiffness on their functions is still debated. We characterize herein how human primary CD4+ T cell functions are affected by stiffness within the physiological Young’s modulus range of 0.5 kPa to 100 kPa. Stiffness modulates T lymphocyte migration and morphological changes induced by TCR/CD3 triggering. Stiffness also increases TCR-induced immune system, metabolism and cell-cycle-related genes. Yet, upon TCR/CD3 stimulation, while cytokine production increases within a wide range of stiffness, from hundreds of Pa to hundreds of kPa, T cell metabolic properties and cell cycle progression are only increased by the highest stiffness tested (100 kPa). Finally, mechanical properties of adherent antigen-presenting cells modulate cytokine production by T cells. Together, these results reveal that T cells discriminate between the wide range of stiffness values found in the body and adapt their responses accordingly. DOI: http://dx.doi.org/10.7554/eLife.23190.001 PMID:28594327

Differences in TCR-Vβ Repertoire and Effector Phenotype between Tumor Infiltrating Lymphocytes and Peripheral Blood Lymphocytes Increase with Age

PubMed Central

Wang, Teng; Shen, Han; Wu, Fenglin; Zhang, Wenfeng; Tao, Changli; Yuan, Yin; Bo, Huaben; Wang, Hui; Huang, Shulin

2014-01-01

Tumor infiltrating lymphocytes (TIL) reflect the host's anti-tumor immune response, and can be a valuable predictor of prognosis. However, many properties of TIL are not fully understood. In the present study, TCR-Vβ repertoires of cancer patients were primarily analyzed by flow cytometry. Abnormally expressed TCR-Vβ subfamilies were generally found in both TIL and peripheral blood lymphocytes (PBL) of each patient. Of note, increased patient age was associated with increasingly biased TCR-Vβ repertoire in TIL but not in PBL, and the dispersion degree of the differences of TCR-Vβ subfamilies between TIL and PBL correlated positively with age (P = 0.007). Utilizing immunoscope analysis, we identified the age-related reduction in TCR-Vβ diversity, but polyclonal pattern was predominant in significantly expanded TCR-Vβ subfamilies. In addition, we found that older patients possessed a decreased ratio of CD8+CD62L+ non-effector cells in TIL compared to PBL, implying age-related increase of CD8+CD62L− effector cells in TIL. The colocalization analysis of CD8 and CD3, however, suggested the suppressed activity of these effector cells in tumor microenvironment. These findings further elucidate the properties of TIL, showing an increasing difference between TIL and PBL with age, which may provide insight for the development of effective immunotherapies for cancer patients of different ages. PMID:25019226

Variability and repertoire size of T-cell receptor V alpha gene segments.

PubMed

Becker, D M; Pattern, P; Chien, Y; Yokota, T; Eshhar, Z; Giedlin, M; Gascoigne, N R; Goodnow, C; Wolf, R; Arai, K

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

TCR Signal Strength Alters T–DC Activation and Interaction Times and Directs the Outcome of Differentiation

PubMed Central

van Panhuys, Nicholas

2016-01-01

The ability of CD4+ T cells to differentiate into effector subsets underpins their ability to shape the immune response and mediate host protection. During T cell receptor-induced activation of CD4+ T cells, both the quality and quantity of specific activatory peptide/MHC ligands have been shown to control the polarization of naive CD4+ T cells in addition to co-stimulatory and cytokine-based signals. Recently, advances in two-­photon microscopy and tetramer-based cell tracking methods have allowed investigators to greatly extend the study of the role of TCR signaling in effector differentiation under in vivo conditions. In this review, we consider data from recent in vivo studies analyzing the role of TCR signal strength in controlling the outcome of CD4+ T cell differentiation and discuss the role of TCR in controlling the critical nature of CD4+ T cell interactions with dendritic cells during activation. We further propose a model whereby TCR signal strength controls the temporal aspects of T–DC interactions and the implications for this in mediating the downstream signaling events, which influence the transcriptional and epigenetic regulation of effector differentiation. PMID:26834747

SDF-1 signaling via the CXCR4-TCR heterodimer requires PLC-β3 and PLC-γ1 for distinct cellular responses 1

PubMed Central

Kremer, Kimberly N.; Clift, Ian C.; Miamen, Alexander G.; Bamidele, Adebowale O.; Qian, Nan-Xin; Humphreys, Troy D.; Hedin, Karen E.

2011-01-01

The CXCR4 chemokine receptor is a G protein-coupled receptor (GPCR) that signals in T lymphocytes by forming a heterodimer with the T cell antigen receptor (TCR). CXCR4 and TCR functions are consequently highly cross-regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAP kinase and downstream AP-1-dependent cytokine transcription in response to SDF-1, the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP-70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate GPCR-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. Here, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-β3 to activate the Ras-ERK pathway and increase intracellular Ca2+ concentrations, while PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-β3, is required for SDF-1-mediated migration, via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-β3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors in order to distinctly regulate migration versus other signaling functions. PMID:21705626

Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy.

PubMed

Raman, Marine C C; Rizkallah, Pierre J; Simmons, Ruth; Donnellan, Zoe; Dukes, Joseph; Bossi, Giovanna; Le Provost, Gabrielle S; Todorov, Penio; Baston, Emma; Hickman, Emma; Mahon, Tara; Hassan, Namir; Vuidepot, Annelise; Sami, Malkit; Cole, David K; Jakobsen, Bent K

2016-01-13

Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics.

TCR signaling by conventional CD4+ T cells is required for optimal maintenance of peripheral regulatory T cell numbers.

PubMed

Leichner, Theresa M; Satake, Atsushi; Kambayashi, Taku

2016-06-01

To maintain immune tolerance, regulatory T cell (Treg) numbers must be closely indexed to the number of conventional T cells (Tconvs) so that an adequate Treg:Tconv ratio can be maintained. Two factors important in this process are the cytokine interleukin-2 (IL-2) and T cell receptor (TCR) stimulation by major histocompatibility complex class II (MHC-II). Here, we report that in addition to TCR stimulation of Tregs themselves, the maintenance of Tregs also requires TCR signaling by Tconvs. We found that Tconvs produce IL-2 in response to self-peptide-MHC-II complexes and that Tconvs possessing more highly self-reactive TCRs express more IL-2 at baseline. Furthermore, selective disruption of TCR signaling in Tconvs led to a trend toward decreased expression of IL-2 and attenuated their ability to maintain Treg numbers. These data suggest that in order to maintain an adequate Treg:Tconv ratio, Tregs are continuously indexed to self-peptide-MHC-II-induced TCR signaling of Tconvs. These results have implications in attempts to modulate immune tolerance, as Treg numbers adjust to the self-reactivity, and ultimately IL-2 production by the T cells around them.

Propensity of a single-walled carbon nanotube-peptide to mimic a KK10 peptide in an HLA-TCR complex

NASA Astrophysics Data System (ADS)

Feng, Mei; Bell, David R.; Zhou, Ruhong

2017-12-01

The application of nanotechnology to improve disease diagnosis, treatment, monitoring, and prevention is the goal of nanomedicine. We report here a theoretical study of a functionalized single-walled carbon nanotube (CNT) mimic binding to a human leukocyte antigen-T cell receptor (HLA-TCR) immune complex as a first attempt of a potential nanomedicine for human immunodeficiency virus (HIV) vaccine development. The carbon nanotube was coated with three arginine residues to imitate the HIV type 1 immunodominant viral peptide KK10 (gag 263-272: KRWIILGLNK), named CNT-peptide hereafter. Through molecular dynamics simulations, we explore the CNT-peptide and KK10 binding to an important HLA-TCR complex. Our results suggest that the CNT-peptide and KK10 bind comparably to the HLA-TCR complex, but the CNT-peptide forms stronger interactions with the TCR. Desorption simulations highlight the innate flexibility of KK10 over the CNT-peptide, resulting in a slightly higher desorption energy required for KK10 over the CNT-peptide. Our findings indicate that the designed CNT-peptide mimic has favorable propensity to activate TCR pathways and should be further explored to understand therapeutic potential.

Regulation of TCR Signaling to NF-kB

DTIC Science & Technology

2013-03-20

applications and benefits to basic research are discussed in detail in Chapter 4.      16   CHAPTER 2: Selective autophagy of the...To confirm the specificity of the chemical inhibitors of autophagy, we employed a genetic approach. Specifically, we in vitro differentiated Th2...MG132 treatment. These data provide strong genetic evidence that TCR-mediated degradation of endogenous Bcl10 is controlled by autophagy, with a

beta-Thalassemia present in cis to a new beta-chain structural variant, Hb Vicksburg [beta 75 (E19)Leu leads to 0].

PubMed

Adams, J G; Steinberg, M H; Newman, M V; Morrison, W T; Benz, E J; Iyer, R

1981-01-01

Hemoglobin Vicksburg was discovered in a 6-year-old Black boy who had been anemic since infancy. Examination of his hemolysate revealed 87.5% Hb F, 2.4% Hb A2, and 7.6% Hb Vicksburg, which had the electrophoretic and chromatographic properties of Hb A. Structural analysis of Hb Vicksburg demonstrated a deletion of leucine at beta 75(E19), a new variant. Hb Vicksburg was neither unstable nor subject to posttranslational degradation. The alpha/non-alpha biosynthetic ratio was 2.6. Because the proband appeared to be a mixed heterozygote for Hb Vicksburg and beta 0-thalassemia, Hb Vicksburg should have comprised the major portion of the hemolysate. Thus, Hb Vicksburg was synthesized at a rate considerably lower than would be expected on the basis of gene dosage. There was no reason to suspect abnormal translation of beta Vicksburg mRNA; in individuals with Hb St. Antoine (beta 74 and beta 75 deleted), the abnormal hemoglobin comprised 25% of the hemolysate in the simple heterozygote yet was unstable. Deletion of beta 75, therefore, would not in itself appear to lead to diminished synthesis. There was a profound deficit of beta Vicksburg mRNA when measured by liquid hybridization analysis with beta cDNA. The most plausible explanation for the low output of Hb Vicksburg is that a mutation for beta +-thalassemia is present in cis to the structural mutation.

Rejection of syngeneic colon carcinoma by CTLs expressing single-chain antibody receptors codelivering CD28 costimulation.

PubMed

Haynes, Nicole M; Trapani, Joseph A; Teng, Michele W L; Jackson, Jacob T; Cerruti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K

2002-11-15

A new strategy to improve the therapeutic utility of redirected T cells for cancer involves the development of novel Ag-specific chimeric receptors capable of stimulating optimal and sustained T cell antitumor activity in vivo. Given that T cells require both primary and costimulatory signals for optimal activation and that many tumors do not express critical costimulatory ligands, modified single-chain Ab receptors have been engineered to codeliver CD28 costimulation. In this study, we have compared the antitumor potency of primary T lymphocytes expressing carcinoembryonic Ag (CEA)-reactive chimeric receptors that incorporate either TCR-zeta or CD28/TCR-zeta signaling. Although both receptor-transduced T cell effector populations demonstrated cytolysis of CEA(+) tumors in vitro, T cells expressing the single-chain variable fragment of Ig (scFv)-CD28-zeta chimera had a far greater capacity to control the growth of CEA(+) xenogeneic and syngeneic colon carcinomas in vivo. The observed enhanced antitumor activity of T cells expressing the scFv-CD28-zeta receptor was critically dependent on perforin and the production of IFN-gamma. Overall, this study has illustrated the ability of a chimeric scFv receptor capable of harnessing the signaling machinery of both TCR-zeta and CD28 to augment T cell immunity against tumors that have lost expression of both MHC/peptide and costimulatory ligands in vivo.

Interaction between two discontiguous chain segments from the beta-sheet of Escherichia coli thioredoxin suggests an initiation site for folding.

PubMed

Tasayco, M L; Fuchs, J; Yang, X M; Dyalram, D; Georgescu, R E

2000-09-05

The approach of comparing folding and folding/binding processes is exquisitely poised to narrow down the regions of the sequence that drive protein folding. We have dissected the small single alpha/beta domain of oxidized Escherichia coli thioredoxin (Trx) into three complementary fragments (N, residues 1-37; M, residues 38-73; and C, residues 74-108) to study them in isolation and upon recombination by far-UV CD and NMR spectroscopy. The isolated fragments show a minimum of ellipticity of ca. 197 nm in their far-UV CD spectra without concentration dependence, chemical shifts of H(alpha) that are close to the random coil values, and no medium- and long-range NOE connectivities in their three-dimensional NMR spectra. These fragments behave as disordered monomers. Only the far-UV CD spectra of binary or ternary mixtures that contain N- and C-fragments are different from the sum of their individual spectra, which is indicative of folding and/or binding of these fragments. Indeed, the cross-peaks corresponding to the rather hydrophobic beta(2) and beta(4) regions of the beta-sheet of Trx disappear from the (1)H-(15)N HSQC spectra of isolated labeled N- and C-fragments, respectively, upon addition of the unlabeled complementary fragments. The disappearing cross-peaks indicate interactions between the beta(2) and beta(4) regions, and their reappearance at lower temperatures indicates unfolding and/or dissociation of heteromers that are predominantly held by hydrophobic forces. Our results argue that the folding of Trx begins by zippering two discontiguous and rather hydrophobic chain segments (beta(2) and beta(4)) corresponding to neighboring strands of the native beta-sheet.

Unbiased Analysis of TCRα/β Chains at the Single-Cell Level in Human CD8+ T-Cell Subsets

PubMed Central

Sun, Xiaoming; Saito, Masumichi; Sato, Yoshinori; Chikata, Takayuki; Naruto, Takuya; Ozawa, Tatsuhiko; Kobayashi, Eiji; Kishi, Hiroyuki; Muraguchi, Atsushi; Takiguchi, Masafumi

2012-01-01

T-cell receptor (TCR) α/β chains are expressed on the surface of CD8+ T-cells and have been implicated in antigen recognition, activation, and proliferation. However, the methods for characterization of human TCRα/β chains have not been well established largely because of the complexity of their structures owing to the extensive genetic rearrangements that they undergo. Here we report the development of an integrated 5′-RACE and multiplex PCR method to amplify the full-length transcripts of TCRα/β at the single-cell level in human CD8+ subsets, including naive, central memory, early effector memory, late effector memory, and effector phenotypic cells. Using this method, with an approximately 47% and 62% of PCR success rate for TCRα and for TCRβ chains, respectively, we were able to analyze more than 1,000 reads of transcripts of each TCR chain. Our comprehensive analysis revealed the following: (1) chimeric rearrangements of TCRδ-α, (2) control of TCRα/β transcription with multiple transcriptional initiation sites, (3) altered utilization of TCRα/β chains in CD8+ subsets, and (4) strong association between the clonal size of TCRα/β chains and the effector phenotype of CD8+ T-cells. Based on these findings, we conclude that our method is a useful tool to identify the dynamics of the TCRα/β repertoire, and provides new insights into the study of human TCRα/β chains. PMID:22792299

beta-Thalassemia present in cis to a new beta-chain structural variant, Hb Vicksburg [beta 75 (E19)Leu leads to 0].

PubMed Central

Adams, J G; Steinberg, M H; Newman, M V; Morrison, W T; Benz, E J; Iyer, R

1981-01-01

Hemoglobin Vicksburg was discovered in a 6-year-old Black boy who had been anemic since infancy. Examination of his hemolysate revealed 87.5% Hb F, 2.4% Hb A2, and 7.6% Hb Vicksburg, which had the electrophoretic and chromatographic properties of Hb A. Structural analysis of Hb Vicksburg demonstrated a deletion of leucine at beta 75(E19), a new variant. Hb Vicksburg was neither unstable nor subject to posttranslational degradation. The alpha/non-alpha biosynthetic ratio was 2.6. Because the proband appeared to be a mixed heterozygote for Hb Vicksburg and beta 0-thalassemia, Hb Vicksburg should have comprised the major portion of the hemolysate. Thus, Hb Vicksburg was synthesized at a rate considerably lower than would be expected on the basis of gene dosage. There was no reason to suspect abnormal translation of beta Vicksburg mRNA; in individuals with Hb St. Antoine (beta 74 and beta 75 deleted), the abnormal hemoglobin comprised 25% of the hemolysate in the simple heterozygote yet was unstable. Deletion of beta 75, therefore, would not in itself appear to lead to diminished synthesis. There was a profound deficit of beta Vicksburg mRNA when measured by liquid hybridization analysis with beta cDNA. The most plausible explanation for the low output of Hb Vicksburg is that a mutation for beta +-thalassemia is present in cis to the structural mutation. PMID:6165992

Diving behaviour and haemoglobin function: the primary structure of the alpha- and beta-chains of the sea turtle (Caretta caretta) and its functional implications.

PubMed

Petruzzelli, R; Aureli, G; Lania, A; Galtieri, A; Desideri, A; Giardina, B

1996-06-15

The amino acid sequence of the alpha- and beta-chains of haemoglobin (Hb) from the loggerhead sea turtle (Caretta caretta) has been determined. Comparison with that of human Hb shows differences in several residues involved in both alpha 1 beta 1 and alpha 1 beta 2 packing contacts. On the whole, in spite of the mutations, the essential characteristics of both interfaces seem to be maintained. The functional properties of the sea turtle Hb have been investigated at different temperatures and as a function of proton, chloride and organic phosphate concentrations. In addition to overall similarities shared with most of the vertebrate Hbs previously described, this molecule shows significant differences which could be related to the life behaviour of the turtle. In fact, while the shape of the Bohr-effect curve is well adapted for gas exchange during prolonged dives, the very small enthalpy change for O2 binding ensures that O2 delivery becomes essentially insensitive to the temperature changes of the environment. Moreover, and similarly to the case of emperor penguin Hb, the small alkaline Bohr effect appears to be only choride-linked, since the pH dependence of the O2 affinity is abolished in the absence of this ion. These functional characteristics are discussed on the basis of the primary structure of alpha- and beta-chains.

Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy

PubMed Central

Raman, Marine C C; Rizkallah, Pierre J; Simmons, Ruth; Donnellan, Zoe; Dukes, Joseph; Bossi, Giovanna; Le Provost, Gabrielle S; Todorov, Penio; Baston, Emma; Hickman, Emma; Mahon, Tara; Hassan, Namir; Vuidepot, Annelise; Sami, Malkit; Cole, David K; Jakobsen, Bent K.

2016-01-01

Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics. PMID:26758806

Unprecedented {sup 1}/{sub {infinity}}[{beta}-Mo{sub 8}O{sub 26}]{sup 4-} polymeric chains and four novel organic-inorganic hybrids based on Mo-POMs and azaheterocycles templates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du Haijuan; Zunzhe Shu; Niu Yunyin, E-mail: niuyy@zzu.edu.cn

2012-06-15

Abstrct: Four novel organic-inorganic hybrid materials based on Mo-POMs and organic templates, namely [DEB] [{beta}-Mo{sub 8}O{sub 26}] [NH{sub 4}]{sub 2} (1), [BMIM] [{beta}-Mo{sub 8}O{sub 26}]{sub 0.5}{center_dot}H{sub 2}O (2), [BMIM] [1D-Mo{sub 8}O{sub 26}]{sub 0.5} (3) and {l_brace}3D-[Cu(DIE){sub 2}] [1D-Mo{sub 8}O{sub 26}]{sub 0.5}{r_brace}{sub {infinity}} (4) [DEB= 1,1 Prime -diethyl-4,4 Prime -bipyridinium, BMIM=1,1 Prime -bis(1-methylimidazolium)methylene, DIE=1,2-diimidazoloethane] have been hydrothermally synthesized and characterized by elemental analyses, IR spectroscopy, thermal gravimetric analysis(TGA) and single-crystal X-ray diffraction. Both compounds 1 and 2 are POMs-based supramolecular compounds consisted of independent [{beta}-Mo{sub 8}O{sub 26}]{sup 4-} anions and [DEB]{sup 2+} or [BMIM]{sup 2+} organic cations. Compound 3 is themore » first external template example of Mo-POMs-based supramolecular network incorporated with novel {sup 1}/{sub {infinity}}[{beta}-Mo{sub 8}O{sub 26}]{sup 4-} polymeric chains. Compound 4 is a rare supramolecular structure that contains octamolybdate {sup 1}/{sub {infinity}}[{beta}-Mo{sub 8}O{sub 26}]{sup 4-} polymeric chains interconnected via DIE ligands to form a 3D net. Moreover, it was indicated that these polyacid compounds had definite catalytic activities on the probe reaction of acetaldehyde oxidation to acetic acid with H{sub 2}O{sub 2}. - Graphical abstract: Four novel organic templated polyoxometalates comprising of 0D, 1D and 3D supramolecular frameworks together with the catalytic activities on the acetaldehyde oxidation to acetic acid were reported. Highlights: Using cation templated self-assembly four novel polyoxometalates were prepared. Compounds 1 and 2 consisted of independent [{beta}-Mo{sub 8}O{sub 26}]{sup 4-} anions and organic cations. Compound 3 is the first external template-assisted POMs with {sup 1}/{sub {infinity}}[{beta}-Mo{sub 8}O{sub 26}]{sup 4-} chain. Compound 4 is a rare 3D

[Mutation analysis of beta myosin heavy chain gene in hypertrophic cardiomyopathy families].

PubMed

Fan, Xin-ping; Yang, Zhong-wei; Feng, Xiu-li; Yang, Fu-hui; Xiao, Bai; Liang, Yan

2011-08-01

To detect the gene mutations of beta-myosin heavy chain gene (MYH7) in Chinese pedigrees with hypertrophic cardiomyopathy (HCM), and to analyze the correlation between the genotype and phenotype. Exons 3, 5, 7-9, 11-16 and 18-23 of the MYH7 gene were amplified with PCR in three Chinese pedigrees with HCM. The products were sequenced. Sequence alignment between the detected and the standard sequences was performed. A missense mutation of Thr441Met in exon 14 was identified in a pedigree, which was not detected in the controls. Several synonymous mutations of MYH7 gene were detected in the three pedigrees. The mutation of Thr441Met, located in the actin binding domain of the globular head, was first identified in Chinese. It probably caused HCM. HCM is a heterogeneous disease. Many factors are involved in the process of its occurrence and development.

KIAA1530 Protein Is Recruited by Cockayne Syndrome Complementation Group Protein A (CSA) to Participate in Transcription-coupled Repair (TCR)

PubMed Central

Fei, Jia; Chen, Junjie

2012-01-01

Transcription-coupled repair (TCR) is the major pathway involved in the removal of UV-induced photolesions from the transcribed strand of active genes. Two Cockayne syndrome (CS) complementation group proteins, CSA and CSB, are important for TCR repair. The molecular mechanisms by which CS proteins regulate TCR remain elusive. Here, we report the characterization of KIAA1530, an evolutionarily conserved protein that participates in this pathway through its interaction with CSA and the TFIIH complex. We found that UV irradiation led to the recruitment of KIAA1530 onto chromatin in a CSA-dependent manner. Cells lacking KIAA1530 were highly sensitive to UV irradiation and displayed deficiency in TCR. In addition, KIAA1530 depletion abrogated stability of the CSB protein following UV irradiation. More excitingly, we found that a unique CSA mutant (W361C), which was previously identified in a patient with UVsS syndrome, showed defective KIAA1530 binding and resulted in a failure of recruiting KIAA1530 and stabilizing CSB after UV treatment. Together, our data not only reveal that KIAA1530 is an important player in TCR but also lead to a better understanding of the molecular mechanism underlying UVsS syndrome. PMID:22902626

Occurrence of the Transferable Copper Resistance Gene tcrB among Fecal Enterococci of U.S. Feedlot Cattle Fed Copper-Supplemented Diets

PubMed Central

Amachawadi, R. G.; Alvarado, C. A.; Mainini, T. R.; Vinasco, J.; Drouillard, J. S.; Nagaraja, T. G.

2013-01-01

Copper, an essential micronutrient, is supplemented in the diet at elevated levels to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance gene (tcrB) borne on a plasmid. The present study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2×2 factorial arrangement of treatments consisting of dietary copper and a commercial linseed meal-based energy protein supplement. A total of 22 isolates, each identified as Enterococcus faecium, were positive for tcrB with an overall prevalence of 3.8% (22/576). The prevalence was higher among the cattle fed diets supplemented with copper (6.9%) compared to normal copper levels (0.7%). The tcrB-positive isolates always contained both erm(B) and tet(M) genes. Median copper MICs for tcrB-positive and tcrB-negative enterococci were 22 and 4 mM, respectively. The transferability of the tcrB gene was demonstrated via a filter-mating assay. Multilocus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci remain potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut. PMID:23666328

Use of beta-methylphenylalanine (beta MeF) residues to probe the nature of the interaction of substance P with its receptor: effects of beta MeF-containing substance P analogs on rabbit iris smooth muscle contraction.

PubMed

Birney, D M; Cole, D C; Crosson, C E; Kahl, B F; Neff, B W; Reid, T W; Ren, K; Walkup, R D

1995-06-23

The effects of substituting (2S,3S)-beta-methylphenylalanine (S-beta MeF) or (2S,3R)-beta-methylphenylalanine (R-beta MeF) for the Phe7 and/or Phe8 residues of the tachykinin substance P (SP, RPKPQQFFGLM-NH2) upon the ability of SP to stimulate contraction of the rabbit iris smooth muscle were investigated. The eight beta MeF-containing SP analogs (four monosubstituted analogs, four disubstituted analogs) 1-8 were synthesized and found to be agonsts of SP in the smooth muscle contraction assay, having EC50 values ranging from 0.15 to 10.0 nM. Three analogs are significantly more active than SP [8R-(beta MeF)SP (4), 7S,8S-(beta MeF)2SP (5), and 7R,8S-(beta MeF)2SP (6)], three analogs are approximately equipotent with SP [7S-(beta MeF)SP (1), 7R-(beta MeF)SP (2), and 7S,8R-(beta MeF)2SP (8)], and two analogs are significantly less active than SP [8S-(beta MeF)SP (3) and 7R,8R-(beta MeF)2SP (7)]. The effects of the beta MeF substitutions upon the activity of SP are not additive and cannot be explained using simple conformational models which focus only on the side chain conformations of the beta MeF residues. It is postulated that the beta MeF residues induce minor distortions in the peptide backbone with resultant consequences upon peptide-receptor binding which are not dictated soley by the side chain conformations. This idea is consistent with 1H-NMR data for the monosubstituted analogs 1-4, which imply that the beta MeF substitutions cause slight distortions in the peptide backbone and that the beta MeF side chains are assuming trans or gauche(-) conformations.

T cells to a dominant epitope of GAD65 express a public CDR3 motif.

PubMed

Quinn, Anthony; McInerney, Marcia; Huffman, Donald; McInerney, Brigid; Mayo, Stella; Haskins, Kathryn; Sercarz, Eli

2006-06-01

Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes, and serve as a model for type 1 diabetes (T1D) and natural autoimmunity. T cell responses to the pancreatic islet antigen glutamic acid decarboxylase 65 (GAD65) can be detected in the spleens of young prediabetic NOD mice, which display a unique MHC class II molecule. Here, we report that a distinct TcR beta chain and CDR3 motif are utilized by all NOD mice in response to a dominant determinant on GAD65, establishing a public repertoire in the spontaneous autoimmunity to an important islet cell antigen. GAD65 530-543 (p530)-reactive T cells preferentially utilize the Vbeta4, Dbeta2.1 and Jbeta2.7 gene segments, with a CDR3 that is characterized by a triad of amino acids, DWG, preceded by a polar residue. In addition, we used CDR3 length spectratyping, CDR3-specific reverse transcriptase-PCR and direct TcR sequencing to show that the TcR beta chain structural patterns associated with p530-specific T cells consistently appeared in the islets of young NOD mice with insulitis, but not in the inflamed islets of streptozotocin-treated C57BL/6 mice, or in inflamed NOD salivary glands. To our knowledge, this is the first report to demonstrate that a public T cell repertoire is used in spontaneous autoimmunity to a dominant self-determinant. These findings suggest that defined clonotypes and repertoires may be preferentially selected in haplotypes predisposed to spontaneous autoimmunity.

Spectroscopic evidence for gas-phase formation of successive beta-turns in a three-residue peptide chain.

PubMed

Chin, Wutharath; Compagnon, Isabelle; Dognon, Jean-Pierre; Canuel, Clélia; Piuzzi, François; Dimicoli, Iliana; von Helden, Gert; Meijer, Gerard; Mons, Michel

2005-02-09

We report the first gas-phase spectroscopic study of a three-residue model of a peptide chain, Ac-Phe-Gly-Gly-NH2 (Ac = acetyl), using the IR/UV double resonance technique. The existence of at least five different conformers under supersonic expansion conditions is established, most of them exhibiting rather strong intramolecular H-bonds. One of the most populated conformers, however, exhibits a different H-bonding network characterized by two weak H-bonds. Comparison of the amide A and I/II experimental data with density functional theory calculations carried out on a series of selected conformations enables us to assign this conformer to two successive beta-turns along the peptide chain, the two H-bonds being of C10 type, i.e., each of them closing a 10-atom ring in the molecule. The corresponding form is found to be more stable than the 310 helix secondary structure (not observed), presumably because of specific effects due to the glycine residues.

How many TCR clonotypes does a body maintain?

PubMed Central

Lythe, Grant; Callard, Robin E.; Hoare, Rollo L.; Molina-París, Carmen

2016-01-01

We consider the lifetime of a T cell clonotype, the set of T cells with the same T cell receptor, from its thymic origin to its extinction in a multiclonal repertoire. Using published estimates of total cell numbers and thymic production rates, we calculate the mean number of cells per TCR clonotype, and the total number of clonotypes, in mice and humans. When there is little peripheral division, as in a mouse, the number of cells per clonotype is small and governed by the number of cells with identical TCR that exit the thymus. In humans, peripheral division is important and a clonotype may survive for decades, during which it expands to comprise many cells. We therefore devise and analyse a computational model of homeostasis of a multiclonal population. Each T cell in the model competes for self pMHC stimuli, cells of any one clonotype only recognising a small fraction of the many subsets of stimuli. A constant mean total number of cells is maintained by a balance between cell division and death, and a stable number of clonotypes by a balance between thymic production of new clonotypes and extinction of existing ones. The number of distinct clonotypes in a human body may be smaller than the total number of naive T cells by only one order of magnitude. PMID:26546971

A HYPOTHESIS ACCOUNTING FOR THE PARADOXICAL EXPRESSION OF THE D GENE SEGMENT IN THE BCR AND THE TCR

PubMed Central

Cohn, Melvin

2009-01-01

The D gene segment expressed in both the TCR and BCR has a challenging behavior that begs interpretation. It is incorporated in three reading frames in the rearranged transcription unit but is expressed in antigen-selected cells in a preferred frame. Why was it so important to waste 2/3 of newborn cells? The hypothesis is presented that the D region is framework playing a role in both the TCR and the BCR by determining whether a signal is transmitted to the cell upon interaction with a cognate ligand. This assumption operates in determining haplotype exclusion for the BCR and in regulating the signaling orientation for the TCR. Relevant data as well as a definitive experiment challenging the validity of this hypothesis, are discussed. PMID:18546143

Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.

PubMed

Utz, U; Banks, D; Jacobson, S; Biddison, W E

1996-02-01

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.

Beta-ketoacyl-acyl carrier protein synthase IV: a key enzyme for regulation of medium-chain fatty acid synthesis in Cuphea lanceolata seeds.

PubMed

Schütt, Burkhardt Siegfried; Abbadi, Amine; Loddenkötter, Brigitte; Brummel, Monika; Spener, Friedrich

2002-09-01

With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a beta-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C. lanceolata seed embryos. The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences. Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity. Further elongation steps were catalysed with distinctly less activity. Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV. Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C. lanceolata is presented.

Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis.

PubMed

Borlido, Joana; Sakuma, Stephen; Raices, Marcela; Carrette, Florent; Tinoco, Roberto; Bradley, Linda M; D'Angelo, Maximiliano A

2018-06-01

Nuclear pore complexes (NPCs) are channels connecting the nucleus with the cytoplasm. We report that loss of the tissue-specific NPC component Nup210 causes a severe deficit of naïve CD4 + T cells. Nup210-deficient CD4 + T lymphocytes develop normally but fail to survive in the periphery. The decreased survival results from both an impaired ability to transmit tonic T cell receptor (TCR) signals and increased levels of Fas, which sensitize Nup210 -/- naïve CD4 + T cells to Fas-mediated cell death. Mechanistically, Nup210 regulates these processes by modulating the expression of Cav2 (encoding Caveolin-2) and Jun at the nuclear periphery. Whereas the TCR-dependent and CD4 + T cell-specific upregulation of Cav2 is critical for proximal TCR signaling, cJun expression is required for STAT3-dependent repression of Fas. Our results uncover an unexpected role for Nup210 as a cell-intrinsic regulator of TCR signaling and T cell homeostasis and expose NPCs as key players in the adaptive immune system.

Maple syrup urine disease: The E1{beta} gene of human branched-chain {alpha}-ketoacid dehydrogenase complex has 11 rather than 10 exons, and the 3{prime} UTR in one of the two E1{beta} mRNAs arises from intronic sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, J.L.; Chuang, D.T.; Cox, R.P.

1996-06-01

Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is caused by a deficiency in the mitochondrial branched-chain {alpha}-ketoacid dehydrogenase (BCKAD) complex. The clinical manifestations are characterized by accumulation of branched chain amino and {alpha}-ketoacids, which leads to severe cerebral edema with seizures, ketoacidosis, and mental retardation. The BCKAD complex comprises three catalytic components, i.e., a decarboxylase (E1) consisting of two E1{alpha} (M{sub r} = 46,000) and two E1{Beta} (M{sub r} = 37,500) subunits, a transacylase (E2) that contains 24 lipoic acid-bearing subunits, and a dehydrogenase (E3), which is a homodimeric flavoprotein. MSUD is genetically heterogeneous, since mutations in the E1{alpha}more » subunit (type IA MSUD), the E1{Beta} subunit (type IB), the E2 subunit (type II) and the E3 subunit (type III) have been described. The functional consequences of certain mutations in the BCKAD complex have been studied. 23 refs., 3 figs.« less

An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8+ T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines

PubMed Central

Guio, Heinner

2018-01-01

Lung cancer is a leading cause of cancer-related death among both men and women in the United States, where non–small cell lung cancer accounts for ∼85% of lung cancer. Lung adenocarcinoma (ADC) is the major histologic subtype. The presence of actionable mutations prompts the use of therapies designed to specifically address the deleterious effects of those cancer-driving mutations; these therapies have already shown promise in cases carrying those actionable mutations (∼30%). Innovative therapeutic approaches are needed for the treatment of 70% of patients suffering from lung ADC. Adoptive transfer of CD8+ T cells specific against cancer/testis (CT) Ags, whose protein expression is restricted to the gonads (testis and ovary) and cancerous cells, is an excellent alternative. In this study, we report the isolation of HLA-A*02:01/CT37 peptide–specific α and β TCR chains from a CD8+ T cell clone obtained from a patient suffering from lung ADC. We also report the development of an innovative CD3ζ construct. With those TCR chains and the engineered (modified) CD3ζ chain, we produced a construct that when transduced into CD8+ T cells is capable of redirecting transduced CD8+ T cell cytotoxic activity and IFN-γ secretion against peptide-pulsed autologous cells and HLA-A*02:01–positive and CT37-expressing lung ADC cell lines. Our findings will launch the development of innovative adoptive transfer immunotherapies for the treatment of lung ADC, targeting the most prevalent HLA molecules and CT37 peptides restricted by these molecules. PMID:29555781

Heterogeneity in acute undifferentiated leukemia.

PubMed

LeMaistre, A; Childs, C C; Hirsch-Ginsberg, C; Reuben, J; Cork, A; Trujillo, J M; Andersson, B; McCredie, K B; Freireich, E; Stass, S A

1988-01-01

From January 1985 to May 1987, we studied 256 adults with newly diagnosed acute leukemia. Acute undifferentiated leukemia (AUL) was diagnosed in 12 of the 256 (4.6%) cases when lineage could not be delineated by light microscopy and light cytochemistry. To further characterize the blasts, immunophenotyping, ultrastructural myeloperoxidase (UMPO), and ultrastructural platelet peroxidase parameters were examined in 10, 11, and 6 of the 12 cases, respectively. Five cases demonstrated UMPO and were reclassified as acute myeloblastic leukemia (AML). Of the six UMPO-negative cases, three had a myeloid and one had a mixed immunophenotype. One UMPO-negative patient with a myeloid immunophenotype was probed for the immunoglobulin heavy chain gene (JH) and the beta chain of the T-cell receptor gene (Tcr beta) with no evidence of rearrangement. Six cases were treated with standard acute lymphoblastic leukemia (ALL) chemotherapy and failed to achieve complete remission (CR). Various AML chemotherapeutic regimens produced CR in only 3 of the 12 cases. One case was treated with gamma interferon and the other 2 with high-dose Ara-C. Our findings indicate a myeloid lineage can be detected by UMPO (5/12) in some cases of AUL. A germline configuration with JH and Tcr beta in one case as well as a myeloid immunophenotype in 3 UMPO-negative cases raises the possibility that myeloid lineage commitment may occur in the absence of myeloid peroxidase (MPO) cytochemical positivity.

Molecular analysis of the beta-thalassemia phenotype associated with inheritance of hemoglobin E (alpha 2 beta2(26)Glu leads to Lys).

PubMed Central

Benz, E J; Berman, B W; Tonkonow, B L; Coupal, E; Coates, T; Boxer, L A; Altman, A; Adams, J G

1981-01-01

Inheritance of the gene for betaE-globin is associated with hypochromia and microcytosis, reminiscent of typical heterozygous beta-thalassemia. Patients with hemoglobin (Hb)E-beta-thalassemia exhibit clinical phenotypes of severe beta-thalassemia, a circumstance not encountered in other compound heterozygous states for structural beta-chain mutations and beta-thalassemia. We have analyzed the kinetics of globin synthesis and the levels of globin messenger (m) RNA accumulation in patients with Hb E-beta-thalassemia and Hb E trait. The initial rate of beta-globin synthesis (betaE/alpha=0.20-0.34) was less than expected on the basis of gene dosage, or comparable studies of other compound heterozygous states for beta-thalassemia and structurally abnormal beta-chains. betaE-globin synthesis was not only reduced during short-term incubations (1-5 min), but also remained relatively unchanged during long-term pulse or chase incubations up to 5h. Analysis of globin mRNA by cell-free translation and molecular hybridization confirmed that the unexpectedly low levels of betaE-globin synthesis were associated with comparable reduction in the levels of beta-globin mRNA. In Hb E-beta-thalassemia the betaA + betaE (alpha globin nRNA ratio observed were substantially lower than those obtained from reticulocytes of patients with heterozygous beta-thalassemia, or Hb S-betaO-thalassemia, while in Hb E trait, the betaA + betaE/alpha mRNA ratio was in the ranged observed for beta-thalassemia trait. The betaE-globin gene specifies reduced accumulation of betaE-globin mRNA, a property characteristic of other forms of beta-thalassemia. The beta-thalassemia phenotype associated with inheritance of Hb E is thus determined at the level of beta-globin mRNA metabolism. PMID:6166632

Mechanism and function of Vav1 localisation in TCR signalling.

PubMed

Ksionda, Olga; Saveliev, Alexander; Köchl, Robert; Rapley, Jonathan; Faroudi, Mustapha; Smith-Garvin, Jennifer E; Wülfing, Christoph; Rittinger, Katrin; Carter, Tom; Tybulewicz, Victor L J

2012-11-15

The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4(+) and CD8(+) T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3(B)) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3(B) domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux.

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

PubMed Central

Tsang, Julia Yuen-Shan; Tanriver, Yakup; Jiang, Shuiping; Xue, Shao-An; Ratnasothy, Kulachelvy; Chen, Daxin; Stauss, Hans J.; Bucy, R. Pat; Lombardi, Giovanna; Lechler, Robert

2008-01-01

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules — a process known as indirect recognition. As CD4+CD25+ Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4+CD25+ cells from C57BL/6 mice (H-2b) with allogeneic DCs from BALB/c mice (H-2d). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2Kd presented by H-2Ab MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential. PMID:18846251

Short-chain fatty acids and poly-beta-hydroxyalkanoates: (New) Biocontrol agents for a sustainable animal production.

PubMed

Defoirdt, Tom; Boon, Nico; Sorgeloos, Patrick; Verstraete, Willy; Bossier, Peter

2009-01-01

Because of the risk of antibiotic resistance development, there is a growing awareness that antibiotics should be used more carefully in animal production. However, a decreased use of antibiotics could result in a higher frequency of pathogenic bacteria, which in its turn could lead to a higher incidence of infections. Short-chain fatty acids (SCFAs) have long been known to exhibit bacteriostatic activity. These compounds also specifically downregulate virulence factor expression and positively influence the gastrointestinal health of the host. As a consequence, there is currently considerable interest in SCFAs as biocontrol agents in animal production. Polyhydroxyalkanoates (PHAs) are polymers of beta-hydroxy short-chain fatty acids. Currently, PHAs are applied as replacements for synthetic polymers. These biopolymers can be depolymerised by many different microorganisms that produce extracellular PHA depolymerases. Interestingly, different studies provided some evidence that PHAs can also be degraded upon passage through the gastrointestinal tract of animals and consequently, adding these compounds to the feed might result in biocontrol effects similar to those described for SCFAs.

Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

PubMed

Langerak, A W; Molina, T J; Lavender, F L; Pearson, D; Flohr, T; Sambade, C; Schuuring, E; Al Saati, T; van Dongen, J J M; van Krieken, J H J M

2007-02-01

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell receptor (TCR) rearrangements can be useful. Here we address the issue of clonality assessment in 106 histologically defined reactive lesions, using the standardized BIOMED-2 Ig/TCR multiplex polymerase chain reaction (PCR) heteroduplex and GeneScan assays. Samples were reviewed nationally, except 10% random cases and cases with clonal results selected for additional international panel review. In total 75% (79/106) only showed polyclonal Ig/TCR targets (type I), whereas another 15% (16/106) represent probably polyclonal cases, with weak Ig/TCR (oligo)clonality in an otherwise polyclonal background (type II). Interestingly, in 10% (11/106) clear monoclonal Ig/TCR products were observed (types III/IV), which prompted further pathological review. Clonal cases included two missed lymphomas in national review and nine cases that could be explained as diagnostically difficult cases or probable lymphomas upon additional review. Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal character in the vast majority of reactive lesions. However, clonality detection in a minority should lead to detailed pathological review, including close interaction between pathologist and molecular biologist.

Harnessing the theoretical foundations of the exponential and beta-Poisson dose-response models to quantify parameter uncertainty using Markov Chain Monte Carlo.

PubMed

Schmidt, Philip J; Pintar, Katarina D M; Fazil, Aamir M; Topp, Edward

2013-09-01

Dose-response models are the essential link between exposure assessment and computed risk values in quantitative microbial risk assessment, yet the uncertainty that is inherent to computed risks because the dose-response model parameters are estimated using limited epidemiological data is rarely quantified. Second-order risk characterization approaches incorporating uncertainty in dose-response model parameters can provide more complete information to decisionmakers by separating variability and uncertainty to quantify the uncertainty in computed risks. Therefore, the objective of this work is to develop procedures to sample from posterior distributions describing uncertainty in the parameters of exponential and beta-Poisson dose-response models using Bayes's theorem and Markov Chain Monte Carlo (in OpenBUGS). The theoretical origins of the beta-Poisson dose-response model are used to identify a decomposed version of the model that enables Bayesian analysis without the need to evaluate Kummer confluent hypergeometric functions. Herein, it is also established that the beta distribution in the beta-Poisson dose-response model cannot address variation among individual pathogens, criteria to validate use of the conventional approximation to the beta-Poisson model are proposed, and simple algorithms to evaluate actual beta-Poisson probabilities of infection are investigated. The developed MCMC procedures are applied to analysis of a case study data set, and it is demonstrated that an important region of the posterior distribution of the beta-Poisson dose-response model parameters is attributable to the absence of low-dose data. This region includes beta-Poisson models for which the conventional approximation is especially invalid and in which many beta distributions have an extreme shape with questionable plausibility. © Her Majesty the Queen in Right of Canada 2013. Reproduced with the permission of the Minister of the Public Health Agency of Canada.

Clonal T-Cell Receptor γ-Chain Gene Rearrangements in Differential Diagnosis of Lymphomatoid Papulosis From Skin Metastasis of Nodal Anaplastic Large-Cell Lymphoma

PubMed Central

Akilov, Oleg E.; Pillai, Raju K.; Grandinetti, Lisa M.; Kant, Jeffrey A.; Geskin, Larisa

2012-01-01

Background In patients with a history of nodal anaplastic large-cell lymphoma (ALCL), differentiation of type C lymphomatoid papulosis from cutaneous involvement of systemic ALCL may be challenging because the 2 entities may exhibit identical histologic features. Although metastatic ALCL generally carries the same clone as the primary lymphoma, expression of a distinct clone likely represents a distinct process. Observations A 54-year-old white man had a history of anaplastic lymphoma kinase 1–negative ALCL in the right inguinal lymph node 6 years ago. A complete response was achieved after 6 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine [Oncovin], and prednisone administered in 21-day cycles) and radiation therapy. After 3½ years, the patient observed waxing and waning papules and nodules. Examination of the biopsy specimen revealed a dense CD30+ lymphocytic infiltrate; no evidence of systemic malignancy was evident on positron emission tomography. Although clinically the presentation was consistent with lymphomatoid papulosis, metastatic ALCL had to be excluded. Polymerase chain reaction analysis with T-cell receptor γ-chain gene rearrangement (TCR-γR) was performed on the original lymph node and new skin lesions. Results of the TCR-γR analysis were positive for clonality in both lesions. However, separate clonal processes were identified. The identification of distinct clones supported the clinical impression of lymphomatoid papulosis. Conclusion Polymerase chain reaction analysis of TCR-γR is a useful method for distinguishing different clonal processes and is recommended when differentiation of primary and secondary lymphoproliferative disorders is required. PMID:21844453

Functional CD1d and/or NKT cell invariant chain transcript in horse, pig, African elephant and guinea pig, but not in ruminants.

PubMed

Looringh van Beeck, Frank A; Reinink, Peter; Hermsen, Roel; Zajonc, Dirk M; Laven, Marielle J; Fun, Axel; Troskie, Milana; Schoemaker, Nico J; Morar, Darshana; Lenstra, Johannes A; Vervelde, Lonneke; Rutten, Victor P M G; van Eden, Willem; Van Rhijn, Ildiko

2009-04-01

CD1d-restricted invariant natural killer T cells (NKT cells) have been well characterized in humans and mice, but it is unknown whether they are present in other species. Here we describe the invariant TCR alpha chain and the full length CD1d transcript of pig and horse. Molecular modeling predicts that porcine (po) invariant TCR alpha chain/poCD1d/alpha-GalCer and equine (eq) invariant TCR alpha chain/eqCD1d/alpha-GalCer form complexes that are highly homologous to the human complex. Since a prerequisite for the presence of NKT cells is the expression of CD1d protein, we performed searches for CD1D genes and CD1d transcripts in multiple species. Previously, cattle and guinea pig have been suggested to lack CD1D genes. The CD1D genes of European taurine cattle (Bos taurus) are known to be pseudogenes because of disrupting mutations in the start codon and in the donor splice site of the first intron. Here we show that the same mutations are found in six other ruminants: African buffalo, sheep, bushbuck, bongo, N'Dama cattle, and roe deer. In contrast, intact CD1d transcripts were found in guinea pig, African elephant, horse, rabbit, and pig. Despite the discovery of a highly homologous NKT/CD1d system in pig and horse, our data suggest that functional CD1D and CD1d-restricted NKT cells are not universally present in mammals.

Molecular basis of beta-thalassemia in the Maldives.

PubMed

Furuumi, H; Firdous, N; Inoue, T; Ohta, H; Winichagoon, P; Fucharoen, S; Fukumaki, Y

1998-03-01

We have systematically analyzed beta-thalassemia genes using polymerase chain reaction-related techniques, dot-blot hybridization with oligonucleotide probes, allele specific-polymerase chain reaction, and sequencing of amplified DNA fragments from 41 unrelated patients, including 37 beta-thalassemia homozygotes, three with beta-thalassemia/Hb E, and one with beta-thalassemia/Hb S. Four different beta-thalassemia mutations were detected in 78 alleles. These are the IVS-I-5 (G-->C), codon 30 (AGG-->ACG) [also indicated as IVS-I (-1)], IVS-I-1 (G-->A), and codons 41/42 (-TTCT) mutations. The distribution of the beta-thalassemia mutations in the Maldives is 58 alleles (74.3%) with the IVS-I-5 (G-->C) mutation, 12 (15.4%) with the codon 30 (AGG-->ACG) mutation, seven (9%) with the IVS-I-1 (G-->A) mutation, and one with the codons 41/42 (-TTCT) mutation. The first three mutations account for 98.7% of the total number of beta-thalassemia chromosomes studied. These mutations are clustered in the region spanning 6 bp around the junction of exon 1 and the first intervening sequence of the beta-globin gene. These observations have significant implications for setting up a thalassemia prevention and control program in the Maldives. Analysis of haplotypes and frameworks of chromosomes bearing each beta-thalassemia mutation suggested that the origin and spread of these mutations were reflected by the historical record.

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

PubMed

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

Synthesis and cytotoxic analysis of some disodium 3beta,6beta-dihydroxysterol disulfates.

PubMed

Cui, Jianguo; Wang, Hui; Huang, Yanmin; Xin, Yi; Zhou, Aimin

2009-01-01

Disodium 3beta,6beta-dihydroxy-5alpha-cholestane disulfate (1) was synthesized in 4 steps with a high overall yield from cholesterol. First, cholesterol (4a) was converted to cholest-4-en-3,6-dione (5a) via oxidation with pyridinium chlorochromate (PCC) and then 5a was reduced by NaBH(4) in the presence of NiCl(2) to produce cholest-3beta,6beta-diol (6a). The reaction of 6a with the triethylamine-sulfur trioxide complex generated diammonium 3beta,6beta-dihydroxy-5alpha-cholestane disulfate (7a) and the treatment of 7a by cation exchange resin 732 (sodium form)(Na(+)) yielded the target steroid 1. Disodium 24-ethyl-3beta,6beta-dihydroxycholest-22-ene disulfate (2) and disodium 24-ethyl-3beta,6beta-dihydroxycholestane disulfate (3) were synthesized using a similar method. The cytotoxicity of these compounds against Sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells was investigated. Our results indicate that presence of a cholesterol-type side chain at position 17 is necessary for their biological activity.

Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study

PubMed Central

Boonyawat, Boonchai; Monsereenusorn, Chalinee; Traivaree, Chanchai

2014-01-01

Background Beta-thalassemia is one of the most common genetic disorders in Thailand. Clinical phenotype ranges from silent carrier to clinically manifested conditions including severe beta-thalassemia major and mild beta-thalassemia intermedia. Objective This study aimed to characterize the spectrum of beta-globin gene mutations in pediatric patients who were followed-up in Phramongkutklao Hospital. Patients and methods Eighty unrelated beta-thalassemia patients were enrolled in this study including 57 with beta-thalassemia/hemoglobin E, eight with homozygous beta-thalassemia, and 15 with heterozygous beta-thalassemia. Mutation analysis was performed by multiplex amplification refractory mutation system (M-ARMS), direct DNA sequencing of beta-globin gene, and gap polymerase chain reaction for 3.4 kb deletion detection, respectively. Results A total of 13 different beta-thalassemia mutations were identified among 88 alleles. The most common mutation was codon 41/42 (-TCTT) (37.5%), followed by codon 17 (A>T) (26.1%), IVS-I-5 (G>C) (8%), IVS-II-654 (C>T) (6.8%), IVS-I-1 (G>T) (4.5%), and codon 71/72 (+A) (2.3%), and all these six common mutations (85.2%) were detected by M-ARMS. Six uncommon mutations (10.2%) were identified by DNA sequencing including 4.5% for codon 35 (C>A) and 1.1% initiation codon mutation (ATG>AGG), codon 15 (G>A), codon 19 (A>G), codon 27/28 (+C), and codon 123/124/125 (-ACCCCACC), respectively. The 3.4 kb deletion was detected at 4.5%. The most common genotype of beta-thalassemia major patients was codon 41/42 (-TCTT)/codon 26 (G>A) or betaE accounting for 40%. Conclusion All of the beta-thalassemia alleles have been characterized by a combination of techniques including M-ARMS, DNA sequencing, and gap polymerase chain reaction for 3.4 kb deletion detection. Thirteen mutations account for 100% of the beta-thalassemia genes among the pediatric patients in our study. PMID:25525381

An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8+ T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines.

PubMed

Flores-Villanueva, Pedro O; Ganachari, Malathesha; Guio, Heinner; Mejia, Jaime A; Granados, Julio

2018-04-15

Lung cancer is a leading cause of cancer-related death among both men and women in the United States, where non-small cell lung cancer accounts for ∼85% of lung cancer. Lung adenocarcinoma (ADC) is the major histologic subtype. The presence of actionable mutations prompts the use of therapies designed to specifically address the deleterious effects of those cancer-driving mutations; these therapies have already shown promise in cases carrying those actionable mutations (∼30%). Innovative therapeutic approaches are needed for the treatment of 70% of patients suffering from lung ADC. Adoptive transfer of CD8 + T cells specific against cancer/testis (CT) Ags, whose protein expression is restricted to the gonads (testis and ovary) and cancerous cells, is an excellent alternative. In this study, we report the isolation of HLA-A*02:01/CT37 peptide-specific α and β TCR chains from a CD8 + T cell clone obtained from a patient suffering from lung ADC. We also report the development of an innovative CD3ζ construct. With those TCR chains and the engineered (modified) CD3ζ chain, we produced a construct that when transduced into CD8 + T cells is capable of redirecting transduced CD8 + T cell cytotoxic activity and IFN-γ secretion against peptide-pulsed autologous cells and HLA-A*02:01 -positive and CT37-expressing lung ADC cell lines. Our findings will launch the development of innovative adoptive transfer immunotherapies for the treatment of lung ADC, targeting the most prevalent HLA molecules and CT37 peptides restricted by these molecules. Copyright © 2018 by The American Association of Immunologists, Inc.

Expression of TGF-beta1, osteonectin, and BMP-4 in mandibular distraction osteogenesis with compression stimulation: reverse transcriptase-polymerase chain reaction study and biomechanical test.

PubMed

Kim, Uk-Kyu; Park, Seong-Jin; Seong, Wook-Jin; Heo, Jun; Hwang, Dae-Seok; Kim, Yong-Deok; Shin, Sang-Hun; Kim, Gyoo-Cheon

2010-09-01

This study compared the levels of transforming growth factor-beta1 (TGF-beta1), osteonectin, and bone morphogenetic protein-4 (BMP-4) expression in regenerated bone in a rabbit mandible that had undergone conventional distraction osteogenesis (DO) with those in regenerated bone from a modified DO technique with compression stimulation. A total of 42 rabbits were used in this reverse transcriptase-polymerase chain reaction study. In the control group, distraction was performed at 1 mm/day for 8 days. In the experimental group, overdistraction was performed for 10 days, followed by a 3-day latency period and 2 days of compression to achieve the same amount of DO. Three rabbits per subgroup were killed at 0, 5, 13, 20, 27, 34, and 41 days after the initial osteotomy. The levels of TGF-beta1, osteonectin, and BMP-4 in the bone regenerates were measured by reverse transcriptase-polymerase chain reaction. A biomechanical microhardness test was also performed in 8 rabbits as a separate experiment. Reverse transcriptase-polymerase chain reaction revealed a greater level of TGF-beta1 in the experimental group immediately after applying the compression force that continued for 2 weeks. The level then decreased to that of the control group at 3 weeks. The greater level of osteonectin in the experimental group after compression than that in the control group continued for 3 weeks. In the experimental group, the level of BMP-4 increased immediately after compression. However, the level in the control group decreased. The microhardness ratio of distracted bone to normal bone on the cortex was statistically different at 0.47 in the control group and 0.80 in the experimental group (P = .049) at 55 days after osteotomy. The effectiveness of the new DO technique with compression stimulation was confirmed by the gene expression study and the biomechanical test findings. Copyright 2010 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights

Prediction of beta-turns from amino acid sequences using the residue-coupled model.

PubMed

Guruprasad, K; Shukla, S

2003-04-01

We evaluated the prediction of beta-turns from amino acid sequences using the residue-coupled model with an enlarged representative protein data set selected from the Protein Data Bank. Our results show that the probability values derived from a data set comprising 425 protein chains yielded an overall beta-turn prediction accuracy 68.74%, compared with 94.7% reported earlier on a data set of 30 proteins using the same method. However, we noted that the overall beta-turn prediction accuracy using probability values derived from the 30-protein data set reduces to 40.74% when tested on the data set comprising 425 protein chains. In contrast, using probability values derived from the 425 data set used in this analysis, the overall beta-turn prediction accuracy yielded consistent results when tested on either the 30-protein data set (64.62%) used earlier or a more recent representative data set comprising 619 protein chains (64.66%) or on a jackknife data set comprising 476 representative protein chains (63.38%). We therefore recommend the use of probability values derived from the 425 representative protein chains data set reported here, which gives more realistic and consistent predictions of beta-turns from amino acid sequences.

Immunoglobulin Heavy Chain Exclusion in the Shark

PubMed Central

Malecek, Karolina; Lee, Victor; Feng, Wendy; Huang, Jing Li; Flajnik, Martin F; Ohta, Yuko; Hsu, Ellen

2008-01-01

The adaptive immune system depends on specific antigen receptors, immunoglobulins (Ig) in B lymphocytes and T cell receptors (TCR) in T lymphocytes. Adaptive responses to immune challenge are based on the expression of a single species of antigen receptor per cell; and in B cells, this is mediated in part by allelic exclusion at the Ig heavy (H) chain locus. How allelic exclusion is regulated is unclear; we considered that sharks, the oldest vertebrates possessing the Ig/TCR-based immune system, would yield insights not previously approachable and reveal the primordial basis of the regulation of allelic exclusion. Sharks have an IgH locus organization consisting of 15–200 independently rearranging miniloci (VH-D1-D2-JH-Cμ), a gene organization that is considered ancestral to the tetrapod and bony fish IgH locus. We found that rearrangement takes place only within a minilocus, and the recombining gene segments are assembled simultaneously and randomly. Only one or few H chain genes were fully rearranged in each shark B cell, whereas the other loci retained their germline configuration. In contrast, most IgH were partially rearranged in every thymocyte (developing T cell) examined, but no IgH transcripts were detected. The distinction between B and T cells in their IgH configurations and transcription reveals a heretofore unsuspected chromatin state permissive for rearrangement in precursor lymphocytes, and suggests that controlled limitation of B cell lineage-specific factors mediate regulated rearrangement and allelic exclusion. This regulation may be shared by higher vertebrates in which additional mechanistic and regulatory elements have evolved with their structurally complex IgH locus. PMID:18578572

Three-dimensional crystal structure of recombinant murine interferon-beta.

PubMed Central

Senda, T; Shimazu, T; Matsuda, S; Kawano, G; Shimizu, H; Nakamura, K T; Mitsui, Y

1992-01-01

The crystal structure of recombinant murine interferon-beta (IFN-beta) has been solved by the multiple isomorphous replacement method and refined to an R-factor of 20.5% against 2.6 A X-ray diffraction data. The structure shows a variant of the alpha-helix bundle with a new chain-folding topology, which seems to represent a basic structural framework of all the IFN-alpha and IFN-beta molecules belonging to the type I family. Functionally important segments of the polypeptide chain, as implied through numerous gene manipulation studies carried out so far, are spatially clustered indicating the binding site(s) to the receptor(s). Comparison of the present structure with those of other alpha-helical cytokine proteins, including porcine growth hormone, interleukin 2 and interferon gamma, indicated either a topological similarity in chain folding or a similar spatial arrangement of the alpha-helices. Images PMID:1505514

Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity.

PubMed

Matsunaga, Toshiyuki; Endo, Satoshi; Maeda, Satoshi; Ishikura, Shuhei; Tajima, Kazuo; Tanaka, Nobutada; Nakamura, Kazuo T; Imamura, Yorishige; Hara, Akira

2008-09-15

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

Mechanism and function of Vav1 localisation in TCR signalling

PubMed Central

Ksionda, Olga; Saveliev, Alexander; Köchl, Robert; Rapley, Jonathan; Faroudi, Mustapha; Smith-Garvin, Jennifer E.; Wülfing, Christoph; Rittinger, Katrin; Carter, Tom; Tybulewicz, Victor L. J.

2012-01-01

Summary The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4+ and CD8+ T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3B) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3B domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux. PMID:22956543

Innate signals overcome acquired TCR signaling pathway regulation and govern the fate of human CD161(hi) CD8α⁺ semi-invariant T cells.

PubMed

Turtle, Cameron J; Delrow, Jeff; Joslyn, Rochelle C; Swanson, Hillary M; Basom, Ryan; Tabellini, Laura; Delaney, Colleen; Heimfeld, Shelly; Hansen, John A; Riddell, Stanley R

2011-09-08

Type 17 programmed CD161(hi)CD8α(+) T cells contribute to mucosal immunity to bacteria and yeast. In early life, microbial colonization induces proliferation of CD161(hi) cells that is dependent on their expression of a semi-invariant Vα7.2(+) TCR. Although prevalent in adults, CD161(hi)CD8α(+) cells exhibit weak proliferative and cytokine responses to TCR ligation. The mechanisms responsible for the dichotomous response of neonatal and adult CD161(hi) cells, and the signals that enable their effector function, have not been established. We describe acquired regulation of TCR signaling in adult memory CD161(hi)CD8α(+) T cells that is absent in cord CD161(hi) cells and adult CD161(lo) cells. Regulated TCR signaling in CD161(hi) cells was due to profound alterations in TCR signaling pathway gene expression and could be overcome by costimulation through CD28 or innate cytokine receptors, which dictated the fate of their progeny. Costimulation with IL-1β during TCR ligation markedly increased proinflammatory IL-17 production, while IL-12-induced Tc1-like function and restored the response to TCR ligation without costimulation. CD161(hi) cells from umbilical cord blood and granulocyte colony stimulating factor-mobilized leukaphereses differed in frequency and function, suggesting future evaluation of the contribution of CD161(hi) cells in hematopoietic stem cell grafts to transplant outcomes is warranted.

Bet v 1-specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1-specific T-cell effector function in an activation-dependent manner.

PubMed

Schmetterer, Klaus G; Haiderer, Daniela; Leb-Reichl, Victoria M; Neunkirchner, Alina; Jahn-Schmid, Beatrice; Küng, Hans J; Schuch, Karina; Steinberger, Peter; Bohle, Barbara; Pickl, Winfried F

2011-01-01

Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αβ-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αβ-chains. cDNAs encoding the α and β-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become

[Characterization of microstructure of ibuprofen-hydroxypropyl-beta-cyclodextrin and ibuprofen-beta-cyclodextrin by atomic force microscope].

PubMed

Wang, Li-juan; Zhu, Zhao-jing; Che, Ke-ke; Ju, Feng-ge

2008-09-01

The microstructures of ibuprofen-hydroxypropyl-bets-cyclodextrin (IBU-HP-beta-CyD) and ibuprofen-beta-cyclodextrin (IBU-beta-CyD) were observed by atomic force microscope (AFM). The high resolving capability of AFM has the tungsten filament probe with the spring constant of 0.06 N x m(-1). Samples were observed in a small scale scanning area of 10.5 nm x 10.5 nm and 800 x 800 pixels. The original scanning images were gained by tapping mode at room temperature. Their three-dimensional reconstruction of microstructure was performed by G3DR software. The outer diameters of HP-beta-CyD and beta-CyD are 1.53 nm. The benzene diameter of IBU is 0.62 nm, fitting to the inner diameters of HP-beta-CyD and beta-CyD. The benzene and hydrophobic chain of IBU enter into the hole of cyclodextrin at 1:1 ratio. The results were evidenced by IR, X-ray diffraction and the phase solubility.

Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6.

PubMed

Draper, Lindsey M; Kwong, Mei Li M; Gros, Alena; Stevanović, Sanja; Tran, Eric; Kerkar, Sid; Raffeld, Mark; Rosenberg, Steven A; Hinrichs, Christian S

2015-10-01

The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV(+) tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV(+) tumor cells. T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV(+) epithelial tumor cells. We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01-restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01-restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16(+) cervical, and head and neck cancer cell lines. These findings demonstrate that HPV-16(+) tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16(+) malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers. ©2015 American Association for Cancer Research.

Infection of human T lymphotropic virus-I-specific immune T cell clones by human T lymphotropic virus-I.

PubMed Central

Mitsuya, H; Jarrett, R F; Cossman, J; Cohen, O J; Kao, C S; Guo, H G; Reitz, M S; Broder, S

1986-01-01

Human T lymphotropic virus-I (HTLV-I)-specific T cell lines were established and cloned. K5, an OKT8+ clone bearing multiple proviral integration sites, retained its HTLV-I-specific cytotoxicity and a normal dependence on interleukin 2 (IL-2), indicating that there is a finite number of transforming integration sites. R2, an OKT4+ HTLV-I-infected clone, initially mounted a proliferative response to HTLV-I; but then its IL-2-independent proliferation increased and the antigen specificity was lost. All HTLV-I-infected clones tested including K7, another OKT8+ transformed cytotoxic clone that had lost its reactivity, expressed comparable levels of T cell receptor beta-chain (TCR-beta) messenger (m)RNA. Although clones K5 and K7 had different functional properties, they had the same rearrangement of the TCR-beta gene, suggesting that they had the same clonal origin. These data indicate that HTLV-I-specific T cells retain their immune reactivity for variable periods of time following infection, but then usually lose it; in some cases, however, no alteration in function can be detected. The data also suggest that different consequences can take place in the same clone depending on the pattern of retroviral infection. Images PMID:2877011

TCR-engineered, customized, antitumor T cells for cancer immunotherapy: advantages and limitations.

PubMed

Chhabra, Arvind

2011-01-05

The clinical outcome of the traditional adoptive cancer immunotherapy approaches involving the administration of donor-derived immune effectors, expanded ex vivo, has not met expectations. This could be attributed, in part, to the lack of sufficient high-avidity antitumor T-cell precursors in most cancer patients, poor immunogenicity of cancer cells, and the technological limitations to generate a sufficiently large number of tumor antigen-specific T cells. In addition, the host immune regulatory mechanisms and immune homeostasis mechanisms, such as activation-induced cell death (AICD), could further limit the clinical efficacy of the adoptively administered antitumor T cells. Since generation of a sufficiently large number of potent antitumor immune effectors for adoptive administration is critical for the clinical success of this approach, recent advances towards generating customized donor-specific antitumor-effector T cells by engrafting human peripheral blood-derived T cells with a tumor-associated antigen-specific transgenic T-cell receptor (TCR) are quite interesting. This manuscript provides a brief overview of the TCR engineering-based cancer immunotherapy approach, its advantages, and the current limitations.

Genes del receptor variable beta de células T en células circulantes de pacientes con lupus eritematoso generalizado y sus familiares sanos.

PubMed

Jakez-Ocampo, Juan; Paulín-Vera, Carmen María; Rivadeneyra-Espinoza, Liliana; Gómez-Martín, Diana; Carrillo-Maravilla, Eduardo; Lima, Guadalupe; Vargas-Rojas, María Inés; Pérez-Romano, Beatriz; Calva-Cevenini, Gabriella; García-Carrasco, Mario; Ruiz-Argüelles, Alejandro; Llorente, Luis

Se investigó la proporción de la expresión génica del receptor variable beta de células T (Vβ TCR) en linfocitos periféricos CD3+ en pacientes con lupus eritematoso generalizado (LEG) familiar y no familiar. El repertorio de Vβ TCR se estudió en 14 familias que presentaban más de un miembro con LEG. El uso de Vβ TCR en pacientes con LEG (n = 27) se comparó con el de los miembros sanos de estas familias (n = 47), con 37 pacientes con LEG esporádico y con 15 controles sanos. La expresión del repertorio de Vβ TCR se estudió por citometría de flujo multiparamétrica utilizando un arreglo de 24 diferentes anticuerpos monoclonales específicos de genes familiares para Vβ TCR. Se encontró el mismo perfil de expresión en las comparaciones entre los casos de LEG esporádico y familiar, así como en los consanguíneos sanos de las familias multicasos, que incluía una expresión incrementada de Vβ 5.2, Vβ 11 y Vβ 16, y una menor expresión de Vβ 3, Vβ4, Vβ 7.1 y Vβ 7. De manera interesante, solo Vβ 17 se expresó de modo diferente entre casos familiares y esporádicos de LEG. Igualmente, la expresión incrementada de Vβ 9 fue el distintivo entre los casos de LEG familiar (casos y consanguíneos sanos) y los controles sanos. Estos resultados refuerzan la noción de que el perfil final del repertorio Vβ TCR observado en LEG familiar y no familiar parece surgir de la interacción de factores genéticos, ambientales e inmunorreguladores, además de que pueden explicar las alteraciones inmunitarias que se observan en los consanguíneos sanos de pacientes con LEG. Copyright: © 2018 SecretarÍa de Salud

Natural polypeptide scaffolds: beta-sheets, beta-turns, and beta-hairpins.

PubMed

Rotondi, Kenneth S; Gierasch, Lila M

2006-01-01

This paper provides an introduction to fundamental conformational states of polypeptides in the beta-region of phi,psi space, in which the backbone is extended near to its maximal length, and to more complex architectures in which extended segments are linked by turns and loops. There are several variants on these conformations, and they comprise versatile scaffolds for presentation of side chains and backbone amides for molecular recognition and designed catalysts. In addition, the geometry of these fundamental folds can be readily mimicked in peptidomimetics. Copyright 2005 Wiley Periodicals, Inc.

Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths.

PubMed Central

Snyder, S W; Ladror, U S; Wade, W S; Wang, G T; Barrett, L W; Matayoshi, E D; Huffaker, H J; Krafft, G A; Holzman, T F

1994-01-01

One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and

New developments and prospective applications for beta (1,3) glucans.

PubMed

Laroche, Celine; Michaud, Philippe

2007-01-01

Publications and patents relative to newly observed functions of beta-(1,3)-D-glucans have notably increased in the last few years with the exploitation of their biological activities. The term beta-(1,3)-D-glucans includes a very large number of polysaccharides from bacterial, fungal and vegetable sources. Their structures have a common backbone of beta-(1,3) linked glucopyranosyl residues but the polysaccharidic chain can be beta-(1,6) branched with glucose or integrate some beta-(1,4) linked glucopyranosyl residues in the main chain. Except for the curdlan, a bacterial linear beta-(1,3)-D-glucans, and for the scleroglucan produced by Sclerotium rolfsii, the main drawback limiting the development of these polysaccharides is the lack of efficient processes for their extraction and purification and their cost. However new applications in agronomy, foods, cosmetic and therapeutic could in a next future accentuate the effort of research for their development. So this review focuses on these beta-(1,3)-D-glucans with the objective to detail the strategies employed for their extraction and the relation structure-functions identified when they induce biological activities.

Molecular analysis of abnormal hemoglobins in beta chain in Aegean region of Turkey and first reports of hemoglobin Andrew-Minneapolis and Hb Hinsdale from Turkey.

PubMed

Aykut, Ayça; Onay, Hüseyin; Durmaz, Asude; Karaca, Emin; Vergin, Canan; Aydınok, Yeşim; Özkınay, Ferda

2015-07-01

The Agean is one of the regions in Turkey where thalassemias and abnormal hemoglobins (Hbs) are prevalent. Combined heterozygosity of thalassemia mutations with a variety of structural Hb variants lead to an extremely wide spectrum of clinical and hematological phenotypes which is of importance for prenatal diagnosis. One hundred and seventeen patients and carriers diagnosed by hemoglobin electrophoresis (HPLC), at risk for abnormal hemoglobinopathies were screened for mutational analysis of the beta-globin gene. The full coding the 5' UTR, and the 3' UTR sequences of beta-globin gene (GenBank accession no. U01317) were amplified and sequenced. In this study, a total of 118 (12.24%) structural Hb variant alleles were identified in 1341 mutated beta-chain alleles in Medical Genetics Department of Ege University between January 2006 and November 2013. Here, we report the mutation spectrum of abnormal Hbs associated with the beta-globin gene in Aegean region of Turkey. In the present study, the Hb Hinsdale and Hb Andrew-Minneapolis variants are demonstrated for the first time in the Turkish population.

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

PubMed Central

Nunes-Alves, Cláudio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

2015-01-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

PubMed

Nunes-Alves, Cláudio; Booty, Matthew G; Carpenter, Stephen M; Rothchild, Alissa C; Martin, Constance J; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M

2015-05-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

beta-Methyl-15-p-iodophenylpentadecanoic acid metabolism and kinetics in the isolated rat heart.

PubMed

DeGrado, T R; Holden, J E; Ng, C K; Raffel, D M; Gatley, S J

1989-01-01

The use of 15-p-iodophenyl-beta-methyl-pentadecanoic acid (beta Me-IPPA) as an indicator of long chain fatty acid (LCFA) utilization in nuclear medicine studies was evaluated in the isolated, perfused, working rat heart. Time courses of radioactivity (residue curves) were obtained following bolus injections of both beta Me-IPPA and its straight chain counterpart 15-p-iodophenyl-pentadecanoic acid (IPPA). IPPA kinetics clearly indicated flow independent impairment of fatty acid oxidation caused by the carnitine palmitoyltransferase I inhibitor 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA). In contrast, beta Me-IPPA kinetics were insensitive to changes in fatty acid oxidation rate and net utilization of long chain fatty acid. Analysis of radiolabeled species in coronary effluent and heart homogenates showed the methylated fatty acid to be readily incorporated into complex lipids but a poor substrate for oxidation. POCA did not significantly alter metabolism of the tracer, suggesting that the tracer is poorly metabolized beyond beta Me-IPPA-CoA in the oxidative pathway.

TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

PubMed

Hawse, William F; Boggess, William C; Morel, Penelope A

2017-07-15

The Akt/mTOR pathway is a key driver of murine CD4 + T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. Copyright © 2017 by The American Association of Immunologists, Inc.

TCR-pMHC encounter differentially regulates transcriptomes of tissue-resident CD8 T cells.

PubMed

Yoshizawa, Akihiro; Bi, Kevin; Keskin, Derin B; Zhang, Guanglan; Reinhold, Bruce; Reinherz, Ellis L

2018-01-01

To investigate the role of TCR-pMHC interaction in regulating lung CD8 tissue-resident T cell (T R ) differentiation, polyclonal responses were compared against NP 366-374 /D b and PA 224-233 /D b , two immunodominant epitopes that arise during influenza A infection in mice. Memory niches distinct from iBALTs develop within the lamina propria, supporting CD103 + and CD103 - CD8 T R generation and intraepithelial translocation. Gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) identify dominant TCR, adherens junction, RIG-I-like and NOD-like pattern recognition receptor as well as TGF-β signaling pathways and memory signatures among PA 224-233 /D b T cells consistent with T resident memory (T RM ) status. In contrast, NP 366-374 /D b T cells exhibit enrichment of effector signatures, upregulating pro-inflammatory mediators even among T RM . While NP 366-374 /D b T cells manifest transcripts linked to canonical exhaustion pathways, PA 224-233 /D b T cells exploit P2rx7 purinoreceptor attenuation. The NP 366-374 /D b CD103 + subset expresses the antimicrobial lactotransferrin whereas PA 224-233 /D b CD103 + utilizes pore-forming mpeg-1, with <22% of genes correspondingly upregulated in CD103 + (or CD103 - ) subsets of both specificities. Thus, TCR-pMHC interactions among T R and antigen presenting cells in a tissue milieu strongly impact CD8 T cell biology. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Clostridium perfringens enterotoxin is a superantigen reactive with human T cell receptors V beta 6.9 and V beta 22

PubMed Central

1992-01-01

Candidate superantigens were screened for their ability to induce lysis of human histocompatibility leukocyte antigen class II-positive targets by human CD8+ influenza-specific cytotoxic T cell (CTL) lines. Clostridium perfringens enterotoxin (CPET) induced major histocompatibility complex unrestricted killing by some but not all CTL lines. Using "anchored" polymerase chain reactions, CPET was shown to selectively stimulate peripheral blood lymphocytes bearing T cell receptor V beta 6.9 and V beta 22 in five healthy donors. V beta 24, V beta 21, V beta 18, V beta 5, and V beta 6.1-5 appeared to be weakly stimulated. Antigen processing was not required for CPET to induce proliferation. Like the staphylococcal enterotoxins, CPET is a major cause of food poisoning. These data suggest that superantigenic and enterotoxigenic properties may be closely linked. PMID:1512551

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation.

PubMed

Abraham, Libin; Bankhead, Peter; Pan, Xiaoyu; Engel, Ulrike; Fackler, Oliver T

2012-08-15

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

Mutation of Arg723Gly in beta-myosin heavy chain gene in five Chinese families with hypertrophic cardiomyopathy.

PubMed

Yang, Jun-hua; Zheng, Dong-dong; Dong, Ning-zheng; Yang, Xiang-jun; Song, Jian-ping; Jiang, Ting-bo; Cheng, Xu-jie; Li, Hong-xia; Zhou, Bing-yuan; Zhao, Cai-ming; Jiang, Wen-ping

2006-11-05

Hypertrophic cardiomyopathy (HCM) is a form of cardiomyopathy with an autosomal dominant inherited disease, which is caused by mutations in at least one of the sarcomeric protein genes. Mutations in the beta-myosin heavy chain (beta-MHC) are the most common cause of HCM. This study was to reveal the disease-causing gene mutations in Chinese population with HCM, and to analyze the correlation between the genotype and phenotype. The exons 3 to 26 of MYH7 were amplified by PCR, and the PCR products were sequenced in five non-kin HCM patients. A 17-year-old patient was detected to be an Arg723Gly mutation carrier. Then his family was gene-screened, and the correlation between genotype and phenotype was analyzed. The mutation of Arg723Gly in a Chinese family with HCM was detected for the first time. With a C-G transversion in nucleotide 13,619 of the MYH7 gene, located at the essential light chain interacting region in S1, the replacement of arginine by glycine took place at amino acid residue 723. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atria enlargement. There was no obstruction in the left ventricular outflow tract. In his family, a total of 13 individuals were diagnosed HCM and 5 of them were dead of congestive heart failure at a mean age of 66-year-old. Eight living members were all detected to carry the mutation, in which 3 developed progressive heart failure. Moreover, the heart function of the people evidently deteriorates when their age are older than 50. The mutation and the disease show co-separated. The Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.

Platypus TCRμ provides insight into the origins and evolution of a uniquely mammalian TCR locus.

PubMed

Wang, Xinxin; Parra, Zuly E; Miller, Robert D

2011-11-15

TCRμ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and nonmammals. In this study, we show that TCRμ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRμ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRμ is expressed in a form containing double V domains. These V domains more closely resemble Ab V than that of conventional TCR. Platypus TCRμ differs from its marsupial homolog by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRμ but it has been lost from this lineage.

Overproduction of alpha chains provides a proton-insensitive component to the bluefish hemoglobin system.

PubMed

Bonaventura, Celia; Godette, Gerald; Stevens, Robert; Brenowitz, Michael; Henkens, Robert

2005-12-09

Expression of alpha and beta chains and their post-translational assembly into alpha(2)beta(2) tetramers is fundamental to the formation and function of most vertebrate hemoglobins. There is a strong evolutionary bias that favors expression of equal amounts of the two types of chains, because cooperativity, pH sensitivity, and anionic control of function occurs only for the alpha(2)beta(2) tetramers. Remarkably, an over-production of alpha chains, as in the pathological condition known as beta thalassemia in humans, is adaptive rather than pathological in the bluefish hemoglobin system. The thalassemia of the bluefish is a novel means of providing for oxygen uptake and delivery when low pH conditions incapacitate the highly pH-sensitive Root effect hemoglobins of the fish. Although fish often have pH-insensitive along with highly pH-sensitive hemoglobins, having pH-insensitive alpha chain monomers in circulation is an unusual structural variation. The role of bluefish alpha chains in oxygen transport is enabled by their remarkably lower oxygen affinity relative to human alpha chains. This is the first reported case of a thalassemic condition that is maintained in a species as an adaptive advantage.

Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus.

PubMed

Ott, Jeannine A; Castro, Caitlin D; Deiss, Thaddeus C; Ohta, Yuko; Flajnik, Martin F; Criscitiello, Michael F

2018-04-17

Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates. © 2018, Ott et al.

Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus

PubMed Central

Ott, Jeannine A; Castro, Caitlin D; Deiss, Thaddeus C; Ohta, Yuko; Flajnik, Martin F

2018-01-01

Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates. PMID:29664399

Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

PubMed

Jin, Jianjian; Gkitsas, Nikolaos; Fellowes, Vicki S; Ren, Jiaqiang; Feldman, Steven A; Hinrichs, Christian S; Stroncek, David F; Highfill, Steven L

2018-01-24

Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1

PubMed Central

Côte, Marjorie; Fos, Camille; Canonigo-Balancio, Ann J.; Ley, Klaus; Bécart, Stéphane; Altman, Amnon

2015-01-01

ABSTRACT SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca2+ signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4+ T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4+ T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6−/−) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling. PMID:26483383

SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1.

PubMed

Côte, Marjorie; Fos, Camille; Canonigo-Balancio, Ann J; Ley, Klaus; Bécart, Stéphane; Altman, Amnon

2015-12-01

SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca(2+) signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4(+) T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4(+) T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6(-/-)) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling. © 2015. Published by The Company of Biologists Ltd.

The activation threshold of CD4+ T cells is defined by TCR/peptide-MHC class II interactions in the thymic medulla.

PubMed

Stephen, Tom Li; Tikhonova, Anastasia; Riberdy, Janice M; Laufer, Terri M

2009-11-01

Immature thymocytes that are positively selected based upon their response to self-peptide-MHC complexes develop into mature T cells that are not overtly reactive to those same complexes. Developmental tuning is the active process through which TCR-associated signaling pathways of single-positive thymocytes are attenuated to respond appropriately to the peptide-MHC molecules that will be encountered in the periphery. In this study, we explore the mechanisms that regulate the tuning of CD4(+) single-positive T cells to MHC class II encountered in the thymic medulla. Experiments with murine BM chimeras demonstrate that tuning can be mediated by MHC class II expressed by either thymic medullary epithelial cells or thymic dendritic cells. Tuning does not require the engagement of CD4 by MHC class II on stromal cells. Rather, it is mediated by interactions between MHC class II and the TCR. To understand the molecular changes that distinguish immature hyperactive T cells from tuned mature CD4(+) T cells, we compared their responses to TCR stimulation. The altered response of mature CD4 single-positive thymocytes is characterized by the inhibition of ERK activation by low-affinity self-ligands and increased expression of the inhibitory tyrosine phosphatase SHP-1. Thus, persistent TCR engagement by peptide-MHC class II on thymic medullary stroma inhibits reactivity to self-Ags and prevents autoreactivity in the mature repertoire.

Beta-structures in fibrous proteins.

PubMed

Kajava, Andrey V; Squire, John M; Parry, David A D

2006-01-01

The beta-form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the beta-conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer's Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X-ray crystallography studies have revealed the high incidence of the beta-fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the beta-fold, summarize the many different new forms of beta-structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.

Functional overload increases beta-MHC promoter activity in rodent fast muscle via the proximal MCAT (betae3) site.

PubMed

Giger, Julia M; Haddad, Fadia; Qin, Anqi X; Baldwin, Kenneth M

2002-03-01

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, beta-MHC isoform. Different length rat beta-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of -3,500, -914, -408, and -215 bp promoters increased in response to 1 wk of OL. The smallest -171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the -171 and -408 bp region were performed. The -408 bp promoters containing mutations of the betae1, distal muscle CAT (MCAT; betae2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (betae3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the betae3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the betae3 site functions as a putative OL-responsive element in the rat beta-MHC gene promoter.

Platypus TCRμ provides insight into the origins and evolution of a uniquely mammalian TCR locus1

PubMed Central

Wang, Xinxin; Parra, Zuly E.; Miller, Robert D.

2011-01-01

TCRμ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and non-mammals. Here we show that TCRμ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRμ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRμ is expressed in a form containing double V domains. These V domains more closely resemble antibody V than that of conventional TCR. Platypus TCRμ differs from its marsupial homologue by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRμ but it has been lost from this lineage. PMID:21976776

Sarcomere protein gene mutations and inherited heart disease: a beta-cardiac myosin heavy chain mutation causing endocardial fibroelastosis and heart failure.

PubMed

Kamisago, Mitsuhiro; Schmitt, Joachim P; McNamara, Dennis; Seidman, Christine; Seidman, J G

2006-01-01

Inherited human cardiomyopathies often lead to heart failure. A common feature of these conditions is that affected individuals can express the disease causing mutations for many years without showing clinical signs of the disease. Previous studies have demonstrated that sarcomere protein gene mutations can cause either dilated cardiomyopathy or hypertrophic cardiomyopathy. Here we demonstrate that the Arg442His missense mutation in beta-cardiac myosin heavy chain (betaMHC) causes dilated cardiomyopathy, endocardial fibroelastosis and heart failure at a very early age. Using standard genetic engineering tools we and others have made murine models by introducing human disease causing mutations into mice. The central hypothesis of these studies has been that by identifying the pathophysiological pathways activated by these mutations we can define enzymatic activities that are modified during the disease process and which may be involved in pathways that involve more common forms of cardiac disease. Murine models bearing different mutant myosins are being used to address whether each disease causing mutant betaMHC activates the same or different cellular pathways. Dissecting the molecular pathways modulated by mutations in sarcomere protein genes as well as other genes has already demonstrated that there are multiple pathways leading to cardiac remodelling and heart failure. Defining the mechanisms by which mutations in the same genes activate different cellular pathways remains an important question.

Synthesis and studies of polypeptide materials: Enantioselective polymerization of gamma-benzyl glutamate-N-carboxyanhydride and synthesis of optically active poly(beta-peptides)

NASA Astrophysics Data System (ADS)

Cheng, Jianjun

A class of zero-valent transition metal complexes have been developed by Deming et al for the controlled polymerization of alpha-aminoacid-N-carboxyanhydrides (alpha-NCAs). This discovery provided a superior starting point for the development of enantioselective polymerizations of racemic alpha-NCAs. Bidentate chiral ligands were synthesized and tested for their abilities to induce enantioselective polymerization of gamma-benzyl-glutamate NCA (Glu NCA) when they were coordinated to zero-valent nickel complexes. When optically active 2-pyridinyl oxazoline ligands were mixed with bis(1,5-cyclooctadiene)nickel in THF, chiral nickel complexes were formed that selectively polymerized one enantiomer of Glu NCA over the other. The highest selectivity was observed with the nickel complex of (S)-4-tert-butyl-2-pyridinyl oxazoline, which gave a ratio of enantiomeric polymerization rate constants (kD/kL) of 5.2. It was found that subtle modification of this ligand by incorporation of additional substituents had a substantial impact on initiator enantioselectivities. In separate efforts, methodology was developed for the general synthesis of optically active beta-aminoacid-N-carboxyanhydrides (beta-NCAs) via cyclization of Nbeta-Boc- or Nbeta-Cbz-beta-amino acids using phosphorus tribromide. The beta-NCA molecules could be polymerized in good yields using strong bases or transition metal complexes to give optically active poly(beta-peptides) bearing proteinogenic side chains. The resulting poly(beta-peptides), which have moderate molecular weights, adopt stable helical conformations in solution. Poly(beta-homoglutamate and poly(beta-homolysine), the side-chain deprotected polymers, were found to display pH dependent helix-coil conformation transitions in aqueous solution, similar to their alpha-analogs. A novel method for poly(beta-aspartate) synthesis was developed via the polymerization of L-aspartate alkyl ester beta lactams using metal-amido complexes. Poly(beta

Clonal immunoglobulin heavy chain and T-cell receptor γ gene rearrangements in primary gastric lymphoma.

PubMed

Shan, Guo-Dong; Hu, Feng-Ling; Yang, Ming; Chen, Hong-Tan; Chen, Wen-Guo; Wang, Yun-Gui; Chen, Li-Hua; Li, You-Ming; Xu, Guo-Qiang

2013-09-14

To study the diagnostic value of immunoglobulin heavy chain (IgH) and T-cell receptor γ (TCR-γ) gene monoclonal rearrangements in primary gastric lymphoma (PGL). A total of 48 patients with suspected PGL at our hospital were prospectively enrolled in this study from January 2009 to December 2011. The patients were divided into three groups (a PGL group, a gastric linitis plastica group, and a benign gastric ulcer group) based on the pathological results (gastric mucosal specimens obtained by endoscopy or surgery) and follow-up. Endoscopic ultrasonography (EUS) and EUS-guided biopsy were performed in all the patients. The tissue specimens were used for histopathological examination and for IgH and TCR-γ gene rearrangement polymerase chain reaction analyses. EUS and EUS-guided biopsy were successfully performed in all 48 patients. In the PGL group (n = 21), monoclonal IgH gene rearrangements were detected in 14 (66.7%) patients. A positive result for each set of primers was found in 12 (57.1%), 8 (38.1%), and 4 (19.0%) cases using FR1/JH, FR2/JH, and FR3/JH primers, respectively. Overall, 12 (75%) patients with mucosal-associated lymphoid tissue lymphoma (n = 16) and 2 (40%) patients with diffuse large B-cell lymphoma (n = 5) were positive for monoclonal IgH gene rearrangements. No patients in the gastric linitis plastica group (n = 17) and only one (10%) patient in the benign gastric ulcer group (n = 10) were positive for a monoclonal IgH gene rearrangement. No TCR-γ gene monoclonal rearrangements were detected. The sensitivity of monoclonal IgH gene rearrangements was 66.7% for a PGL diagnosis, and the specificity was 96.4%. In the PGL group, 8 (100%) patients with stage IIE PGL (n = 8) and 6 (46.1%) patients with stage IE PGL (n = 13) were positive for monoclonal IgH gene rearrangements. IgH gene rearrangements may be associated with PGL staging and may be useful for the diagnosis of PGL and for differentiating between PGL and gastric linitis plastica.

In silico study of full-length amyloid beta 1-42 tri- and penta-oligomers in solution.

PubMed

Masman, Marcelo F; Eisel, Ulrich L M; Csizmadia, Imre G; Penke, Botond; Enriz, Ricardo D; Marrink, Siewert Jan; Luiten, Paul G M

2009-08-27

Amyloid oligomers are considered to play causal roles in the pathogenesis of amyloid-related degenerative diseases including Alzheimer's disease. Using MD simulation techniques, we explored the contributions of the different structural elements of trimeric and pentameric full-length Abeta1-42 aggregates in solution to their stability and conformational dynamics. We found that our models are stable at a temperature of 310 K, and converge toward an interdigitated side-chain packing for intermolecular contacts within the two beta-sheet regions of the aggregates: beta1 (residues 18-26) and beta2 (residues 31-42). MD simulations reveal that the beta-strand twist is a characteristic element of Abeta-aggregates, permitting a compact, interdigitated packing of side chains from neighboring beta-sheets. The beta2 portion formed a tightly organized beta-helix, whereas the beta1 portion did not show such a firm structural organization, although it maintained its beta-sheet conformation. Our simulations indicate that the hydrophobic core comprising the beta2 portion of the aggregate is a crucial stabilizing element in the Abeta aggregation process. On the basis of these structure-stability findings, the beta2 portion emerges as an optimal target for further antiamyloid drug design.

Hb L'Aquila [beta106(G8)Leu-->Val, CTG-->GTG]: a novel thalassemic hemoglobin variant.

PubMed

Amato, Antonio; Cappabianca, Maria Pia; Ponzini, Donatella; Rinaldi, Silvana; Biagio, Paola Di; Foglietta, Enrica; Grisanti, Paola; Mastropietro, Fabrizio

2007-01-01

A new beta-globin variant at codon 106 (CTG-->GTG), and which we named Hb L'Aquila [beta106(G8)Leu-->Val], was detected by DNA analysis. The proband and her father presented with the features of a mild beta(+)-thalassemia (thal), confirmed by their alpha/beta-globin chain biosynthesis ratios.

Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency

PubMed Central

Wong, Gabriel K.; Millar, David; Penny, Sarah; Heather, James M.; Mistry, Punam; Buettner, Nico; Bryon, Jane; Huissoon, Aarnoud P.

2016-01-01

Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRβ sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21lo B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease. PMID:27481850

A novel mutation of the beta myosin heavy chain gene responsible for familial hypertrophic cardiomyopathy.

PubMed

Wang, Juan; Xu, Shi-Jie; Zhou, Hua; Wang, Li-Jie; Hu, Bo; Fang, Fang; Zhang, Xu-Min; Luo, Yi-Wei; He, Xiao-Yan; Zhuang, Shao-Wei; Li, Xin-Ming; Liu, Zhong-Ming; Hu, Da-Yi

2009-09-01

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder and shows high variability in genetic heterogeneity and phenotypic characteristics. The genetic etiology responsible for HCM in many individuals remains unclear. This instigation was sought to identify novel genetic determinants for familial hypertrophic cardiomyopathy. Six unrelated Chinese families with HCM were studied. For each of the 13 established HCM-susceptibility genes, 3 to 5 microsatellite markers were selected to perform genotyping and haplotype analysis. The linked genes were sequenced. Haplotype analyses on candidate genetic loci revealed cosegregation of the gene beta-myosin heavy chain (MYH7) with HCM in a single family. A novel double heterozygous missense mutation of Ala26Val plus Arg719Trp in MYH7 was subsequently identified by sequencing in this family and was associated with a severe phenotype of HCM. The novel double mutation of Ala26Val plus Arg719Trp in MYH7 identified in a Chinese family highlights the remarkable genetic heterogeneity of HCM, which provides important information for genetic counseling, accurate diagnosis, prognostic evaluation, and appropriate clinical management. Copyright 2009 Wiley Periodicals, Inc.

Generation of TCR-Expressing Innate Lymphoid-like Helper Cells that Induce Cytotoxic T Cell-Mediated Anti-leukemic Cell Response.

PubMed

Ueda, Norihiro; Uemura, Yasushi; Zhang, Rong; Kitayama, Shuichi; Iriguchi, Shoichi; Kawai, Yohei; Yasui, Yutaka; Tatsumi, Minako; Ueda, Tatsuki; Liu, Tian-Yi; Mizoro, Yasutaka; Okada, Chihiro; Watanabe, Akira; Nakanishi, Mahito; Senju, Satoru; Nishimura, Yasuharu; Kuzushima, Kiyotaka; Kiyoi, Hitoshi; Naoe, Tomoki; Kaneko, Shin

2018-06-05

CD4 + T helper (Th) cell activation is essential for inducing cytotoxic T lymphocyte (CTL) responses against malignancy. We reprogrammed a Th clone specific for chronic myelogenous leukemia (CML)-derived b3a2 peptide to pluripotency and re-differentiated the cells into original TCR-expressing T-lineage cells (iPS-T cells) with gene expression patterns resembling those of group 1 innate lymphoid cells. CD4 gene transduction into iPS-T cells enhanced b3a2 peptide-specific responses via b3a2 peptide-specific TCR. iPS-T cells upregulated CD40 ligand (CD40L) expression in response to interleukin-2 and interleukin-15. In the presence of Wilms tumor 1 (WT1) peptide, antigen-specific dendritic cells (DCs) conditioned by CD4-modified CD40L high iPS-T cells stimulated WT1-specific CTL priming, which eliminated WT1 peptide-expressing CML cells in vitro and in vivo. Thus, CD4 modification of CD40L high iPS-T cells generates innate lymphoid helper-like cells inducing bcr-abl-specific TCR signaling that mediates effectiveanti-leukemic CTL responses via DC maturation, showing potential for adjuvant immunotherapy against leukemia. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Activated PLC-γ1 is Catalytically Induced at LAT but Activated PLC-γ1 is Localized at both LAT- and TCR-Containing Complexes

PubMed Central

Cruz-Orcutt, Noemi; Vacaflores, Aldo; Connolly, Sean F.; Bunnell, Stephen C.; Houtman, Jon C.D.

2014-01-01

Phospholipase C-γ1 (PLC-γ1) is a key regulator of T cell receptor (TCR)-induced signaling. Activation of the TCR enhances PLC-γ1 enzymatic function, resulting in calcium influx and the activation of PKC family members and RasGRP. The current model is that phosphorylation of LAT tyrosine 132 facilitates the recruitment of PLC-γ1, leading to its activation and function at the LAT complex. In this study, we examined the phosphorylation kinetics of LAT and PLC-γ1 and the cellular localization of activated PLC-γ1. We observed that commencement of the phosphorylation of LAT tyrosine 132 and PLC-γ1 tyrosine 783 occurred simultaneously, supporting the current model. However, once begun, PLC-γ1 activation occurred more rapidly than LAT tyrosine 132. The association of LAT and PLC-γ1 was more transient than the interaction of LAT and Grb2 and a pool of activated PLC-γ1 translocated away from LAT to cellular structures containing the TCR. These studies demonstrate that LAT and PLC-γ1 form transient interactions that catalyze the activation of PLC-γ1, but that activated PLC-γ1 resides in both LAT and TCR clusters. Together, this work highlights that our current model is incomplete and the activation and function of PLC-γ1 in T cells is highly complex. PMID:24412752

Peptide design using alpha,beta-dehydro amino acids: from beta-turns to helical hairpins.

PubMed

Mathur, Puniti; Ramakumar, S; Chauhan, V S

2004-01-01

Incorporation of alpha,beta-dehydrophenylalanine (DeltaPhe) residue in peptides induces folded conformations: beta-turns in short peptides and 3(10)-helices in larger ones. A few exceptions-namely, alpha-helix or flat beta-bend ribbon structures-have also been reported in a few cases. The most favorable conformation of DeltaPhe residues are (phi,psi) approximately (-60 degrees, -30 degrees ), (-60 degrees, 150 degrees ), (80 degrees, 0 degrees ) or their enantiomers. DeltaPhe is an achiral and planar residue. These features have been exploited in designing DeltaPhe zippers and helix-turn-helix motifs. DeltaPhe can be incorporated in both right and left-handed helices. In fact, consecutive occurrence of three or more DeltaPhe amino acids induce left-handed screw sense in peptides containing L-amino acids. Weak interactions involving the DeltaPhe residue play an important role in molecular association. The C--H.O==C hydrogen bond between the DeltaPhe side-chain and backbone carboxyl moiety, pi-pi stacking interactions between DeltaPhe side chains belonging to enantiomeric helices have shown to stabilize folding. The unusual capability of a DeltaPhe ring to form the hub of multicentered interactions namely, a donor in aromatic C--H.pi and C--H.O==C and an acceptor in a CH(3).pi interaction suggests its exploitation in introducing long-range interactions in the folding of supersecondary structures. Copyright 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004

Intermediates of peroxisomal beta-oxidation. A study of the fatty acyl-CoA esters which accumulate during peroxisomal beta-oxidation of [U-14C]hexadecanoate.

PubMed Central

Bartlett, K; Hovik, R; Eaton, S; Watmough, N J; Osmundsen, H

1990-01-01

1. 14C-labelled fatty acyl-CoA esters resulting from beta-oxidation of [U-14C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50 microM), decanoyl-CoA was present in lowest amounts. At higher concentrations of [U-14C]hexadecanoate (greater than 100 microM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of beta-oxidation now resembling chain-shortening of hexadecanoate by two cycles of beta-oxidation. 3. In the absence of an NAD(+)-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1.1.28)] 2-enoyl- and 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal beta-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (greater than 100 microM), 3-oxohexadecanoyl-CoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9) can become rate-limiting for peroxisomal beta-oxidation. Images Fig. 2. Fig. 3. Fig. 4. PMID:2396977

TGF-beta inhibits IL-1beta-activated PAR-2 expression through multiple pathways in human primary synovial cells.

PubMed

Tsai, Shin-Han; Sheu, Ming-Thau; Liang, Yu-Chih; Cheng, Hsiu-Tan; Fang, Sheng-Shiung; Chen, Chien-Ho

2009-10-23

To investigate the mechanism how Transforming growth factor-beta(TGF-beta) represses Interleukin-1beta (IL-1beta)-induced Proteinase-Activated Receptor-2 (PAR-2) expression in human primary synovial cells (hPSCs). Human chondrocytes and hPSCs isolated from cartilages and synovium of Osteoarthritis (OA) patients were cultured with 10% fetal bovine serum media or serum free media before treatment with IL-1beta, TGF-beta1, or Connective tissue growth factor (CTGF). The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Collagen zymography was performed to assess the activity of Matrix metalloproteinases-13 (MMP-13). It was demonstrated that IL-1beta induces PAR-2 expression via p38 pathway in hPSCs. This induction can be repressed by TGF-beta and was observed to persist for at least 48 hrs, suggesting that TGF-beta inhibits PAR-2 expression through multiple pathways. First of all, TGF-beta was able to inhibit PAR-2 activity by inhibiting IL-1beta-induced p38 signal transduction and secondly the inhibition was also indirectly due to MMP-13 inactivation. Finally, TGF-beta was able to induce CTGF, and in turn CTGF represses PAR-2 expression by inhibiting IL-1beta-induced phospho-p38 level. TGF-beta could prevent OA from progression with the anabolic ability to induce CTGF production to maintain extracellular matrix (ECM) integrity and to down regulate PAR-2 expression, and the anti-catabolic ability to induce Tissue inhibitors of metalloproteinase-3 (TIMP-3) production to inhibit MMPs leading to avoid PAR-2 over-expression. Because IL-1beta-induced PAR-2 expressed in hPSCs might play a significantly important role in early phase of OA, PAR-2 repression by exogenous TGF-beta or other agents might be an ideal therapeutic target to prevent OA from progression.

Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

López-Sagaseta, Jacinto; Sibener, Leah V.; Kung, Jennifer E.

2014-10-02

Invariant Natural Killer T (iNKT) cells use highly restricted {alpha}{beta} T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted {alpha}1 helix resulting in an open A pocket. Binding of the iNKT TCR requires a 7-{angstrom} displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint onmore » CD1d-LPC is anchored by the conserved positioning of the CDR3{alpha} loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3{beta} and J{beta} segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.« less

Polymorphisms of the IL-1beta and IL-1beta-inducible genes in ulcerative colitis.

PubMed

Nohara, Hiroaki; Saito, Yuki; Higaki, Singo; Okayama, Naoko; Hamanaka, Yuichiro; Okita, Kiwamu; Hinoda, Yuji

2002-11-01

Ulcerative colitis (UC) is a chronic disorder of undetermined etiology, but a genetic predisposition to UC is well recognized. Among cytokines induced in UC, interleukin 1 (IL-1) appears to have a central role because of its immunological upregulatory and proinflammatory activities. The aim of this study was to assess whether UC is associated with polymorphisms of the IL-1beta gene and three additional genes inducible with IL-1beta in Japanese subjects. A total of 96 patients with UC and 106 ethnically matched controls were genotyped at polymorphic sites in IL-1beta, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3), and inducible nitric oxide synthase (iNOS) genes, using polymerase chain reaction (PCR)-based methods. There was no significant difference in genotype distributions of IL-1beta, MMP-1, MMP-3, and iNOS genes between controls and UC patients in a Japanese population. Also, no significant association of those polymorphisms with various clinical parameters of the patients was found. However, concerning association of age at onset with clinical factors in UC, the frequency of pancolitis was significantly higher in UC patients with age at onset being less than 30 years than in those more than 30 years of age (P = 0.049). No association of the IL-1beta and three IL-1beta-inducible gene polymorphisms with UC was observed in a Japanese population.

Structural analyses of polymorphic transitions of sn-1, 3-distearoyl-2-oleoylglycerol (SOS) and sn-1, 3-dioleoyl-2-stearoylglycerol (OSO): assessment on steric hindrance of unsaturated and saturated acyl chain interactions.

PubMed

Yano, J; Sato, K; Kaneko, F; Small, D M; Kodali, D R

1999-01-01

Polymorphic transformations in two saturated-unsaturated mixed acid triacylglycerols, SOS (sn -1,3-distearoyl-2-oleoylglycerol) and OSO (sn -1,3-dioleoyl-2-stearoylglycerol), have been studied by FT-IR spectroscopy using deuterated specimens in which stearoyl chains are fully deuterated. A reversible phase transition between sub alpha and alpha and a series of irreversible transitions (alpha-->gamma-->beta'-->beta (beta2, beta1) for SOS and alpha-->beta'-->beta for OSO) were studied with an emphasis on the conformational ordering process of stearoyl and oleoyl chains. The alpha-->sub alpha reversible transition was due to the orientational change of stearoyl chains in the lateral directions from the hexagonal subcell to a perpendicularly packed one. As the first stage of the series of irreversible transitions from alpha to beta, the conformational ordering of saturated chains took place in the alpha-->gamma transition of SOS and in the alpha-->beta' transition of OSO; one stearoyl chain in SOS and OSO takes the all-trans conformation and the second stearoyl chain in SOS takes the bent conformation like those observed in the most stable beta-type. As the final stage, the ordering of unsaturated chains occurred in the beta'-->beta transition both for SOS and OSO. A conversion in the layered structure from bilayer to trilayer was also accompanied by the conformational ordering in the alpha-->gamma transition of SOS and in the beta'-->beta transition of OSO.

Differential effects of Phe19 and Phe20 on fibril formation by amyloidogenic peptide A beta 16-22 (Ac-KLVFFAE-NH2).

PubMed

Inouye, Hideyo; Gleason, Katherine A; Zhang, Dong; Decatur, Sean M; Kirschner, Daniel A

2010-08-01

The sequence KLVFFAE (A beta 16-22) in Alzheimer's beta-amyloid is thought to be a core beta-structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in beta-sheet. To elucidate the mechanism of the initial folding process, we undertook combined X-ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized A beta 16-22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild-type (WT) A beta 16-22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab-like beta-crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30-A period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side-chain dispositions for WT and 20CN, but not for 19CN. These X-ray results and modeling imply that in the assembly of WT A beta 16-22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation. (c) 2010 Wiley-Liss, Inc.

WC1 is a hybrid γδ TCR coreceptor and pattern recognition receptor for pathogenic bacteria.

PubMed

Hsu, Haoting; Chen, Chuang; Nenninger, Ariel; Holz, Lauren; Baldwin, Cynthia L; Telfer, Janice C

2015-03-01

WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. While present in variable, and sometimes high, numbers in the genomes of mammals and birds, in cattle there are 13 distinct genes (WC1-1 to WC1-13). All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation dependent manner, and some are required for the γδ T cell response to Leptospira. We hypothesized that individual WC1 receptors encode Ag specificity via coligation of bacteria with the γδ TCR. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species, L. borgpetersenii, and L. interrogans. Three to five of eleven WC1-3 SRCR domains, but none of the eleven WC1-4 SRCR domains, interacted with Leptospira spp. and Borrelia burgdorferi, but not with Escherichia coli or Staphylococcus aureus. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is composed of amino acids in three discontinuous regions. Recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. Our data suggest that WC1 gene arrays play a multifaceted role in the γδ T cell response to bacteria, including acting as hybrid pattern recognition receptors and TCR coreceptors, and they may function as antimicrobials. Copyright © 2015 by The American Association of Immunologists, Inc.

Genetic basis of human complement C8[beta] deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaufmann, T.; Rittner, C.; Schneider, P.M.

1993-06-01

The eighth component of human complement (c8) is a serum protein consisting of three chains ([alpha], [beta], and [gamma]) and encoded by three different genes, C8A, C8B, and C8G. C8A and C8B are closely linked on chromosome 1p, whereas C8G is located on chromosome 9q. In the serum the [beta] subunit is non-covalently bound to the disulfide-linked [alpha]-[gamma] subunit. Patients with C8[beta] deficiency suffer from recurrent neisserial infections such as meningitis. Exon-specific polymerase chain reaction (PCR) amplification with primer pairs from the flanking intron sequences was used to amplify all 12 C8B exons separately. No difference regarding the exon sizesmore » was observed in a C8[beta]-deficient patient compared with a normal person. Therefore, direct sequence analysis of all exon-specific PCR products from normal and C8[beta]-deficient individuals was carried out. As a cause for C8[beta] deficiency, we found a single C-T exchange in exon 9 leading to a stop codon. An allele-specific PCR system was designed to detect the normal and the deficiency allele simultaneously. Using this approach as well as PCR typing of the Taql polymorphism located in intron 11, five families with 7 C8[beta]-deficient members were investigated. The mutation was not found to be restricted to one of the two Taql RFLP alleles. The mutant allele was observed in all families investigated and can therefore be regarded as a major cause of C8[beta] deficiency in the Caucasian population. In addition, two C8[beta]-deficient patients were found to be heterozygous for the C-T exchange. The molecular basis of the alleles without this point mutation also causing deficiency has not yet been defined. 23 refs., 4 figs., 3 tabs.« less

Immunoglobulin heavy and light chains and T-cell receptor beta and gamma chains PCR assessment on cytological samples. A study comparing FTA cards and cryopreserved lymph node fine-needle cytology.

PubMed

Peluso, A L; Cozzolino, I; Bottiglieri, A; Lucchese, L; Di Crescenzo, R M; Langella, M; Selleri, C; Zeppa, P

2017-06-01

To evaluate and compare the DNA yield and quality extracted from lymph node fine needle cytology (FNC) samples stored on FTA cards to those cryopreserved, and to assess the immunoglobulin heavy and light chains (IGHK) and T-Cell receptor beta and gamma chains (TCRBG) PCR tests. DNA extractions were performed on FNC of 80 non-Hodgkin lymphomas (NHL), four myelomas and 56 benign reactive hyperplasias (BRH) cryopreserved and stored on FTA cards. The JAK2 gene was amplified to assess the DNA integrity and the IGHK/TCRBG clonality status was tested. IGHK monoclonality was found in 99% of B-cell NHL and 100% of myeloma. TCRBG monoclonality was found in 100% of T-cell NHL. TCRBG polyclonality was detected in 97% of B-cell NHL, 100% of myeloma and 96% of BRH. IGHK/TCRBG PCR data were confirmed by histological and/or follow-up controls. No differences were found in the DNA quality between cryopreservation and FTA cards storage methods. IGHK/TCRBG PCR of the lymphoproliferative process on FTA cards is comparable to those cryopreserved. FTA cards can be used to store lymph node FNC for further molecular investigations. © 2016 John Wiley & Sons Ltd.

Complementary DNA sequences encoding the multimammate rat MHC class II DQ alpha and beta chains and cross-species sequence comparison in rodents.

PubMed

de Bellocq, J Goüy; Leirs, H

2009-09-01

Sequences of the complete open reading frame (ORF) for rodents major histocompatibility complex (MHC) class II genes are rare. Multimammate rat (Mastomys natalensis) complementary DNA (cDNA) encoding the alpha and beta chains of MHC class II DQ gene was cloned from a rapid amplifications of cDNA Emds (RACE) cDNA library. The ORFs consist of 801 and 771 bp encoding 266 and 256 amino acid residues for DQB and DQA, respectively. The genomic structure of Mana-DQ genes is globally analogous to that described for other rodents except for the insertion of a serine residue in the signal peptide of Mana-DQB, which is unique among known rodents.

The effect of smoking cessation pharmacotherapies on pancreatic beta cell function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woynillowicz, Amanda K.; Raha, Sandeep; Nicholson, Catherine J.

The goal of our study was to evaluate whether drugs currently used for smoking cessation (i.e., nicotine replacement therapy, varenicline [a partial agonist at nicotinic acetylcholine receptors (nAChR)] and bupropion [which acts in part as a nAChR antagonist]) can affect beta cell function and determine the mechanism(s) of this effect. INS-1E cells, a rat beta cell line, were treated with nicotine, varenicline and bupropion to determine their effects on beta cell function, mitochondrial electron transport chain enzyme activity and cellular/oxidative stress. Treatment of INS-1E cells with equimolar concentrations (1 μM) of three test compounds resulted in an ablation of normalmore » glucose-stimulated insulin secretion by the cells. This disruption of normal beta cell function was associated with mitochondrial dysfunction since all three compounds tested significantly decreased the activity of mitochondrial electron transport chain enzyme activity. These results raise the possibility that the currently available smoking cessation pharmacotherapies may also have adverse effects on beta cell function and thus glycemic control in vivo. Therefore whether or not the use of nicotine replacement therapy, varenicline and bupropion can cause endocrine changes which are consistent with impaired pancreatic function warrants further investigation. -- Highlights: ► Smoking cessation drugs have the potential to disrupt beta cell function in vitro. ► The effects of nicotine, varenicline and bupropion are similar. ► The impaired beta cell function is mediated by mitochondrial dysfunction. ► If similar effects are seen in vivo, these drugs may increase the risk of diabetes.« less

Predicting beta-turns and their types using predicted backbone dihedral angles and secondary structures.

PubMed

Kountouris, Petros; Hirst, Jonathan D

2010-07-31

Beta-turns are secondary structure elements usually classified as coil. Their prediction is important, because of their role in protein folding and their frequent occurrence in protein chains. We have developed a novel method that predicts beta-turns and their types using information from multiple sequence alignments, predicted secondary structures and, for the first time, predicted dihedral angles. Our method uses support vector machines, a supervised classification technique, and is trained and tested on three established datasets of 426, 547 and 823 protein chains. We achieve a Matthews correlation coefficient of up to 0.49, when predicting the location of beta-turns, the highest reported value to date. Moreover, the additional dihedral information improves the prediction of beta-turn types I, II, IV, VIII and "non-specific", achieving correlation coefficients up to 0.39, 0.33, 0.27, 0.14 and 0.38, respectively. Our results are more accurate than other methods. We have created an accurate predictor of beta-turns and their types. Our method, called DEBT, is available online at http://comp.chem.nottingham.ac.uk/debt/.

Using support vector machine to predict beta- and gamma-turns in proteins.

PubMed

Hu, Xiuzhen; Li, Qianzhong

2008-09-01

By using the composite vector with increment of diversity, position conservation scoring function, and predictive secondary structures to express the information of sequence, a support vector machine (SVM) algorithm for predicting beta- and gamma-turns in the proteins is proposed. The 426 and 320 nonhomologous protein chains described by Guruprasad and Rajkumar (Guruprasad and Rajkumar J. Biosci 2000, 25,143) are used for training and testing the predictive model of the beta- and gamma-turns, respectively. The overall prediction accuracy and the Matthews correlation coefficient in 7-fold cross-validation are 79.8% and 0.47, respectively, for the beta-turns. The overall prediction accuracy in 5-fold cross-validation is 61.0% for the gamma-turns. These results are significantly higher than the other algorithms in the prediction of beta- and gamma-turns using the same datasets. In addition, the 547 and 823 nonhomologous protein chains described by Fuchs and Alix (Fuchs and Alix Proteins: Struct Funct Bioinform 2005, 59, 828) are used for training and testing the predictive model of the beta- and gamma-turns, and better results are obtained. This algorithm may be helpful to improve the performance of protein turns' prediction. To ensure the ability of the SVM method to correctly classify beta-turn and non-beta-turn (gamma-turn and non-gamma-turn), the receiver operating characteristic threshold independent measure curves are provided. (c) 2008 Wiley Periodicals, Inc.

T-bet Down-Modulation in Tolerized Th1 Effector CD4 Cells Confers a TCR-Distal Signaling Defect That Selectively Impairs IFN-γ Expression1

PubMed Central

Long, Meixiao; Slaiby, Aaron M.; Hagymasi, Adam T.; Mihalyo, Marianne A.; Lichtler, Alexander C.; Reiner, Steven L.; Adler, Adam J.

2010-01-01

When Th1 effector CD4 cells encounter tolerizing Ag in vivo, their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than noneffector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with Ag vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and -distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ -expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggested that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure. PMID:16393991

Extraordinary TCR in Carbon Nanotube-Polymer Composites and Device Implications in Bolometric Infrared Detection

DTIC Science & Technology

2015-03-24

were made via filtration of 1 mL of 1 mg/L SWCNT solution (Nanointegris Inc.) through a 20 nm pore anodized aluminum oxide ( AAO ) membrane filter to...prepared by attaching a rectangular piece cut from the AAO membrane filter onto a glass substrate. Contacts were made with silver paste to the extremities...NUMBER(S) 14. ABSTRACT _ The development of high TCR materials, such as vanadium oxide (VOx), has enabled the introduction of bolometric infrared

The quantum chemical causality of pMHC-TCR biological avidity: Peptide atomic coordination data and the electronic state of agonist N termini.

PubMed

Antipas, Georgios S E; Germenis, Anastasios E

2015-06-01

The quantum state of functional avidity of the synapse formed between a peptide-Major Histocompatibility Complex (pMHC) and a T cell receptor (TCR) is a subject not previously touched upon. Here we present atomic pair correlation meta-data based on crystalized tertiary structures of the Tax (HTLV-1) peptide along with three artificially altered variants, all of which were presented by the (Class I) HLA-A201 protein in complexation with the human (CD8(+)) A6TCR. The meta-data reveal the existence of a direct relationship between pMHC-TCR functional avidity (agonist/antagonist) and peptide pair distribution function (PDF). In this context, antagonist peptides are consistently under-coordinated in respect to Tax. Moreover, Density Functional Theory (DFT) datasets in the BLYP/TZ2P level of theory resulting from relaxation of the H species on peptide tertiary structures reveal that the coordination requirement of agonist peptides is also expressed as a physical observable of the protonation state of their N termini: agonistic peptides are always found to retain a stable ammonium (NH3 (+)) terminal group while antagonist peptides are not.

T-cell receptor repertoire of human peripheral CD161hiTRAV1-2+ MAIT cells revealed by next generation sequencing and single cell analysis.

PubMed

Held, Kathrin; Beltrán, Eduardo; Moser, Markus; Hohlfeld, Reinhard; Dornmair, Klaus

2015-09-01

Mucosal-associated invariant T (MAIT) cells are a T-cell subset that expresses a conserved TRAV1-2 (Vα7.2) T-cell receptor (TCR) chain and the surface marker CD161. They are involved in the defence against microbes as they recognise small organic molecules of microbial origin that are presented by the non-classical MHC molecule 1 (MR1). MAIT cells express a semi-restricted TCR α chain with TRAV1-2 preferentially linked to TRAJ33, TRAJ12, or TRAJ20 which pairs with a limited set of β chains. To investigate the TCR repertoire of human CD161(hi)TRAV1-2(+) T cells in depth we analysed the α and β chains of this T-cell subset by next generation sequencing. Concomitantly we analysed 132 paired α and β chains from single cells to assess the αβ pairing preferences. We found that the CD161(hi)TRAV1-2(+) TCR repertoire in addition to the typical MAIT TCRs further contains polyclonal elements reminiscent of classical αβ T cells. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Mimicry by asx- and ST-turns of the four main types of beta-turn in proteins.

PubMed

Duddy, William J; Nissink, J Willem M; Allen, Frank H; Milner-White, E James

2004-11-01

Hydrogen-bonded beta-turns in proteins occur in four categories: type I (the most common), type II, type II', and type I'. Asx-turns resemble beta-turns, in that both have an NH. . .OC hydrogen bond forming a ring of 10 atoms. Serine and threonine side chains also commonly form hydrogen-bonded turns, here called ST-turns. Asx-turns and ST-turns can be categorized into four classes, based on side chain rotamers and the conformation of the central turn residue, which are geometrically equivalent to the four types of beta-turns. We propose asx- and ST-turns be named using the type I, II, I', and II' beta-turn nomenclature. Using this, the frequency of occurrence of both asx- and ST-turns is: type II' > type I > type II > type I', whereas for beta-turns it is type I > type II > type I' > type II'. Almost all type II asx-turns occur as a recently described three residue feature named an asx-nest.

Site preference, magnetism and lattice vibrations of intermetallics Lu₂Fe 17–xT x (T=Cr, Mn, Ru)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jin-Chun; Qian, Ping, E-mail: qianpinghu@sohu.com; Zhang, Zhen-Feng

We present an atomistic study on the phase stability, site preference and lattice constants of the rare earth intermetallics Lu₂Fe 17–xT x (T=Cr, Mn, Ru). The calculated preferential occupation site of ternary element T is found to be the 4f site. The order of site preference is given as 4f, 12k, 12j and 6g for Lu₂Fe 17–xT x. The calculated lattice parameters are corresponding to the experimental results. We have calculated the magnetic moments of Lu₂Fe 17–xT x compounds. Results show that the calculated total magnetic moment of Lu₂Fe₁₇ compound is M=37.34 μ B/f.u. In addition, the total and partialmore » phonon densities of states are evaluated first for these complicated structures. - Graphical abstract: The vibrational modes are mostly excited by Fe atoms, Lu contributes to the lower frequencies modes, and the contribution of Ru atoms is the same as Fe atoms. Highlights: • There are no reports on lattice vibrations of Lu₂(Fe, T) 17–x (T=Cr, Mn, Ru) compounds. • The phase stability and site preference are evaluated first for the complex structures of Lu₂(Fe, T) 17–x (T=Cr, Mn, Ru) compounds. • The lattice inversion method to obtain the interatomic pair potential is the unique one.« less

CDR3 analysis of TCR Vβ repertoire of CD8⁺ T cells from chickens infected with Eimeria maxima.

PubMed

Ren, Chao; Yin, Guangwen; Qin, Mei; Suo, Jingxia; Lv, Qiyao; Xie, Li; Wang, Yunzhou; Huang, Xiaoxi; Chen, Yuchen; Liu, Xianyong; Suo, Xun

2014-08-01

CD8(+) T cells play a major role in the immune protection of host against the reinfection of Eimeria maxima, the most immunogenic species of eimerian parasites in chickens. To explore the dominant complementarity-determining regions 3 (CDR3) of CD8(+) T cell populations induced by the infection of this parasite, sequence analysis was performed in this study for CDR3 of CD8(+) T cells from E. maxima infected chickens. After 5 days post the third or forth infection, intraepithelial lymphocytes were isolated from the jejunum of bird. CD3(+)CD8(+) T cells were sorted and subjected to total RNA isolation and cDNA preparation. PCR amplification and cloning of the loci between Vβ1 and Cβ was conducted for the subsequent sequencing of CDR3 of T cell receptor (TCR). After the forth infection, 2 birds exhibited two same frequent TCR CDR3 sequences, i.e., AKQDWGTGGYSNMI and AGRVLNIQY; while the third bird showed two different frequent TCR CDR3 sequences, AKQGARGHTPLN and AKQDIEVRGPNTPLN. No frequent CDR3 sequence was detected from uninfected birds, though AGRVLNIQY was also found in two uninfected birds. Our result preliminarily demonstrates that frequent CDR3 sequences may exist in E. maxima immunized chickens, encouraging the mining of the immunodominant CD8(+) T cells against E. maxima infection. Copyright © 2014 Elsevier Inc. All rights reserved.

Beta-papillomaviruses in anogenital hairs plucked from healthy individuals.

PubMed

Potocnik, Marko; Kocjan, Bostjan J; Seme, Katja; Luzar, Bostjan; Babic, Dunja Z; Poljak, Mario

2006-12-01

A total of 150 specimens of anogenital hairs plucked from the scrotal, pubic, and perianal region of 51 immunocompetent healthy male individuals were tested for the presence of beta-papillomaviruses (beta-HPV) using the nested M(a)/H(a) polymerase chain reaction. Beta-HPV were found in a total of 38 (25.3%) of 150 hair samples. According to the sampling sites, beta-HPV were detected in 18/51 (35.3%), 13/50 (26.0%), and 7/49 (14.3%) plucked hair samples obtained from the pubic, scrotal, and perianal region, respectively. The prevalence of beta-HPV in the plucked pubic hairs was significantly higher than in the perianal hairs (P = 0.013). In contrast, the difference in the prevalence of beta-HPV in the pubic and scrotal hairs as well as in scrotal and perianal hairs did not reach statistical significance (P = 0.302 and P = 0.227, respectively). The difference in the lifetime-cumulative sun exposure is the most likely explanation for the differences obtained on beta-HPV prevalence. Beta-HPV genotype HPV-38 was detected most frequently, followed by HPV-36, HPV-15, and HPV-14D. In addition to the beta-HPV recognized officially five partial DNA sequences suggesting putative new HPV genotypes were identified. (c) 2006 Wiley-Liss, Inc.

Alkyl gallates, intensifiers of beta-lactam susceptibility in methicillin-resistant Staphylococcus aureus.

PubMed

Shibata, Hirofumi; Kondo, Kyoko; Katsuyama, Ryo; Kawazoe, Kazuyoshi; Sato, Yoichi; Murakami, Kotaro; Takaishi, Yoshihisa; Arakaki, Naokatu; Higuti, Tomihiko

2005-02-01

We found that ethyl gallate purified from a dried pod of tara (Caesalpinia spinosa) intensified beta-lactam susceptibility in methicillin-resistant and methicillin-sensitive strains of Staphylococcus aureus (MRSA and MSSA strains, respectively). This compound and several known alkyl gallates were tested with MRSA and MSSA strains to gain new insights into their structural functions in relation to antimicrobial and beta-lactam susceptibility-intensifying activities. The maximum activity of alkyl gallates against MRSA and MSSA strains occurred at 1-nonyl and 1-decyl gallate, with an MIC at which 90% of the isolates tested were inhibited of 15.6 microg/ml. At concentrations lower than the MIC, alkyl gallates synergistically elevated the susceptibility of MRSA and MSSA strains to beta-lactam antibiotics. Such a synergistic activity of the alkyl gallates appears to be specific for beta-lactam antibiotics, because no significant changes were observed in the MICs of other classes of antibiotics examined in this study. The length of the alkyl chain was also associated with the modifying activity of the alkyl gallates, and the optimum length was C5 to C6. The present work clearly demonstrates that the length of the alkyl chain has a key role in the elevation of susceptibility to beta-lactam antibiotics.

Paired analysis of TCRα and TCRβ chains at the single-cell level in mice

PubMed Central

Dash, Pradyot; McClaren, Jennifer L.; Oguin, Thomas H.; Rothwell, William; Todd, Brandon; Morris, Melissa Y.; Becksfort, Jared; Reynolds, Cory; Brown, Scott A.; Doherty, Peter C.; Thomas, Paul G.

2010-01-01

Characterizing the TCRα and TCRβ chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRβ chain utilization is very limited for pathogen- and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ chains with single-cell resolution. Using CD8+ CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease. PMID:21135507

Microvascular pericytes express platelet-derived growth factor-beta receptors in human healing wounds and colorectal adenocarcinoma.

PubMed Central

Sundberg, C.; Ljungström, M.; Lindmark, G.; Gerdin, B.; Rubin, K.

1993-01-01

The expression of platelet-derived growth factor- beta (PDGF-beta) receptors in the microvasculature of human healing wounds and colorectal adenocarcinoma was investigated. Frozen sections were subjected to double immunofluorescence staining using monoclonal antibodies (MAbs) specific for pericytes (MAb 225.28 recognizing the high-molecular weight-melanoma-associated antigen, expressed by activated pericytes during angiogenesis), endothelial cells (MAb PAL-E), laminin, as well as PDGF-beta receptors (MAb PDGFR-B2) and its ligand PDGF-B chain (MAb PDGF 007). Stained sections were analyzed by computer-aided imaging processing that allowed for a numerical quantification of the degree of colocalization of the investigated antigens. An apparent background colocalization, varying between 23 and 35%, between markers for cells not expected to co-localize was recorded. This background could be due to limitations of camera resolution, to out-of-focus fluorescence, and to interdigitations of the investigated structures. In all six tumor specimens, co-localization of PDGF-beta receptors and PAL-E was not different from the background co-localization, whereas that of PDGF-beta receptors and high-molecular weight-melanoma-associated antigen was significantly higher with mean values between 57 and 71%. Qualitatively, the same pattern was obtained in the two investigated healing wounds. PDGF-B chain did not co-localize with either PAL-E or high-molecular weight-melanoma-associated antigen, but PDGF-B chain-expressing cells were, however, frequently found juxtaposed to the microvasculature. The expression of PDGF-beta receptors on pericytes in activated microvessels and the presence of PDGF-B chain-expressing cells in close proximity to the microvasculature of healing wounds and colorectal adenocarcinoma is compatible with a role for PDGF in the physiology of the microvasculature in these conditions. Images Figure 1 p1381-a Figure 3 Figure 4 PMID:8238254

A Cuphea beta-ketoacyl-ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases.

PubMed

Leonard, J M; Knapp, S J; Slabaugh, M B

1998-03-01

Acyl-acyl carrier protein (ACP) thioesterases with specificities on medium chain substrates (C8-C14) are requisite enzymes in plants that produce 8:0, 10:0, 12:0 and 14:0 seed oils, but they may not be the sole enzymatic determinants of chain length. The contribution to chain length regulation of a beta-ketoacyl-ACP synthase, Cw KAS A1, derived from Cuphea wrightii, a species that accumulates 30% 10:0 and 54% 12:0 in seed oils, was investigated. Expression of Cw KAS A1 in Arabidopsis seeds reduced 16:0 from 8.2 to 6.2 mol%, suggesting a KAS II-type activity. In the presence of the KAS I inhibitor cerulenin, however, transgenic seed extracts extended 6:0- and 8:0-ACP at a rate four- to fivefold greater than extracts from untransformed plants, whereas no difference was observed in extension of 14:0- and 16:0-ACP. The effect of KAS A1 on seed oils was tested by combining it with the C. wrightii medium chain-specific thioesterases, Cw FatB1 and Cw FatB2, in crosses of transformed plants. Fatty acid synthesis thesis shifted towards shorter chains in progeny expressing both classes of enzymes. KasA1/FatB1 homozygotes produced threefold more 12:0 than the FatB1 parent while 14:0 and 16:0 were reduced by one-third and one-half, respectively. F2 progeny expressing KasA1 and FatB2 produced twofold more 10:0 and 1.4-fold more 12:0 than the FatB2 parent, and the double-transgenic progeny produced one-quarter less 14:0 and one-half less 16:0 than the FatB2 parent. It is hypothesized that the shift towards production of shorter chains resulted from increased pools of medium chain acyl-ACP resulting from KAS A1 activity. The combined activities of KAS A1 and FatB thioesterases appear to determine the C. wrightii phenotype.

Determining Beta Sheet Crystallinity in Fibrous Proteins by Thermal Analysis and Infrared Spectroscopy

NASA Astrophysics Data System (ADS)

Hu, Xiao; Kaplan, David; Cebe, Peggy

2007-03-01

We report a study of self-assembled beta pleated sheets in Bombyx mori silk fibroin films using thermal analysis and infrared spectroscopy. Crystallization of beta pleated sheets was effected either by heating the films above the glass transition temperature (Tg) and holding isothermally, or by exposure to methanol. The fractions of secondary structural components including random coils, alpha helices, beta pleated sheets, turns, and side chains, were evaluated using Fourier self-deconvolution (FSD) of the infrared absorbance spectra. As crystalline beta sheets form, the heat capacity increment from the TMDSC trace at Tg is systematically decreased and is linearly well correlated with beta sheet content determined from FSD. This analysis of beta sheet content can serve as an alternative to X-ray methods and may have wide applicability to other crystalline beta sheet forming proteins.

Soluble donor HLA class I and beta 2m-free heavy chain in serum of lung transplant recipients: steady-state levels and increases in patients with recurrent CMV infection, acute rejection episodes, and poor outcome.

PubMed

DeVito-Haynes, L D; Jankowska-Gan, E; Meyer, K C; Cornwell, R D; Zeevi, A; Griffith, B; Dauber, J; Iacono, A; Burlingham, W J; Love, R B

2000-12-01

We determined the concentration of donor sHLA/beta(2)m and total beta(2)m-free heavy chain (HC) in the serum of lung transplant recipients with ELISA assays. While we were unable to detect specific donor beta(2)m-free HCs due to a lack of available antibodies, we could determine if events that led to an increase in the release of beta(2)m-free HC also led to an increase in the release of donor sHLA/beta(2)m, particularly the 36 kDa, proteolytically cleaved form. We found that lung transplants constituitively release donor sHLA/beta(2)m at ng/ml levels. The levels (both of donor sHLA/beta(2)m and total beta(2)m-free HC) were significantly increased in CMV-sero-negative recipients (but not in CMV-sero-positive recipients) at the onset of post-transplant CMV disease. Acute rejection episodes were also associated with an increased release of donor sHLA/beta(2)m, but not of beta(2)m-free HC. However, in patients with particularly poor outcome (i.e., graft loss within 1 year) there was a significant release of beta(2)m-free HC. Analysis of one such patient showed a predominance of 36 kDa forms of donor-sHLA/beta(2)m. Our data are consistent with the hypothesis that the metalloproteinase that cleaves beta(2)m-free HC is active during uncontrolled CMV infection and acute rejection. However, recall responses to CMV and controlled immune responses to donor may result in little or no activation of sHLA class I release.

Gamma delta lymphocytes in endocrine autoimmunity: evidence of expansion in Graves' disease but not in type 1 diabetes.

PubMed Central

Roura-Mir, I C; Alcalde, L; Vargas, F; Tolosa, E; Obiols, G; Foz, M; Jaraquemada, D; Pujol-Borrell, R

1993-01-01

Endocrine autoimmune disorders are mediated by T cell-dependent responses to organ-specific antigens, but the mechanisms initiating the process remain unknown. Lymphocytes which use the gamma delta heterodimer as T cell receptor (TCR) for antigen constitute a distinct subset of T cells whose function remains elusive. In order to investigate their possible involvement in endocrine autoimmunity we have determined the proportion of gamma delta T cells in the peripheral blood of 23 patients with type 1 (insulin-dependent) diabetes mellitus (type-1 DM) and 30 patients with autoimmune thyrotoxicosis (Graves' disease). T lymphocyte TCR expression was assessed by fluorescence-activated flow cytometry on peripheral blood mononuclear cells using MoAbs UCHT1 (CD3), TCR delta 1 (gamma delta TCR), WT31 and beta F1 (alpha beta TCR) and both the percentage of T cells expressing gamma delta and the ratio gamma delta/alpha beta were calculated. In the diabetic patients gamma delta cells were not significantly different from the control group (7.7 +/- 54% versus 8.0 +/- 5.5% of T cells, P NS). There was no relation between the proportion of gamma delta lymphocytes and the presence of islet cell antibodies (ICA) in the sera. The Graves' patients showed a tendency towards a higher proportion of gamma delta T lymphocytes than the controls (gamma delta/alpha beta ratios: 0.095 +/- 0.047 versus 0.063 +/- 0.022, P = 0.03). In 14 Graves' patients the number of gamma delta were measured in paired samples of peripheral and intrathyroidal lymphocytes, demonstrating an expansion of gamma delta within the thyroid glands (0.21 +/- 0.3 versus 0.095 +/- 0.047, P = 0.032). Immunohistochemical studies showed that gamma delta cells were scattered among the predominant alpha beta lymphocytes infiltrating the thyroid gland and that they account for 10% of intraepithelial lymphocytes. No relation was found between the increase of gamma delta lymphocytes and any clinical features. PMID:8485915

Rapid model building of beta-sheets in electron-density maps.

PubMed

Terwilliger, Thomas C

2010-03-01

A method for rapidly building beta-sheets into electron-density maps is presented. beta-Strands are identified as tubes of high density adjacent to and nearly parallel to other tubes of density. The alignment and direction of each strand are identified from the pattern of high density corresponding to carbonyl and C(beta) atoms along the strand averaged over all repeats present in the strand. The beta-strands obtained are then assembled into a single atomic model of the beta-sheet regions. The method was tested on a set of 42 experimental electron-density maps at resolutions ranging from 1.5 to 3.8 A. The beta-sheet regions were nearly completely built in all but two cases, the exceptions being one structure at 2.5 A resolution in which a third of the residues in beta-sheets were built and a structure at 3.8 A in which under 10% were built. The overall average r.m.s.d. of main-chain atoms in the residues built using this method compared with refined models of the structures was 1.5 A.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition

PubMed Central

Nicholls, Sarah; Piper, Karen P.; Mohammed, Fiyaz; Dafforn, Timothy R.; Tenzer, Stefan; Salim, Mahboob; Mahendra, Premini; Craddock, Charles; van Endert, Peter; Schild, Hansjörg; Cobbold, Mark; Engelhard, Victor H.; Moss, Paul A. H.; Willcox, Benjamin E.

2009-01-01

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1H versus HA-1R) in the HMHA1 gene. The HA-1H peptide is restricted by HLA-A2 and is immunogenic in HA-1R/R into HA-1H transplants, while HA-1R has been suggested to be a “null allele” in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1R variant. To understand these findings, we determined the structure of an HLA-A2-HA-1H complex to 1.3Å resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1H-specific T-cells bound HA-1H peptide with moderate affinity but failed to bind HA-1R, indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition. PMID:19234124

Genetic engineering with T cell receptors.

PubMed

Zhang, Ling; Morgan, Richard A

2012-06-01

In the past two decades, human gene transfer research has been translated from a laboratory technology to clinical evaluation. The success of adoptive transfer of tumor-reactive lymphocytes to treat the patients with metastatic melanoma has led to new strategies to redirect normal T cells to recognize tumor antigens by genetic engineering with tumor antigen-specific T cell receptor (TCR) genes. This new strategy can generate large numbers of defined antigen-specific cells for therapeutic application. Much progress has been made to TCR gene transfer systems by optimizing gene expression and gene transfer protocols. Vector and protein modifications have enabled excellent expression of introduced TCR chains in human lymphocytes with reduced mis-pairing between the introduced and endogenous TCR chains. Initial clinical studies have demonstrated that TCR gene-engineered T cells could mediate tumor regression in vivo. In this review, we discuss the progress and prospects of TCR gene-engineered T cells as a therapeutic strategy for treating patients with melanoma and other cancers. Published by Elsevier B.V.

Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

PubMed Central

Phee, Hyewon; Au-Yeung, Byron B; Pryshchep, Olga; O'Hagan, Kyle Leonard; Fairbairn, Stephanie Grace; Radu, Maria; Kosoff, Rachelle; Mollenauer, Marianne; Cheng, Debra; Chernoff, Jonathan; Weiss, Arthur

2014-01-01

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation. DOI: http://dx.doi.org/10.7554/eLife.02270.001 PMID:24843022

{beta}-Catenin regulates airway smooth muscle contraction.

PubMed

Jansen, Sepp R; Van Ziel, Anna M; Baarsma, Hoeke A; Gosens, Reinoud

2010-08-01

beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

Molecular cloning of a small prostate protein, known as beta-microsemenoprotein, PSP94 or beta-inhibin, and demonstration of transcripts in non-genital tissues.

PubMed

Ulvsbäck, M; Lindström, C; Weiber, H; Abrahamsson, P A; Lilja, H; Lundwall, A

1989-11-15

In order to study the gene expression of the seminal plasma protein beta-microseminoprotein, also known as PSP94 and beta-inhibin, clones encoding this protein were isolated from a cDNA library constructed in lambda gt11. Nucleotide sequencing confirmed the structure of a previously cloned cDNA. By northern blot analysis identical sized transcripts were demonstrated in the prostate, the respiratory (tracheal, bronchial and lung) tissues and the antrum part of the gastric mucosa. Thus, the protein is not primarily associated with male reproductive function. Although probably of no physiological significance, a slight structural similarity to the ovarian inhibin beta-chains was identified in the C-terminal half of the molecule.

Comprehensive analysis of all triple helical repeats in beta-spectrins reveals patterns of selective evolutionary conservation.

PubMed

Baines, Anthony J

2003-01-01

The spectrin superfamily (spectrin, alpha-actinin, utrophin and dystrophin) has in common a triple helical repeating unit of ~106 amino acid residues. In spectrin, alpha and beta chains contain multiple copies of this repeat. beta-spectrin chains contain the majority of binding activities in spectrin and are essential for animal life. Canonical beta-spectrins have 17 repeats; beta-heavy spectrins have 30. Here, the repeats of five human beta-spectrins, plus beta-spectrins from several other vertebrates and invertebrates, have been analysed. Repeats 1, 2, 14 and 17 in canonical beta are highly conserved between invertebrates and vertebrates, and repeat 8 in some isoforms. This is consistent with conservation of critical functions, since repeats 1, 2 and 17 bind alpha-spectrin. Repeats 1 of beta-spectrins are not always detected by SMART or Pfam tools. A profile hidden Markov model of beta-spectrin repeat 1 detects alpha-actinins, but not utrophin or dystrophin. Novel examples of repeat 1 were detected in the spectraplakins MACF1, BPAG1 and plectin close to the actin-binding domain. Ankyrin binds to the C-terminal portion of repeat 14; the high conservation of this entire repeat may point to additional, undiscovered ligand-binding activities. This analysis indicates that the basic triple helical repeat pattern was adapted early in the evolution of the spectrin superfamily to encompass essential binding activities, which characterise individual repeats in proteins extant today.

Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing

PubMed Central

Li, Zhoufang; Liu, Guangjie; Tong, Yin; Zhang, Meng; Xu, Ying; Qin, Li; Wang, Zhanhui; Chen, Xiaoping; He, Jiankui

2015-01-01

Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys. PMID:25961410

Mesalamine inhibits epithelial beta-catenin activation in chronic ulcerative colitis.

PubMed

Brown, Jeffrey B; Lee, Goo; Managlia, Elizabeth; Grimm, Gery R; Dirisina, Ramanarao; Goretsky, Tatiana; Cheresh, Paul; Blatner, Nichole R; Khazaie, Khashayarsha; Yang, Guang-Yu; Li, Linheng; Barrett, Terrence A

2010-02-01

Mesalamine is a mainstay therapeutic agent in chronic ulcerative colitis (CUC) in which condition it reverses crypt architectural changes and reduces colitis-associated cancer (CAC). The present study addressed the possibility that mesalamine reduces beta-catenin-associated progenitor cell activation, Akt-phosphorylated beta-catenin(Ser552) (P-beta-catenin), and colitis-induced dysplasia (CID). Effects of mesalamine on P-beta-catenin staining and function were assessed by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in biopsy specimens of CUC in mild or "refractory" severe mucosal inflammation. Effects of mesalamine on epithelial proliferation and activation of Akt and beta-catenin were assessed in interleukin (IL)-10(-/-) colitis and CID by immunohistochemistry and Western blotting. Dysplasia was assessed by counting the number and lengths of lesions per colon. Data from IL-10(-/-) and human colitis samples show that mesalamine reduced Akt activation and P-beta-catenin levels in the middle and upper crypt. Reductions in P-beta-catenin in CUC biopsy specimens with severe inflammation suggested that mesalamine reduced P-beta-catenin levels in tissue refractory to mesalamine's anti-inflammatory effects. In IL-10(-/-) mice, mesalamine reduced CID concordant with inhibition of crypt Akt and beta-catenin signaling. The results are consistent with the model that mesalamine contributes to chemoprevention in CAC by reducing beta-catenin signaling within intestinal progenitors.

Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase.

PubMed

Guo, Kunde; Lukacik, Petra; Papagrigoriou, Evangelos; Meier, Marc; Lee, Wen Hwa; Adamski, Jerzy; Oppermann, Udo

2006-04-14

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.

Study of a large Anglo-Saxon family with beta-thalassaemia trait.

PubMed

Raik, E; Powell, E; Gordon, S

1976-01-01

Study of a large Anglo-Saxon family with beta-thalassaemia trait revealed evidence of consanguinity, moreover both branches of the family shared a Spanish ancestor. The manifestations of the disorder were varied in severity and yet the degree of severity appeared to breed true within any individual part of the family. Our explanation for the inheritance pattern observed in the family was to postulate the existence of two non-allelic genes influencing the rate of beta-chain synthesis.

Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

NASA Astrophysics Data System (ADS)

Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

1987-10-01

The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

Increasing protein stability by improving beta-turns.

PubMed

Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

2009-11-15

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis.

PubMed

Carpenter, Stephen M; Nunes-Alves, Cláudio; Booty, Matthew G; Way, Sing Sing; Behar, Samuel M

2016-01-01

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

PubMed Central

Carpenter, Stephen M.; Nunes-Alves, Cláudio; Booty, Matthew G.; Way, Sing Sing; Behar, Samuel M.

2016-01-01

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection. PMID:26745507

Lewis type 1 antigen synthase (beta3Gal-T5) is transcriptionally regulated by homeoproteins.

PubMed

Isshiki, Soichiro; Kudo, Takashi; Nishihara, Shoko; Ikehara, Yuzuru; Togayachi, Akira; Furuya, Akiko; Shitara, Kenya; Kubota, Tetsuro; Watanabe, Masahiko; Kitajima, Masaki; Narimatsu, Hisashi

2003-09-19

The type 1 carbohydrate chain, Galbeta1-3GlcNAc, is synthesized by UDP-galactose:beta-N-acetylglucosamine beta1,3-galactosyltransferase (beta3Gal-T). Among six beta3Gal-Ts cloned to date, beta3Gal-T5 is an essential enzyme for the synthesis of type 1 chain in epithelium of digestive tracts or pancreatic tissue. It forms the type 1 structure on glycoproteins produced from such tissues. In the present study, we found that the transcriptional regulation of the beta3Gal-T5 gene is controlled by homeoproteins, i.e. members of caudal-related homeobox protein (Cdx) and hepatocyte nuclear factor (HNF) families. We found an important region (-151 to -121 from the transcription initiation site), named the beta3Gal-T5 control element (GCE), for the promoter activity. GCE contained the consensus sequences for members of the Cdx and HNF families. Mutations introduced into this sequence abolished the transcriptional activity. Four factors, Cdx1, Cdx2, HNF1alpha, and HNF1beta, could bind to GCE and transcriptionally activate the beta3Gal-T5 gene. Transcriptional regulation of the beta3Gal-T5 gene was consistent with that of members of the Cdx and HNF1 families in two in vivo systems. 1) During in vitro differentiation of Caco-2 cells, transcriptional up-regulation of beta3Gal-T5 was observed in correlation with the increase in transcripts for Cdx2 and HNF1alpha. 2) Both transcript and protein levels of beta3Gal-T5 were determined to be significantly reduced in colon cancer. This down-regulation was correlated with the decrease of Cdx1 and HNF1beta expression in cancer tissue. This is the first finding that a glycosyltransferase gene is transcriptionally regulated under the control of homeoproteins in a tissue-specific manner. beta3Gal-T5, controlled by the intestinal homeoproteins, may play an important role in the specific function of intestinal cells by modifying the carbohydrate structure of glycoproteins.

Structural and sequence features of two residue turns in beta-hairpins.

PubMed

Madan, Bharat; Seo, Sung Yong; Lee, Sun-Gu

2014-09-01

Beta-turns in beta-hairpins have been implicated as important sites in protein folding. In particular, two residue β-turns, the most abundant connecting elements in beta-hairpins, have been a major target for engineering protein stability and folding. In this study, we attempted to investigate and update the structural and sequence properties of two residue turns in beta-hairpins with a large data set. For this, 3977 beta-turns were extracted from 2394 nonhomologous protein chains and analyzed. First, the distribution, dihedral angles and twists of two residue turn types were determined, and compared with previous data. The trend of turn type occurrence and most structural features of the turn types were similar to previous results, but for the first time Type II turns in beta-hairpins were identified. Second, sequence motifs for the turn types were devised based on amino acid positional potentials of two-residue turns, and their distributions were examined. From this study, we could identify code-like sequence motifs for the two residue beta-turn types. Finally, structural and sequence properties of beta-strands in the beta-hairpins were analyzed, which revealed that the beta-strands showed no specific sequence and structural patterns for turn types. The analytical results in this study are expected to be a reference in the engineering or design of beta-hairpin turn structures and sequences. © 2014 Wiley Periodicals, Inc.

Estimating TCR using an integrated model-observation framework that accounts for spatio-temporal variability and pre-industrial radiative imbalances.

NASA Astrophysics Data System (ADS)

Haustein, K.; Schurer, A. P.; Venema, V.

2016-12-01

Apart from a few exceptions (e.g. Aldrin et al. 2012, Skeie et al. 2013) TCR estimates with EBMs are based on global data. Since these estimates don't represent the true spatial-temporal behaviour for observed temperature as well as external forcing (Marvel et al. 2015), we have developed a two-box EBM framework that accounts for these effects. In addition, external forcing from anthropogenic aerosol and GHGs tends to have different response times in comparison to volcanic stratospheric aerosols. Using PMIP3 and an extended ensemble of HadCM3 simulations (Euro500; Schurer et al. 2014) GCM simulations for the pre-industrial period, we obtain the fast and slow response time constants required in the EBM. With the most recent anthropogenic and natural forcing estimates, we test a range of TCR values against observations. The TCR/ECS ratio necessary to achieve that goal is taken from CMIP5 as observationally OHC-based estimates are notoriously unreliable. Given that observed and modelled OHC estimates are in agreement (Cheng et al. 2016), we argue that this should be the standard procedure the make inferences about ECS. Alternatively, it should be distinguished between equilibrium and effective climate sensitivity. The preliminary best estimate for TCR is 1.6K (1.1-2.2K) with an associated ECS value of 2.9K (2.0-4.0K). This is in good agreement with other D&A techniques that do use spatio-temporal patterns as well (e.g. Jones et al. 2016, Gillet et al. 2013). Correcting for natural ENSO variability and tas/tos-related inaccuracies (Richardson et al. 2016) further increases the robustness of the estimated sensitivity range. Our results also indicate that the small radiative imbalance caused by the period of very strong volcanic eruptions just before the CMIP5 historical period starts (1809-1840) has noteworthy implications for the response to later volcanic eruptions and the temperature evolution after 1850. Simply put, CMIP5-type simulations are slightly more sensitive

[The Val606Met mutation of human beta myosin heavy chain in a Chinese familial hypertrophic cardiomyopathy family].

PubMed

Yuan, Jian-song; Qiao, Shu-bin; Wang, Shu-xia; Teng, Si-yong; You, Shi-jie; Yang, Wei-xian; Gao, Run-lin; Chen, Ji-lin; Yang, Yue-jin

2008-04-01

To explore the disease-causing gene mutation in Chinese families with hypertrophic cardiomyopathy (HCM) and to analyze the correlation between the genotype and phenotype. Samples of peripheral blood were collected from three Chinese families with HCM (at least two HCM patients existed/family). The exons in the functional regions of the beta myosin heavy chain gene (MYH7) were amplified with PCR and the products were sequenced. A Val606Met missen mutation was identified in the exon 16 of MYH7 gene in a Chinese family and this mutation was identified in all HCM patients (n = 4) and there was also a 15-years-old young mutation carrier who was not HCM patient now (penetrance of 80%). This mutation was not identified in other healthy family members in this family, in other 2 Chinese familiar HCM families and in 120 non-HCM control patients. The Val606Met missen mutation is closely associated with familiar HCM in a Chinese family which is associated with clinical phenotype with a penetrance of 80%.

Evaluation of pharmacological induction of fatty acid beta-oxidation in X-linked adrenoleukodystrophy.

PubMed

McGuinness, M C; Zhang, H P; Smith, K D

2001-01-01

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder associated with elevated levels of saturated unbranched very-long-chain fatty acids (VLCFA; C > 22:0) in plasma and tissues, and reduced VLCFA beta-oxidation in fibroblasts, white blood cells, and amniocytes from X-ALD patients. The X-ALD gene (ABCD1) at Xq28 encodes the adrenoleukodystrophy protein (ALDP) that is related to the peroxisomal ATP-binding cassette (ABCD) transmembrane half-transporter proteins. The function of ALDP is unknown and its role in VLCFA accumulation unresolved. Previously, our laboratory has shown that sodium 4-phenylbutyrate (4PBA) treatment of X-ALD fibroblasts results in increased peroxisomal VLCFA beta-oxidation activity and increased expression of the X-ALD-related protein, ALDRP, encoded by the ABCD2 gene. In this study, the effect of various pharmacological agents on VLCFA beta-oxidation in ALD mouse fibroblasts is tested. 4PBA, styrylacetate and benzyloxyacetate (structurally related to 4PBA), and trichostatin A (functionally related to 4PBA) increase both VLCFA (peroxisomal) and long-chain fatty acid [LCFA (peroxisomal and mitochondrial)] beta-oxidation. Isobutyrate, zaprinast, hydroxyurea, and 5-azacytidine had no effect on VLCFA or LCFA beta-oxidation. Lovastatin had no effect on fatty acid beta-oxidation under normal tissue culture conditions but did result in an increase in both VLCFA and LCFA beta-oxidation when ALD mouse fibroblasts were cultured in the absence of cholesterol. The effect of trichostatin A on peroxisomal VLCFA beta-oxidation is shown to be independent of an increase in ALDRP expression, suggesting that correction of the biochemical abnormality in X-ALD is not dependent on pharmacological induction of a redundant gene (ABCD2). These studies contribute to a better understanding of the role of ALDP in VLCFA accumulation and may lead to the development of more effective pharmacological therapies. Copyright 2001 Academic Press.

Crystal Structure of the Catalytic Domain of Drosophila [beta]1,4-Galactosyltransferase-7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramakrishnan, Boopathy; Qasba, Pradman K.

2010-11-03

The {beta}1,4-galactosyltransferase-7 ({beta}4Gal-T7) enzyme, one of seven members of the {beta}4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member {beta}4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of {beta}4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 {angstrom} resolution. In the crystalmore » structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of {beta}4Gal-T7 and {beta}4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH{sub 2}OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the {beta}4Gal-T7 crystal structure shows that the aromatic side chain of Tyr{sup 177} interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.« less

MOON for neutrino-less {beta}{beta} decays and {beta}{beta} nuclear matrix elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ejiri, H.

2009-11-09

The MOON project aims at spectroscopic 0v{beta}{beta} studies with the v-mass sensitivity of 100-30 meV by measuring two beta rays from {sup 100}Mo and/or {sup 82}Se. The detector is a compact super-module of multi-layer PL scintillator plates. R and D works made by the pro to-type MOON-1 and the small PL plate show the possible energy resolution of around {sigma}{approx}2.2%, as required for the mass sensitivity. Nuclear matrix elements M{sup 2v} for 2v{beta}{beta} are shown to be given by the sum {sigma}{sub L}M{sub k} of the 2v{beta}{beta} matrix elements M{sub k} through intermediate quasi-particle states in the Fermi-surface, where Mimore » is obtained experimentally by using the GT(J{sup {pi}} = 1{sup +}) matrix elements of M{sub i}(k) and M{sub f}(k) for the successive single-{beta} transitions through the k-th intermediate state.« less

Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

PubMed

Jouvin, M H; Adamczewski, M; Numerof, R; Letourneur, O; Vallé, A; Kinet, J P

1994-02-25

Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

Lineage determination of CD7+ CD5- CD2- and CD7+ CD5+ CD2- lymphoblasts: studies on phenotype, genotype, and gene expression of myeloperoxidase, CD3 epsilon, and CD3 delta.

PubMed

Yoneda, N; Tatsumi, E; Teshigawara, K; Nagata, S; Nagano, T; Kishimoto, Y; Kimura, T; Yasunaga, K; Yamaguchi, N

1994-04-01

The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO

A novel missense mutation, Leu390Val, in the cardiac beta-myosin heavy chain associated with pronounced septal hypertrophy in two families with hypertrophic cardiomyopathy.

PubMed

Havndrup, O; Bundgaard, H; Andersen, P S; Larsen, L A; Vuust, J; Kjeldsen, K; Christiansen, M

2000-12-01

An examination of the genetic background and phenotypic presentation of familial hypertrophic cardiomyopathy (FHC) with respect to specific mutations in the MYH7-gene encoding the cardiac beta-myosin heavy chain. Two families (n = 22) from a cohort of 67 families with FHC were studied at the National University Hospital, Rigshospitalet, Copenhagen. Clinical, non-invasive examinations of all included family members followed by molecular genetic analysis including PCR-single strand conformation polymorphism/heteroduplex (SSCP/HD) analysis and sequencing of exon 3-23 of the MYH7-gene. We found FHC associated with a missense mutation in two families, i.e. a C > G transversion at position g10124 and a G > T transversion at position g10126 causing the change of a leucine residue at codon 390 to a valine residue. The mutation is located in the actin-binding region of the beta-myosin heavy chain. The leucine residue is evolutionarily conserved in vertebrate myosins. In the two families, the phenotypic presentations in the clinically affected were characterized by asymmetric septal hypertrophy (septum diameter 18.8 (5.0) mm (mean (SD)) with only minor involvement of the left ventricular free wall (posterior wall diameter 11.0 (2.2) mm). Furthermore, the left ventricular systolic and diastolic functions were well preserved, even at a high age. The symptomatic status of the clinically affected patients depended on the presence or absence of a concomitant left ventricular outflow tract gradient. We report a novel missense mutation associated with FHC caused by a double nucleotide transversion. The penetrance of the mutation was not complete, but in clinically affected patients the mutation gives rise to an echocardiographic phenotype, predominantly characterized by pronounced septal hypertrophy.

Using steric hindrance to design new inhibitors of class C beta-lactamases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trehan, Indi; Morandi, F.; Blaszczak, L.C.

{beta}-lactamases confer resistance to {beta}-lactam antibiotics such as penicillins and cephalosporins. However, {beta}-lactams that form an acyl-intermediate with the enzyme but subsequently are hindered from forming a catalytically competent conformation seem to be inhibitors of {beta}-lactamases. This inhibition may be imparted by specific groups on the ubiquitous R1 side chain of {beta}-lactams, such as the 2-amino-4-thiazolyl methoxyimino (ATMO) group common among third-generation cephalosporins. Using steric hindrance of deacylation as a design guide, penicillin and carbacephem substrates were converted into effective {beta}-lactamase inhibitors and antiresistance antibiotics. To investigate the structural bases of inhibition, the crystal structures of the acyl-adducts of themore » penicillin substrate amoxicillin and the new analogous inhibitor ATMO-penicillin were determined. ATMO-penicillin binds in a catalytically incompetent conformation resembling that adopted by third-generation cephalosporins, demonstrating the transferability of such sterically hindered groups in inhibitor design.« less

The opposite role of two UBA-UBX containing proteins, p47 and SAKS1 in the degradation of a single ERAD substrate, α-TCR.

PubMed

Park, Eun Sil; Yoo, Yung Joon; Elangovan, Muthukumar

2017-01-01

The UBA-UBX domain-containing proteins can interact with ubiquitinated substrates and p97 during endoplasmic reticulum-associated degradation (ERAD). Here, we found that the expressions of all UBA-UBX genes p47, SAKS1, UBXD8, FAF1, and UBXD7 were elevated upon ER stress, albeit with different levels. Of which p47, SAKS1, and UBXD8 are 'immediate' respondents whereas FAF1 and UBXD7 were 'late' respondents to ER stress. Interestingly, the expression of specific UBA-UBX genes were altered in cells stably expressing three different ERAD substrates such as α-TCR, α1-antitrypsin, and δCD3. We first found that p47 and UBXD8 expression levels were increased in α-TCR and α1-antitrypsin stable cell lines, respectively, whereas SAKS1 expression level was reduced in all the three ERAD substrates tested. Of note, we also found p47 promotes, whereas SASK1 delays the degradation of a single ERAD substrate, α-TCR. Additionally, we found that SAKS1 selectively inhibits the degradation of ERAD substrates without affecting cytosolic proteasomal substrates. Taken together, our results identified that UBA-UBX proteins possess substrate selectivity and opposite role of two different UBA-UBX proteins in the degradation of a single ERAD substrate.

Sequence inversion and phenylalanine surrogates at the beta-turn enhance the antibiotic activity of gramicidin S.

PubMed

Solanas, Concepción; de la Torre, Beatriz G; Fernández-Reyes, María; Santiveri, Clara M; Jiménez, M Angeles; Rivas, Luis; Jiménez, Ana I; Andreu, David; Cativiela, Carlos

2010-05-27

A series of gramicidin S (GS) analogues have been synthesized where the Phe (i + 1) and Pro (i + 2) residues of the beta-turn have been swapped while the respective chiralities (D-, L-) at each position are preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation, and flexibility. Although most analogues preserve the beta-sheet structure, as assessed by NMR, their antibiotic activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative pathogens are observed for several analogues, resulting in improved therapeutic profiles. Data indicate that seemingly minor replacements at the GS beta-turn can have significant impact on antibiotic activity, highlighting this region as a hot spot for modulating GS plasticity and activity.

Positive and Negative Selection in Transgenic Mice Expressing a T-Cell Receptor Specific for Influenza Nucleoprotein and Endogenous Superantigen

PubMed Central

Mamalaki, Clio; Elliott, James; Norton, Trisha; Yannoutsos, Nicholas; Townsend, Alain R.; Chandler, Phillip; Simpson, Elizabeth

1993-01-01

A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8+ and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC Db (Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2b, IE-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2k and BALB/c H-2d) and the endogenous Mtv ligands. Positive selection of CD8+ T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of in vitro culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8+ peripheral T-cells in these (H-2k or H-2d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired. PMID:8281031

Cross-Reactivity among Beta-Lactams.

PubMed

Romano, Antonino; Gaeta, Francesco; Arribas Poves, Maria Francisca; Valluzzi, Rocco Luigi

2016-03-01

Penicillins and cephalosporins are the major classes of beta-lactam (BL) antibiotics in use today and one of the most frequent causes of hypersensitivity reactions to drugs. Monobactams, carbapenems, oxacephems, and beta-lactamase inhibitors constitute the four minor classes of BLs. This review takes into account mainly the prospective studies which evaluated cross-reactivity among BLs in subjects with a well-demonstrated hypersensitivity to a certain class of BLs by performing allergy tests with alternative BLs and, in case of negative results, administering them. In subjects with either IgE-mediated or T-cell-mediated hypersensitivity, cross-reactivity among BLs, particularly among penicillins and among cephalosporins, as well as between penicillins and cephalosporins, seems to be mainly related to structural similarities among their side-chain determinants. Specifically, in penicillin-allergic subjects, cross-reactivity between penicillins and cephalosporins may exceed 30% when they are administered cephalosporins with identical side chains to those of responsible penicillins. In these subjects, a few prospective studies have demonstrated a rate of cross-reactivity between penicillins and both carbapenems and aztreonam lower than 1%. With regard to subjects with an IgE-mediated hypersensitivity to cephalosporins, in a single study, about 25% of the 98 subjects with such hypersensitivity had positive results to penicillins, 3% to aztreonam, 2% to imipenem/cilastatin, and 1% to meropenem. The cross-reactivity related to the selective recognition of the BL ring by IgE or T lymphocytes, which entails positive responses to all BLs tested, appears to be exceptional. Some studies concerning cross-reactivity among BLs have found patterns of allergy-test positivity which cannot be explained by either the common BL ring or by similar or identical side chains, thus indicating the possibility of coexisting sensitivities to different BLs because of prior exposures to them.

Identification and comparative expression analysis of interleukin 2/15 receptor B chain in chickens infected with E. tenella

USDA-ARS?s Scientific Manuscript database

Background: Interleukin (IL) 2 and IL15 receptor beta chain (IL2/15Receptor beta, CD122) play critical roles in signal transduction for the biological activities of IL2 and IL15. Increased knowledge of non-mammalian IL2/15Receptor beta will enhance the understanding of IL2 and IL15 functions. Meth...

Spatial and Time Coincidence Detection of the Decay Chain of Short-Lived Radioactive Nuclei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Granja, Carlos; Jakubek, Jan; Platkevic, Michal

The quantum counting position sensitive pixel detector Timepix with per-pixel energy and time resolution enables to detect radioactive ions and register the consecutive decay chain by simultaneous position-and time-correlation. This spatial and timing coincidence technique in the same sensor is demonstrated by the registration of the decay chain {sup 8}He{yields}{sup {beta} 8}Li and {sup 8}Li{yields}{sup {beta}-} {sup 8}Be{yields}{alpha}+{alpha} and by the measurement of the {beta} decay half-lives. Radioactive ions, selectively obtained from the Lohengrin fission fragment spectrometer installed at the High Flux Reactor of the ILL Grenoble, are delivered to the Timepix silicon sensor where decays of the implanted ionsmore » and daughter nuclei are registered and visualized. We measure decay lifetimes in the range {>=}{mu}s with precision limited just by counting statistics.« less

Heterozygous Hb Hope [beta136(H14)Gly --> Asp] in association with heterozygous beta0-thalassemia with apparent homozygous expression, in a Spanish patient.

PubMed

Beneitez, David; Carrera, Alícia; Duran-Suárez, Joan Ramón; Paz, Victoria; León, Antonio; García Talavera, Juan

2006-01-01

Hb Hope [beta136(H14)Gly --> Asp (GGT --> GAT)] has been found alone or in combination with other globin gene mutations in several African-American families, as well as in Japanese, Thai, Laotian, Cuban and Mauritanian families. We report the hematological and molecular characteristics of a heterozygous association of Hb Hope with beta0-thalassemia (thal) in a Spanish patient, in whom the level of expression of abnormal hemoglobin (Hb) by cation exchange high performance liquid chromatography (HPLC) and electrophoresis suggested initially a homozygous expression of the abnormal Hb, although sequencing of the polymerase chain reaction (PCR)-amplified beta-globin gene demonstrated a heterozygous genotype for Hb Hope. To the best of our knowledge, this is the first description of a case of Hb Hope in a Spanish family.

Crystallographic studies of bovine beta2-microglobulin.

PubMed Central

Becker, J W; Ziffer, J A; Edelman, G M; Cunningham, B A

1977-01-01

Crystals of the bovine milk protein lactollin yield x-ray diffraction data extending to a resolution of 2.8 A. Lactollin is a bovine analogue of beta2-microglobulin, a protein that is homologous in amino acid sequence to the constant domains of immunoglobulins and is the light chain of the human and murine major histocompatability antigens. The protein crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 77.4, b = 47.9, and c = 34.3 A. The unit cell parameters and physical chemical solution studies indicate that the molecule exists in the crystal and in solution as a single polypeptide chain of 12,000 daltons. Images PMID:71731

An enhancer located in a CpG-island 3' to the TCR/CD3-epsilon gene confers T lymphocyte-specificity to its promoter.

PubMed Central

Clevers, H; Lonberg, N; Dunlap, S; Lacy, E; Terhorst, C

1989-01-01

The gene encoding the CD3-epsilon chain of the T cell receptor (TCR/CD3) complex is uniquely transcribed in all T lymphocyte lineage cells. The human CD3-epsilon gene, when introduced into the mouse germ line, was expressed in correct tissue-specific fashion. The gene was then screened for T lymphocyte-specific cis-acting elements in transient chloramphenicol transferase assays. The promoter (-228 to +100) functioned irrespective of cell type. A 1225 bp enhancer with strict T cell-specificity was found in a DNase I hypersensitive site downstream of the last exon, 12 kb from the promoter. This site was present in T cells only. The CD3-epsilon enhancer did not display sequence similarity with the T cell-specific enhancer of CD3-delta, a related gene co-regulated with CD3-epsilon during intrathymic differentiation. The CD3-epsilon enhancer was unusual in that it constituted a CpG island, and was hypomethylated independent of tissue type. Two HTLV I-transformed T cell lines were identified in which the CD3-epsilon gene was not expressed, and in which the enhancer was inactive. Images PMID:2583122

ICI 182,780-regulated gene expression in DU145 prostate cancer cells is mediated by estrogen receptor-beta/NFkappaB crosstalk.

PubMed

Leung, Yuet-Kin; Gao, Ying; Lau, Kin-Mang; Zhang, Xiang; Ho, Shuk-Mei

2006-04-01

Estrogen receptor (ER)-beta is the predominant ER subtype in prostate cancer (PCa). We previously demonstrated that ICI 182,780 (ICI), but not estrogens, exerted dose-dependent growth inhibition on DU145 PCa cells by an ER-beta-mediated pathway. Transcriptional profiling detected a greater than three-fold upregulation of seven genes after a 12-hour exposure to 1 microM ICI. Semiquantitative reverse transcriptase polymerase chain reaction confirmed the upregulation of four genes by ICI: interleukin-12alpha chain, interleukin-8, embryonic growth/differentiation factor, and RYK tyrosine kinase. Treatment with an ER-beta antisense oligonucleotide reduced cellular ER-beta mRNA and induced loss of expression of these genes. Sequence analysis revealed the presence of consensus NFkappaB sites, but not estrogen-responsive elements, in promoters of all four genes. Reporter assay and chromatin immunoprecipitation experiments demonstrated that ICI-induced gene expression could be mediated by crosstalk between ER-beta and the NFkappaB signaling pathway, denoting a novel mechanism of ER-beta-mediated ICI action. Therefore, combined therapies targeting ER-beta and NFkappaB signaling may be synergistic as treatment for PCa.

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

PubMed

Supper, Verena; Schiller, Herbert B; Paster, Wolfgang; Forster, Florian; Boulègue, Cyril; Mitulovic, Goran; Leksa, Vladimir; Ohradanova-Repic, Anna; Machacek, Christian; Schatzlmaier, Philipp; Zlabinger, Gerhard J; Stockinger, Hannes

2016-02-01

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression. Copyright © 2016 by The American Association of Immunologists, Inc.

Influenza virus-specific TCR-transduced T cells as a model for adoptive immunotherapy

PubMed Central

Berdien, Belinda; Reinhard, Henrike; Meyer, Sabrina; Spöck, Stefanie; Kröger, Nicolaus; Atanackovic, Djordje; Fehse, Boris

2013-01-01

Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8+ T-cell clones reacting to the Flu matrix protein (Flu-M) and 6 CD4+ T-cell clones reacting to the Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies. PMID:23428899

Phosphorylation and ubiquitination of the IkappaB kinase complex by two distinct signaling pathways.

PubMed

Shambharkar, Prashant B; Blonska, Marzenna; Pappu, Bhanu P; Li, Hongxiu; You, Yun; Sakurai, Hiroaki; Darnay, Bryant G; Hara, Hiromitsu; Penninger, Josef; Lin, Xin

2007-04-04

The IkappaB kinase (IKK) complex serves as the master regulator for the activation of NF-kappaB by various stimuli. It contains two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKalpha/beta at its activation loop and the K63-linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T-cell receptor (TCR) stimulation. However, the phosphorylation of IKKalpha/beta activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1-independent manner and mediate IKKalpha/beta phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKalpha/beta and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.

Src homology 2-domain containing leukocyte-specific phosphoprotein of 76 kDa is mandatory for TCR-mediated inside-out signaling, but dispensable for CXCR4-mediated LFA-1 activation, adhesion, and migration of T cells.

PubMed

Horn, Jessica; Wang, Xiaoqian; Reichardt, Peter; Stradal, Theresia E; Warnecke, Nicole; Simeoni, Luca; Gunzer, Matthias; Yablonski, Deborah; Schraven, Burkhart; Kliche, Stefanie

2009-11-01

Engagement of the TCR or of chemokine receptors such as CXCR4 induces adhesion and migration of T cells via so-called inside-out signaling pathways. The molecular processes underlying inside-out signaling events are as yet not completely understood. In this study, we show that TCR- and CXCR4-mediated activation of integrins critically depends on the membrane recruitment of the adhesion- and degranulation-promoting adapter protein (ADAP)/Src kinase-associated phosphoprotein of 55 kDa (SKAP55)/Rap1-interacting adapter protein (RIAM)/Rap1 module. We further demonstrate that the Src homology 2 domain containing leukocyte-specific phosphoprotein of 76 kDa (SLP76) is crucial for TCR-mediated inside-out signaling and T cell/APC interaction. Besides facilitating membrane recruitment of ADAP, SKAP55, and RIAM, SLP76 regulates TCR-mediated inside-out signaling by controlling the activation of Rap1 as well as Rac-mediated actin polymerization. Surprisingly, however, SLP76 is not mandatory for CXCR4-mediated inside-out signaling. Indeed, both CXCR4-induced T cell adhesion and migration are not affected by loss of SLP76. Moreover, after CXCR4 stimulation, the ADAP/SKAP55/RIAM/Rap1 module is recruited to the plasma membrane independently of SLP76. Collectively, our data indicate a differential requirement for SLP76 in TCR- vs CXCR4-mediated inside-out signaling pathways regulating T cell adhesion and migration.

The emergence of non-cytolytic NK1.1+ T cells in the long-term culture of murine tumour-infiltrating lymphocytes: a possible role of transforming growth factor-beta.

PubMed

Tamada, K; Harada, M; Ito, O; Takenoyama, M; Mori, T; Matsuzaki, G; Nomoto, K

1996-12-01

The mechanism by which murine tumour-infiltrating lymphocytes (TIL) decreased their anti-tumour activity during an in vitro culture with interleukin-2 (IL-2) was investigated. A phenotype analysis revealed that the TIL cultured for 7 days (TIL-d7) were exclusively NKI.1- CD4- CD8+ CD3+ cells and that this population was replaced by natural killer (NK)1.1+ CD4- CD8 CD3+ cells by day 27 (TIL-d27) during the culture of TIL. The TIL-d7 cells showed a cytolytic activity against B16 melanoma, whereas the TIL-d27 cells had lost this activity, suggesting that the decrease in the anti tumour effect of TIL during the culture with IL-2 was due to their populational change. Analysis on the characteristics of the TIL-d27 cells revealed that they expressed skewed T-cell receptor (TCR) V beta 5 and increased mRNA expression of V alpha 14. In addition, they expressed transforming growth factor beta (TGF-beta) mRNA. Interestingly, TGF-beta augmented the proliferation of TIL-d27 cells under the presence of IL-2, but suppressed that of TIL-d7 cells. Moreover, the proliferation of TIL-d27 cells was suppressed by anti-TGF-beta monoclonal antibody. Collectively, these results suggest that, in contrast to its suppressive effect on anti-tumour effector T cells. TGF-beta could be an autocrine growth factor for NKL1.1+ T cells and thereby induce non-cytolytic NK1.1+ T cells in the long-term culture of TIL.

Genetic heterogeneity of Beta thalassemia in Lebanon reflects historic and recent population migration.

PubMed

Makhoul, N J; Wells, R S; Kaspar, H; Shbaklo, H; Taher, A; Chakar, N; Zalloua, P A

2005-01-01

Beta thalassemia is an autosomal recessive disorder characterized by reduced (beta(+)) or absent (beta(0)) beta-globin chain synthesis. In Lebanon it is the most predominant genetic defect. In this study we investigated the religious and geographic distribution of the beta-thalassemia mutations identified in Lebanon, and traced their precise origins. A total of 520 beta-globin chromosomes from patients of different religious and regional backgrounds was studied. Beta thalassemia mutations were identified using Amplification Refractory Mutation System (ARMS) PCR or direct gene sequencing. Six (IVS-I-110, IVS-I-1, IVS-I-6, IVS-II-1, cd 5 and the C > T substitution at cd 29) out of 20 beta-globin defects identified accounted for more than 86% of the total beta-thalassemia chromosomes. Sunni Muslims had the highest beta-thalassemia carrier rate and presented the greatest heterogeneity, with 16 different mutations. Shiite Muslims followed closely with 13 mutations, whereas Maronites represented 11.9% of all beta-thalassemic subjects and carried 7 different mutations. RFLP haplotype analysis showed that the observed genetic diversity originated from both new mutational events and gene flow from population migration. This study provides information about the types and distribution of beta-thalassemia mutations within each religious group and geographic region, which is essential for the implementation of screening and prevention programs.

Beta-lactam hypersensitivity and cross-reactivity.

PubMed

Terico, Adrienne T; Gallagher, Jason C

2014-12-01

Penicillin is the most frequently reported cause of drug allergy, and cross-reactivity of penicillins with other beta-lactam antibiotics is an area of debate. This review evaluates the available data on immunoglobulin E-mediated penicillin hypersensitivity and cross-reactivity with cephalosporin, carbapenem, and monobactam antibiotics. A MEDLINE search was conducted from 1950 to October 2013, and selected references from review articles were also evaluated. There is a wide variety in reported incidences of cross-reactivity between penicillins and cephalosporins or carbapenems, with early retrospective studies suggesting up to 41.7% and 47.4% cross-reactivity, respectively. Conversely, the use of monobactam antibiotics is frequently employed in the case of a penicillin allergy, as prescribers believe that there is no cross-reactivity between the 2 drug classes. More recent prospective studies suggest that the rates of cross-reactivity with cephalosporins and carbapenems are <5% and <1%, respectively. Similarities in penicillin and cephalosporin side chains may play a role in cross-reactivity between these classes. Cross-reactivity with monobactams is essentially negligible; however, there are some clinical data to support an interaction between ceftazidime and aztreonam, due to the similarity of their side chains. The data reviewed suggest that avoidance of other beta-lactams in patients with type 1 hypersensitivity to penicillins should be reconsidered. © The Author(s) 2014.

Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response.

PubMed

Reichstetter, S; Ettinger, R A; Liu, A W; Gebe, J A; Nepom, G T; Kwok, W W

2000-12-15

The 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 VP16(369-379)-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 VP16(369-379) 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/VP16(369-379) tetramer at either 23 or 37 degrees C. Weak staining was also observed at 4 degrees C. Clone 5 showed weaker staining compared with clone 48 at 37 degrees C, and no staining was observed at 23 degrees 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/VP16(369-379) 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.

TCR and IL-7 Signaling Are Altered in the Absence of Functional GTPase of the Immune Associated Nucleotide Binding Protein 5 (GIMAP5)

PubMed Central

Chen, Xi-Lin; Serrano, Daniel; Ghobadi, Farnaz; Mayhue, Marian; Hoebe, Kasper; Ilangumaran, Subburaj; Ramanathan, Sheela

2016-01-01

GTPase of the immune associated nucleotide binding protein (GIMAP) family of proteins are expressed essentially in cells of the hematopoietic system. Mutation in the founding member of this gene family, Gimap5, results in the lymphopenic phenotype in Bio-Breeding diabetes prone rats. In mice, deletion of functional Gimap5 gene affects the survival and renewal of hematopoietic stem cells in addition to the defects observed in T cells. Here we show that T cells from OTII TCR-transgenic Gimap5sph/sph mice do not proliferate in response to its cognate antigen. Furthermore, T cells from Gimap5 mutant rats and mice show decreased phosphorylation of STAT5 following stimulation with IL-7. Our results suggest that functional Gimap5 is required for optimal signaling through TCR and IL-7R in T cells. PMID:27023180

Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

NASA Technical Reports Server (NTRS)

Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

1995-01-01

The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

Immune alterations in male and female mice after 2-deoxy-D-glucose administration

NASA Technical Reports Server (NTRS)

Dreau, D.; Morton, D. S.; Foster, M.; Swiggett, J. P.; Sonnenfeld, G.

1997-01-01

Administration of 2-deoxy-D-glucose (2-DG) induces acute cellular glucoprivation. In the current study, we examined differences in immune parameters after 2-DG administration in both sexes. Male and female BDF1 mice were injected three times, 48 h apart, either with a saline solution (control group) or with 2-DG in saline (500 mg/kg). Two hours after the last injection, blood and spleens were collected. Plasma levels of interleukin-1beta, and interferon-gamma levels were measured. Additionally, the levels of the specific leukocyte antigens CD3, CD4, CD8, T cell receptor (TCR) alpha/beta, I-Ad, and H-2Ld/H-2Db were evaluated by flow cytometry on both blood and spleen cells. The blastogenic response of leukocytes from both tissues to mitogens was assessed. Levels of glucose, corticosterone, testosterone, progesterone, 17beta-estradiol, follicle-stimulating hormone, and luteinizing hormone were also determined. Increases in the percentage of cells bearing TCR alpha/beta and I-Ad in the blood and H-2Ld/H-2Db in the spleen were observed in the 2-DG-treated group for both sexes. In contrast, higher corticosterone and IL-1beta plasma concentrations, as well as higher percentages of splenocytes bearing TCR alpha/beta and I-Ad, and lower mitogen-induced proliferation of mature T splenocytes (79%) were observed in female but not in male mice injected with 2-DG compared with those injected with saline (p < 0.05). Taken together, these results suggest that female mice are more sensitive than male mice to immune alterations induced by 2-DG administration.

The Cish SH2 domain is essential for PLC-γ1 regulation in TCR stimulated CD8+ T cells.

PubMed

Guittard, Geoffrey; Dios-Esponera, Ana; Palmer, Douglas C; Akpan, Itoro; Barr, Valarie A; Manna, Asit; Restifo, Nicholas P; Samelson, Lawrence E

2018-03-28

Cish, participates within a multi-molecular E3 ubiquitin ligase complex, which ubiquitinates target proteins. It has an inhibitory effect on T cell activation mediated by PLC-γ1 regulation, and it functions as a potent checkpoint in CD8 + T cell tumor immunotherapy. To study the structural and functional relationships between Cish and PLC-γ1 during CD8 + T cell activation, we tested mutants of the Cish-SH2 (R107K) and D/BC (L222Q, C226Q) domains. We confirmed that Cish-SH2-specific binding was essential for PLC-γ1 ubiquitination and degradation. This domain was essential for the Cish-mediated inhibition of Ca 2+ release upon TCR stimulation. No effect on inhibition of cytokine release was observed with SH2 or D/BC mutants, although the absence of Cish led to an increased release of IFN-γ and TNF-α. Using imaging we showed that Cish was expressed mostly in the cytoplasm and we did not see any Cish clustering at the plasma membrane upon stimulation. We conclude that the Cish-SH2 domain is essential for PLC-γ1 regulation in TCR-stimulated CD8 + T cells.

Allelic polymorphism in the T cell receptor and its impact on immune responses.

PubMed

Gras, Stephanie; Chen, Zhenjun; Miles, John J; Liu, Yu Chih; Bell, Melissa J; Sullivan, Lucy C; Kjer-Nielsen, Lars; Brennan, Rebekah M; Burrows, Jacqueline M; Neller, Michelle A; Khanna, Rajiv; Purcell, Anthony W; Brooks, Andrew G; McCluskey, James; Rossjohn, Jamie; Burrows, Scott R

2010-07-05

In comparison to human leukocyte antigen (HLA) polymorphism, the impact of allelic sequence variation within T cell receptor (TCR) loci is much less understood. Particular TCR loci have been associated with autoimmunity, but the molecular basis for this phenomenon is undefined. We examined the T cell response to an HLA-B*3501-restricted epitope (HPVGEADYFEY) from Epstein-Barr virus (EBV), which is frequently dominated by a TRBV9*01(+) public TCR (TK3). However, the common allelic variant TRBV9*02, which differs by a single amino acid near the CDR2beta loop (Gln55-->His55), was never used in this response. The structure of the TK3 TCR, its allelic variant, and a nonnaturally occurring mutant (Gln55-->Ala55) in complex with HLA-B*3501(HPVGEADYFEY) revealed that the Gln55-->His55 polymorphism affected the charge complementarity at the TCR-peptide-MHC interface, resulting in reduced functional recognition of the cognate and naturally occurring variants of this EBV peptide. Thus, polymorphism in the TCR loci may contribute toward variability in immune responses and the outcome of infection.

Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.

PubMed Central

Vachon, P H; Xu, H; Liu, L; Loechel, F; Hayashi, Y; Arahata, K; Reed, J C; Wewer, U M; Engvall, E

1997-01-01

Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders. PMID:9312189

TCR-contacting residues orientation and HLA-DRβ* binding preference determine long-lasting protective immunity against malaria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alba, Martha P.; Suarez, Carlos F.; Universidad del Rosario, Bogotá D. C.

Fully-protective, long-lasting, immunological (FPLLI) memory against Plasmodium falciparum malaria regarding immune protection-inducing protein structures (IMPIPS) vaccinated into monkeys previously challenged and re-challenged 60 days later with a lethal Aotus monkey-adapted P. falciparum strain was found to be associated with preferential high binding capacity to HLA-DRβ1* allelic molecules of the major histocompatibility class II (MHC-II), rather than HLA-DRβ3*, β4*, β5* alleles. Complete PPII{sub L} 3D structure, a longer distance (26.5 Å ± 1.5 Å) between residues perfectly fitting into HLA-DRβ1*PBR pockets 1 and 9, a gauche{sup −} rotamer orientation in p8 TCR-contacting polar residue and a larger volume of polar p2 residues was also found. Thismore » data, in association with previously-described p3 and p7 apolar residues having gauche{sup +} orientation to form a perfect MHC-II-peptide-TCR complex, determines the stereo-electronic and topochemical characteristics associated with FPLLI immunological memory. - Highlights: • Stereo-electronic and topochemical rules associated with FPLLI immunological memory. • Presence of very high long-lasting antibody titres against Plasmodium falciparum Spz. • Protective memory induction associated with a binding capacity to HLA-DRβ1*. • gauche{sup −} rotamer orientation in p8 polar residue is related to is related to immunological memory.« less

Hemoglobin Hiroshima (β143 histidine → aspartic acid): a newly identified fast moving beta chain variant associated with increased oxygen affinity and compensatory erythremia

PubMed Central

Hamilton, Howard B.; Iuchi, Iwao; Miyaji, Takaoki; Shibata, Susumu

1969-01-01

During a survey for hemoglobinopathies in over 9000 residents of Hiroshima Prefecture, Japan, a fast moving hemoglobin was identified in eight members of three generations in a Japanese family. The abnormal hemoglobin, named Hb Hiroshima, constitutes about 50% of the total hemoglobin in hemolysates from the carriers who have a mild erythremia but are otherwise apparently clinically unaffected. All preparations of Hb Hiroshima have increased affinity for oxygen, by either tonometric or oxygen electrode determinations. At pH 7.0, the oxygen pressure, P50 required to half saturate an unfractionated hemolysate from a carrier was one-half that of Hb A, and the P50 of a purified sample containing no Hb A was one-fourth that of Hb A. The pH dependence of the oxygen equilibrium (Bohr effect) is below normal, as shown by the absolute value of the Bohr effect factor which is about half that of Hb A, in the pH range between 7.0 and 7.4. The Hill constant, n, for Hb Hiroshima between pH 7.0 and 7.4 is 2-2.4, compared to 2.8-3 for Hb A under the same conditions, indicating reduction of, but not complete abolition of heme-heme interaction. Urea dissociation and canine hybridization tests located the biochemical lesion in the beta chain. Fingerprints (Ingram), carboxypeptidase digestion, and amino acid analysis demonstrated that the substitution was at residue 143 in the beta chain, where histidine was replaced by aspartic acid. In contrast to other recently described high oxygen affinity mutants that show intact Bohr effects, all three of the major characteristics of the reversible combination of hemoglobin with oxygen (oxygen equilibrium, heme-heme interaction, and pH dependence) are affected in Hb Hiroshima. A tentative interpretation of these effects, relating structure to function, is offered in terms of recently developed models of normal hemoglobin. Images PMID:5773089

Differential usage of T-cell receptor V beta gene families by CD4+ and CD8+ T cells in patients with CD8hi common variable immunodeficiency: evidence of a post-thymic effect.

PubMed Central

Duchmann, R; Jaffe, J; Ehrhardt, R; Alling, D W; Strober, W

1996-01-01

In this study, we report that differences between T-cell receptor (TCR) V beta gene family usage in CD4+ and CD8+ T cells are significantly greater in a subgroup of patients with common variable immunodeficiency (CVI) and high levels of activated CD8+ T cells (CD8hi CVI) than in controls (P < 0.001). In CD8hi CVI patients, such differences were also significantly greater for V beta 12 than for other V beta families. As the causes of the differential usage of V beta gene families by CD4+ and CD8+ T cells are under investigation, it was interesting that the combined differences between V beta gene family usage in the CD4+ and CD8+ T-cell subpopulations as a whole were significantly lower than the combined differences between individual V beta gene family usage in either CD4+ or CD8+ T-cell subpopulations (P < 0.001 in both control and CD8hi CVI patients). Further, the pattern of V beta gene family usage in CD4+ T cells was remarkably similar to that in CD8+ T cells in both groups. These data strongly suggest that differences in V beta gene family usage arising from coselection by major histocompatibility complex (MHC) class I versus MHC class II restriction elements do not fundamentally distort 'basic' V beta gene family usage patterns. They also support the concept that differences in CD4+ and CD8+ T-cell V beta gene family usage, which were increased in CD8hi CVI, can arise from high-affinity interactions between disease-associated antigens or superantigens and T cells in the post-thymic T-cell compartment. Images Figure 6 PMID:8666443

Hb taradale [beta82(EF6)Lys-->Arg]: a novel mutation at a 2,3-diphosphoglycerate binding site.

PubMed

Brennan, Stephen O; Sheen, Campbell; Chan, Tim; George, Peter M

2005-01-01

Hb Taradale [beta82(EF6)Lys-->Arg] was initially detected as a split Hb A0 peak on Hb A1c, monitoring. Red cell parameters, hemoglobin (Hb) electrophoresis and stability tests were normal. Mass spectrometry (ms) clearly identified a variant beta chain with a mass increase of 28 Da and peptide mapping located the mutation site to peptide betaT-9. DNA sequencing confirmed the presence of a novel beta82(EF6)Lys-->Arg mutation. This conservative substitution at a 2,3-diphosphoglycerate (2,3-DPG) binding site did not, however, appear to affect the P50 for oxygen binding.

Prediction and analysis of beta-turns in proteins by support vector machine.

PubMed

Pham, Tho Hoan; Satou, Kenji; Ho, Tu Bao

2003-01-01

Tight turn has long been recognized as one of the three important features of proteins after the alpha-helix and beta-sheet. Tight turns play an important role in globular proteins from both the structural and functional points of view. More than 90% tight turns are beta-turns. Analysis and prediction of beta-turns in particular and tight turns in general are very useful for the design of new molecules such as drugs, pesticides, and antigens. In this paper, we introduce a support vector machine (SVM) approach to prediction and analysis of beta-turns. We have investigated two aspects of applying SVM to the prediction and analysis of beta-turns. First, we developed a new SVM method, called BTSVM, which predicts beta-turns of a protein from its sequence. The prediction results on the dataset of 426 non-homologous protein chains by sevenfold cross-validation technique showed that our method is superior to the other previous methods. Second, we analyzed how amino acid positions support (or prevent) the formation of beta-turns based on the "multivariable" classification model of a linear SVM. This model is more general than the other ones of previous statistical methods. Our analysis results are more comprehensive and easier to use than previously published analysis results.

Automated main-chain model building by template matching and iterative fragment extension.

PubMed

Terwilliger, Thomas C

2003-01-01

An algorithm for the automated macromolecular model building of polypeptide backbones is described. The procedure is hierarchical. In the initial stages, many overlapping polypeptide fragments are built. In subsequent stages, the fragments are extended and then connected. Identification of the locations of helical and beta-strand regions is carried out by FFT-based template matching. Fragment libraries of helices and beta-strands from refined protein structures are then positioned at the potential locations of helices and strands and the longest segments that fit the electron-density map are chosen. The helices and strands are then extended using fragment libraries consisting of sequences three amino acids long derived from refined protein structures. The resulting segments of polypeptide chain are then connected by choosing those which overlap at two or more C(alpha) positions. The fully automated procedure has been implemented in RESOLVE and is capable of model building at resolutions as low as 3.5 A. The algorithm is useful for building a preliminary main-chain model that can serve as a basis for refinement and side-chain addition.

Expression of beta 3-adrenoceptor mRNA in rat tissues.

PubMed

Evans, B A; Papaioannou, M; Bonazzi, V R; Summers, R J

1996-01-01

1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with

Neutrophil chemotaxis in response to TGF-beta isoforms (TGF-beta 1, TGF-beta 2, TGF-beta 3) is mediated by fibronectin.

PubMed

Parekh, T; Saxena, B; Reibman, J; Cronstein, B N; Gold, L I

1994-03-01

TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of extracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs), suggesting that TGF-beta s may play a role in the recruitment of PMNs during the initial phase of the inflammatory response. In our current studies, we demonstrate that the maximal chemotactic response was attained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of the PMNs: TGF-beta 2 (556 microM) > TGF-beta 3 (463 microM) > TGF-beta 1 (380 microM) (beta 2: beta 3, p < or = 0.010; beta 3: beta 1, p < or = 0.04; beta 2: beta 1, p < or = 0.0012). A mAb to the cell binding domain (CBD) of Fn inhibited the chemotactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (< 1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results indicate that chemotaxis of PMNs in response to TGF-beta isoforms is mediated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 integrin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated cells (173.6 ng/ml). The concentration of TGF-beta required to cause maximal release of Fn from PMNs (4000 fM) is a concentration at which TGF-beta

Tumor vessel-injuring ability improves antitumor effect of cytotoxic T lymphocytes in adoptive immunotherapy.

PubMed

Kanagawa, N; Yanagawa, T; Nakagawa, T; Okada, N; Nakagawa, S

2013-01-01

Angiogenesis is required for normal physiologic processes, but it is also involved in tumor growth, progression and metastasis. Here, we report the development of an immune-based antiangiogenic strategy based on the generation of T lymphocytes that possess killing specificity for cells expressing vascular endothelial growth factor receptor 2 (VEGFR2). To target VEGFR2-expressing cells, we engineered cytotoxic T lymphocyte (CTL) expressing chimeric T-cell receptors (cTCR-CTL) comprised of a single-chain variable fragment (scFv) against VEGFR2 linked to an intracellular signaling sequence derived from the CD3ζ chain of the TCR and CD28 by retroviral gene transduction methods. The cTCR-CTL exhibited efficient killing specificity against VEGFR2 and a tumor-targeting function in vitro and in vivo. Reflecting such abilities, we confirmed that the cTCR-CTL strongly inhibited the growth of a variety of syngeneic tumors after adoptive transfer into tumor-bearing mice without consequent damage to normal tissue. In addition, CTL expressing both cTCR and tumor-specific TCR induced complete tumor regression due to enhanced tumor infiltration by the CTL and long-term antigen-specific function. These findings provide evidence that the tumor vessel-injuring ability improved the antitumor effect of CTLs in adoptive immunotherapy for a broad range of cancers by inducing immune-mediated destruction of the tumor neovasculature.

Inhibition of glycogen synthase kinase 3beta during heart failure is protective.

PubMed

Hirotani, Shinichi; Zhai, Peiyong; Tomita, Hideharu; Galeotti, Jonathan; Marquez, Juan Pablo; Gao, Shumin; Hong, Chull; Yatani, Atsuko; Avila, Jesús; Sadoshima, Junichi

2007-11-26

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3beta in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta (Tg-GSK-3beta-DN) and tetracycline-regulatable wild-type GSK-3beta. GSK-3beta-DN significantly reduced the kinase activity of endogenous GSK-3beta, inhibited phosphorylation of eukaryotic translation initiation factor 2B epsilon, and induced accumulation of beta-catenin and myeloid cell leukemia-1, confirming that GSK-3beta-DN acts as a dominant negative in vivo. Tg-GSK-3beta-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the alpha-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3beta induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3beta-DN and nontransgenic mice, Tg-GSK-3beta-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3beta transgene in tetracycline-regulatable wild-type GSK-3beta mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3beta-DN in cardiac myocytes inhibited tumor necrosis factor-alpha-induced apoptosis, and the antiapoptotic effect of GSK-3beta-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3beta induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3beta inhibition is mediated by myeloid cell leukemia-1. Thus

The emergence of non-cytolytic NK1.1+ T cells in the long-term culture of murine tumour-infiltrating lymphocytes: a possible role of transforming growth factor-beta.

PubMed Central

Tamada, K; Harada, M; Ito, O; Takenoyama, M; Mori, T; Matsuzaki, G; Nomoto, K

1996-01-01

The mechanism by which murine tumour-infiltrating lymphocytes (TIL) decreased their anti-tumour activity during an in vitro culture with interleukin-2 (IL-2) was investigated. A phenotype analysis revealed that the TIL cultured for 7 days (TIL-d7) were exclusively NKI.1- CD4- CD8+ CD3+ cells and that this population was replaced by natural killer (NK)1.1+ CD4- CD8 CD3+ cells by day 27 (TIL-d27) during the culture of TIL. The TIL-d7 cells showed a cytolytic activity against B16 melanoma, whereas the TIL-d27 cells had lost this activity, suggesting that the decrease in the anti tumour effect of TIL during the culture with IL-2 was due to their populational change. Analysis on the characteristics of the TIL-d27 cells revealed that they expressed skewed T-cell receptor (TCR) V beta 5 and increased mRNA expression of V alpha 14. In addition, they expressed transforming growth factor beta (TGF-beta) mRNA. Interestingly, TGF-beta augmented the proliferation of TIL-d27 cells under the presence of IL-2, but suppressed that of TIL-d7 cells. Moreover, the proliferation of TIL-d27 cells was suppressed by anti-TGF-beta monoclonal antibody. Collectively, these results suggest that, in contrast to its suppressive effect on anti-tumour effector T cells. TGF-beta could be an autocrine growth factor for NKL1.1+ T cells and thereby induce non-cytolytic NK1.1+ T cells in the long-term culture of TIL. Images Figure 4 Figure 6 PMID:9014832

Analysis of betaS and betaA genes in a Mexican population with African roots.

PubMed

Magaña, María Teresa; Ongay, Zoyla; Tagle, Juan; Bentura, Gilberto; Cobián, José G; Perea, F Javier; Casas-Castañeda, Maricela; Sánchez-López, Yoaly J; Ibarra, Bertha

2002-01-01

To investigate the origin of the beta(A) and beta(S) genes in a Mexican population with African roots and a high frequency of hemoglobin S, we analyzed 467 individuals (288 unrelated) from different towns in the states of Guerrero and Oaxaca in the Costa Chica region. The frequency of the sickle-cell trait was 12.8%, which may represent a public health problem. The frequencies of the beta-haplotypes were determined from 350 nonrelated chromosomes (313 beta(A) and 37 beta(S)). We observed 15 different beta(A) haplotypes, the most common of which were haplotypes 1 (48.9%), 2 (13.4%), and 3 (13.4%). The calculation of pairwise distributions and Nei's genetic distance analysis using 32 worldwide populations showed that the beta(A) genes are more closely related to those of Mexican Mestizos and North Africans. Bantu and Benin haplotypes and haplotype 9 were related to the beta(S) genes, with frequencies of 78.8, 18.2, and 3.0%, respectively. Comparison of these haplotypes with 17 other populations revealed a high similitude with the population of the Central African Republic. These data suggest distinct origins for the beta(A) and beta(S) genes in Mexican individuals from the Costa Chica region.

Isolation and characterization of a novel endo-beta-galactofuranosidase from Bacillus sp.

PubMed

Ramli, N; Fujinaga, M; Tabuchi, M; Takegawa, K; Iwahara, S

1995-10-01

A soil bacterium capable of growing on a polysaccharide-containing beta(1-->6)galactofuranoside residues derived from the acidic polysaccharide of Fusarium sp. as a carbon source has been isolated. From various bacteriological characteristics, the organism was identified as a Bacillus sp. The bacterium produced beta-galactofuranosidase inductively in the culture media. The most effective inducer for the beta-galactofuranosidase production was a polysaccharide containing beta(1-->5) or beta(1-->6)-linked galactofuranoside residues, but gum arabic, gum guar, gum ghati, arabinogalactam, araban, and pectic acid did not induce the enzyme. The enzyme had three different molecular weight forms. The low molecular-weight form was purified by a combination of Toyopearl HW-55 and DEAE-Toyopearl 650S column chromatographies, and preparative polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 67,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6 and 37 degrees C, and was stable between pH 4 to 8 at 5 degrees C. The action of the enzyme was inhibited by the addition of Cd2+, Co2+, Hg2+, Zn2+, iodoacetic acid, and EDTA. The purified enzyme cleaved beta(1-->5) and beta(1-->6)-linked galactofuranosyl chains. Based upon the mode of liberation of galactofuranosyl residues from pyridylamino-beta(1-->6)-linked galactofuranoside oligomers, the enzyme can be classified as an endo-beta-galactofuranosidase that randomly hydrolyzes the linkage.

Interaction with beta-arrestin determines the difference in internalization behavor between beta1- and beta2-adrenergic receptors.

PubMed

Shiina, T; Kawasaki, A; Nagao, T; Kurose, H

2000-09-15

The beta(1)-adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. As beta-arrestin is important for internalization, we examine the interaction of beta-arrestin with beta(1)AR with three different methods: intracellular trafficking of beta-arrestin, binding of in vitro translated beta-arrestin to intracellular domains of beta(1)- and beta(2)ARs, and inhibition of betaAR-stimulated adenylyl cyclase activities by beta-arrestin. The green fluorescent protein-tagged beta-arrestin 2 translocates to and stays at the plasma membrane by beta(2)AR stimulation. Although green fluorescent protein-tagged beta-arrestin 2 also translocates to the plasma membrane, it returns to the cytoplasm 10-30 min after beta(1)AR stimulation. The binding of in vitro translated beta-arrestin 1 and beta-arrestin 2 to the third intracellular loop and the carboxyl tail of beta(1)AR is lower than that of beta(2)AR. The fusion protein of beta-arrestin 1 with glutathione S-transferase inhibits the beta(1)- and beta(2)AR-stimulated adenylyl cyclase activities, although inhibition of the beta(1)AR-stimulated activity requires a higher concentration of the fusion protein than that of the beta(2)AR-stimulated activity. These results suggest that weak interaction of beta(1)AR with beta-arrestins explains the resistance to agonist-induced internalization. This is further supported by the finding that beta-arrestin can induce internalization of beta(1)AR when beta-arrestin 1 does not dissociate from beta(1)AR by fusing to the carboxyl tail of beta(1)AR.

Structural re-alignment in an immunologic surface region of ricin A chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zemla, A T; Zhou, C E

2007-07-24

We compared structure alignments generated by several protein structure comparison programs to determine whether existing methods would satisfactorily align residues at a highly conserved position within an immunogenic loop in ribosome inactivating proteins (RIPs). Using default settings, structure alignments generated by several programs (CE, DaliLite, FATCAT, LGA, MAMMOTH, MATRAS, SHEBA, SSM) failed to align the respective conserved residues, although LGA reported correct residue-residue (R-R) correspondences when the beta-carbon (Cb) position was used as the point of reference in the alignment calculations. Further tests using variable points of reference indicated that points distal from the beta carbon along a vector connectingmore » the alpha and beta carbons yielded rigid structural alignments in which residues known to be highly conserved in RIPs were reported as corresponding residues in structural comparisons between ricin A chain, abrin-A, and other RIPs. Results suggest that approaches to structure alignment employing alternate point representations corresponding to side chain position may yield structure alignments that are more consistent with observed conservation of functional surface residues than do standard alignment programs, which apply uniform criteria for alignment (i.e., alpha carbon (Ca) as point of reference) along the entirety of the peptide chain. We present the results of tests that suggest the utility of allowing user-specified points of reference in generating alternate structural alignments, and we present a web server for automatically generating such alignments.« less

Biosynthetic processing of the oligosaccharide chains of cellular fibronectin.

PubMed

Olden, K; Hunter, V A; Yamada, K M

1980-10-15

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-3H]mannose of [35S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-beta-N-acetylgluosaminidase H, which specifically cleaves the 'high-mannose' class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing. This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a 'high-mannose' oligosaccharide precursor and a 'complex' carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (Mr 2100) comparable to (Man)9GlcNAc (Mr 2080), and was sensitive to endo-beta-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistant to endo-beta-N-acetylglucosaminidase H cleavage. The final 'complex' or processed oligosaccharide structure contained approximately two-thirds less [3H]mannose, was insensitive to endo-beta-N-acetylglucosaminidase H and had an apparent Mr of 2300 as estimated by gel filtration. We conclude that the carbohydrate portion of fibronectin is synthesized as a 'high-mannose' intermediate and is subsequently processed to give the characteristic 'complex' oligosaccharide chains of fibronectin.

Synthesis of anatase TiO2 nanoparticles with beta-cyclodextrin as a supramolecular shell.

PubMed

Li, Landong; Sun, Xiaohong; Yang, Yali; Guan, Naijia; Zhang, Fuxiang

2006-11-20

We report a novel, green hydrothermal-synthesis route to well-dispersed anatase TiO2 nanoparticles with particle sizes of 9-16 nm in the presence of beta-CD (beta-cyclodextrin). During the synthesis process, the CD-containing synthesis mixture assembled in both longitudinal and latitudinal directions. Driven by the interaction between molecules, the beta-CDs assembled in the longitudinal direction to form long-chain compounds, whereas in the latitudinal direction, they tended to form regular aggregates through coordination with the Ti species from the hydrolysis of tetrabutyl titanate. In view of the effect of the coordination and the steric hindrance of beta-CDs as a supramolecular shell, homogeneous nuclei and slow growth of TiO2 crystals during the synthesis process was observed, which was responsible for the formation of uniform TiO2 nanoparticles. The low beta-CD dosage and the high product yield (>90%) demonstrated well the potential of this synthesis route in the large-scale industrial production of anatase nanoparticles.

Penicillin and beta-lactam allergy: epidemiology and diagnosis.

PubMed

Macy, Eric

2014-11-01

Penicillin is the most common beta-lactam antibiotic allergy and the most common drug class allergy, reported in about 8% of individuals using health care in the USA. Only about 1% of individuals using health care in the USA have a cephalosporin allergy noted in their medical record, and other specific non-penicillin, non-cephalosporin beta-lactam allergies are even rarer. Most reported penicillin allergy is not associated with clinically significant IgE-mediated reactions after penicillin rechallenge. Un-verified penicillin allergy is a significant and growing public health problem. Clinically significant IgE-mediated penicillin allergy can be safely confirmed or refuted using skin testing with penicilloyl-poly-lysine and native penicillin G and, if skin test is negative, an oral amoxicillin challenge. Acute tolerance of an oral therapeutic dose of a penicillin class antibiotic is the current gold standard test for a lack of clinically significant IgE-mediated penicillin allergy. Cephalosporins and other non-penicillin beta-lactams are widely, safely, and appropriately used in individuals, even with confirmed penicillin allergy. There is little, if any, clinically significant immunologic cross-reactivity between penicillins and other beta-lactams. Routine cephalosporin skin testing should be restricted to research settings. It is rarely needed clinically to safely manage patients and has unclear predictive value at this time. The use of alternative cephalosporins, with different side chains, is acceptable in the setting of a specific cephalosporin allergy. Carbapenems and monobactams are also safely used in individuals with confirmed penicillin allergy. A certain predictable, but low, rate of adverse reactions will occur with all beta-lactam antibiotic use both pre- and post-beta-lactam allergy evaluations.

Spontaneous healing and growth of locked magnetic island chains in toroidal plasmas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fitzpatrick, R.; Waelbroeck, F. L.

2012-11-15

Recent experiments have demonstrated that locked magnetic island chains in stellarator plasmas spontaneously heal under certain conditions, and spontaneously grow under others. A formalism initially developed to study magnetic island dynamics in tokamak plasmas is employed to investigate this phenomenon. It is found that island healing/growth transitions can be caused either by a breakdown in torque balance in the vicinity of the island chain, or by an imbalance between the various terms in the island width evolution equation. The scaling of the healing/growth thresholds with the standard dimensionless plasma parameters {beta}, {nu}{sub *}, and {rho}{sub *} is determined. In accordancemore » with the experimental data, it is found that island healing generally occurs at high {beta} and low {nu}{sub *}, and island growth at low {beta} and high {nu}{sub *}. In further agreement, it is found that island healing is accompanied an ion poloidal velocity shift in the electron diamagnetic direction, and island growth by a velocity shift in the ion diamagnetic direction. Finally, it is found that there is considerable hysteresis in the healing/growth cycle, as is also seen experimentally.« less

Diacylglycerol kinase α inactivation is an integral component of the costimulatory pathway that amplifies TCR signals.

PubMed

Arranz-Nicolás, Javier; Ogando, Jesús; Soutar, Denise; Arcos-Pérez, Raquel; Meraviglia-Crivelli, Daniel; Mañes, Santos; Mérida, Isabel; Ávila-Flores, Antonia

2018-06-01

The arsenal of cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. T cells rely on diacylglycerol (DAG) to carry out their functions. DAG availability and signaling are regulated by the enzymes diacylglycerol kinase (DGK) α and ζ, whose excess function drives T cells into hyporesponsive states. Targeting DGKα is a promising strategy for coping with cancer; its blockade could reinstate T-cell attack on tumors while limiting tumor growth, due to positive DGKα functions in several oncogenic pathways. Here, we made a side-by-side comparison of the effects of commercial pharmacological DGK inhibitors on T-cell responses with those promoted by DGKα and DGKζ genetic deletion or silencing. We show the specificity for DGKα of DGK inhibitors I and II and the structurally similar compound ritanserin. Inhibitor treatment promoted Ras/ERK (extracellular signal-regulated kinase) signaling and AP-1 (Activator protein-1) transcription, facilitated DGKα membrane localization, reduced the requirement for costimulation, and cooperated with enhanced activation following DGKζ silencing/deletion. DGKiII and ritanserin had similar effects on TCR proximal signaling, but ritanserin counteracted long-term T-cell activation, an effect that was potentiated in DGKα -/- cells. In contrast with enhanced activation triggered by pharmacological inhibition, DGKα silencing/genetic deletion led to impaired Lck (lymphocyte-specific protein tyrosine kinase) activation and limited costimulation responses. Our results demonstrate that pharmacological inhibition of DGKα downstream of the TCR provides a gain-of-function effect that amplifies the DAG-dependent signaling cascade, an ability that could be exploited therapeutically to reinvigorate T cells to attack tumors.

Hb Alesha [beta67(E11)Val-->Met, GTG-->ATG] in an Argentinean girl.

PubMed

Eberle, Silvia Eandi; Noguera, Nélida I; Sciuccati, Gabriela; Bonduel, Mariana; Díaz, Lilian; Staciuk, Raquel; Targovnik, Héctor M; Feliu-Torres, Aurora

2007-01-01

Hb Alesha is caused by a GTG-->ATG mutation at codon 67 of the beta-globin gene, resulting in abnormal beta-globin chains in which the normal beta67(E11) valine is changed to methionine. This hemoglobin (Hb) is also known as Hb Bristol, the first unstable Hb described, since in a fraction of the variant the methionine is modified into an aspartic acid by a posttranslational modification. This replacement disrupts the apolar bonds between the valine and the heme group, producing an unstable Hb and severe hemolysis. We have identified this rare hemoglobinopathy in an Argentinean girl with severe hemolytic anemia, splenomegaly and frequent requirement for red blood cell transfusions.

Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morant, Marc

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

TLR4 signaling in effector CD4+ T cells regulates TCR activation and experimental colitis in mice

PubMed Central

González-Navajas, José M.; Fine, Sean; Law, Jason; Datta, Sandip K.; Nguyen, Kim P.; Yu, Mandy; Corr, Maripat; Katakura, Kyoko; Eckman, Lars; Lee, Jongdae; Raz, Eyal

2010-01-01

TLRs sense various microbial products. Their function has been best characterized in DCs and macrophages, where they act as important mediators of innate immunity. TLR4 is also expressed on CD4+ T cells, but its physiological function on these cells remains unknown. Here, we have shown that TLR4 triggering on CD4+ T cells affects their phenotype and their ability to provoke intestinal inflammation. In a model of spontaneous colitis, Il10–/–Tlr4–/– mice displayed accelerated development of disease, with signs of overt colitis as early as 8 weeks of age, when compared with Il10–/– and Il10–/–Tlr9–/– mice, which did not develop colitis by 8 months. Similar results were obtained in a second model of colitis in which transfer of naive Il10–/–Tlr4–/– CD4+ T cells into Rag1–/– recipients sufficient for both IL-10 and TLR4 induced more aggressive colitis than the transfer of naive Il10–/– CD4+ T cells. Mechanistically, LPS stimulation of TLR4-bearing CD4+ T cells inhibited ERK1/2 activation upon subsequent TCR stimulation via the induction of MAPK phosphatase 3 (MKP-3). Our data therefore reveal a tonic inhibitory role for TLR4 signaling on subsequent TCR-dependent CD4+ T cell responses. PMID:20051628

A Tc-99m-labeled long chain fatty acid derivative for myocardial imaging.

PubMed

Magata, Yasuhiro; Kawaguchi, Takayoshi; Ukon, Misa; Yamamura, Norio; Uehara, Tomoya; Ogawa, Kazuma; Arano, Yasushi; Temma, Takashi; Mukai, Takahiro; Tadamura, Eiji; Saji, Hideo

2004-01-01

C-11- and I-123-labeled long chain fatty acid derivatives have been reported as useful radiopharmaceuticals for the estimation of myocardial fatty acid metabolism. We have reported that Tc-99m-labeled N-[[[(2-mercaptoethyl)amino]carbonyl]methyl]-N-(2-mercaptoethyl)-6-aminohexanoic acid ([(99m)Tc]MAMA-HA), a medium chain fatty acid derivative, is metabolized by beta-oxidation in the liver and that the MAMA ligand is useful for attaching to the omega-position of fatty acid derivatives as a chelating group for Tc-99m. On the basis of these findings, we focused on developing a Tc-99m-labeled long chain fatty acid derivative that reflected fatty acid metabolism in the myocardium. In this study, we synthesized a dodecanoic acid derivative, MAMA-DA, and a hexadecanoic acid derivative, MAMA-HDA, and performed radiolabeling and biodistribution studies. [(99m)Tc]MAMA-DA and [(99m)Tc]MAMA-HDA were prepared using a ligand-exchange reaction. Biodistribution studies were carried out in normal mice and rats. Then, a high initial uptake of Tc-99m was observed, followed by a rapid clearance from the heart. The maximum heart/blood ratio was 3.6 at 2 min postinjection of [(99m)Tc]MAMA-HDA. These kinetics were similar to those with postinjection of p-[(125)I]iodophenylpentadecanoic acid. Metabolite analysis showed [(99m)Tc]MAMA-HDA was metabolized by beta-oxidation in the body. In conclusion, [(99m)Tc]MAMA-HDA is a promising compound as a long chain fatty acid analogue for estimating beta-oxidation of fatty acid in the heart.

Three exopolysaccharides of the beta-(1-->6)-D-glucan type and a beta-(1-->3;1-->6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit.

PubMed

Vasconcelos, Ana Flora D; Monteiro, Nilson K; Dekker, Robert F H; Barbosa, Aneli M; Carbonero, Elaine R; Silveira, Joana L M; Sassaki, Guilherme L; da Silva, Roberto; de Lourdes Corradi da Silva, Maria

2008-09-22

Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha, and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and (13)C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1-->3) linkages substituted at O-6 as shown in the putative structure below: [carbohydrate structure: see text]. The EPS of the other fungal isolates consisted of a linear chain of (1-->6)-linked glucopyranosyl residues of the following structure: [carbohydrate structure: see text]. FTIR spectra showed one band at 891 cm(-1), and (13)C NMR spectroscopy showed that all glucosidic linkages were of the beta-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. beta-(1-->6)-d-Glucans produced as exocellular polysaccharides by fungi are uncommon.

18{beta}-Glycyrrhetinic acid inhibits adipogenic differentiation and stimulates lipolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moon, Myung-Hee; Jeong, Jae-Kyo; Lee, You-Jin

2012-04-20

Highlights: Black-Right-Pointing-Pointer 18{beta}-GA inhibits adipogenic differentiation in 3T3-L1 preadipocytes and stimulates lipolysis in differentiated adipocytes. Black-Right-Pointing-Pointer Anti-adipogenic effect of 18{beta}-GA is caused by down-regulation of PPAR{gamma} and inactivation of Akt signalling. Black-Right-Pointing-Pointer Lipolytic effect of 18{beta}-GA is mediated by up-regulation of HSL, ATGL and perilipin and activation of HSL. -- Abstract: 18{beta}-Glycyrrhetinic acid (18{beta}-GA) obtained from the herb liquorice has various pharmacological properties including anti-inflammatory and anti-bacterial activities. However, potential biological anti-obesity activities are unclear. In this study, novel biological activities of 18{beta}-GA in the adipogenesis of 3T3-L1 preadipocytes and in lipolysis of differentiated adipocytes were identified. Mouse 3T3-L1 cellsmore » were used as an in vitro model of adipogenesis and lipolysis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. The amount of lipid droplet accumulation was determined by an AdipoRed assay. The expression of several adipogenic transcription factors and enzymes was investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. 18{beta}-GA dose-dependently (1-40 {mu}M) significantly decreased lipid accumulation in maturing preadipocytes. In 3T3-L1 preadipocytes, 10 {mu}M of 18{beta}-GA down-regulated the transcriptional levels of the peroxisome proliferator-activated receptor {gamma}, CCAAT/enhancer-binding protein {alpha} and adiponectin, which are markers of adipogenic differentiation via Akt phosphorylation. Also, in differentiated adipocytes, 18{beta}-GA increased the level of glycerol release and up-regulated the mRNA of hormone-sensitive lipase, adipose TG lipase and perilipin, as well as the phosphorylation of hormone-sensitive lipase at Serine 563. The results indicate that 18

Coexistence of Southeast Asian ovalocytosis and beta-thalassemia: a molecular and hematological analysis.

PubMed

Fucharoen, Goonnapa; Fucharoen, Supan; Singsanan, Sanita; Sanchaisuriya, Kanokwan

2007-05-01

We describe hematological and molecular characterization of a Thai female who had Southeast Asian ovalocytosis (SAO) associated with beta+-thalassemia trait. The proband had mild microcytosis with Hb 12.9 g/dl, Hct 35.8%, MCV 74.4 fl, MCH 26.8 pg, MCHC 36.0 g/dl, and elevated Hb A2 (5.6%), characteristics of beta-thalassemia trait. Peripheral blood film examination revealed prominent ovalocytosis. However, a one-tube osmotic fragility (OF) test commonly used for thalassemia screening was negative and a normal OF curve was observed. Further polymerase chain reaction (PCR) analyses identified the beta(-28A-G) mutation in the beta-globin gene and a 27 bp deletion in erythrocyte band 3 protein gene, indicating a genetically compound heterozygote. Hematological data of the proband was comparatively presented with those of eight female and 15 male carriers of pure beta-thalassemia with the same mutation. The finding demonstrates that although the association of the SAO and beta-thalassemia does not produce a more severe clinical picture, this could lead to a mis-screening of beta-thalassemia using an OF test as a primary screening test. Additional blood film examination followed by PCR could help in the detection of this unusual genetic interaction in the region. (c) 2006 Wiley-Liss, Inc.

Enhanced production of longer side-chain polyhydroxyalkanoic acid with omega-aromatic group substitution in phaZ-disrupted Pseudomonas fluorescens BM07 mutant through unrelated carbon source cometabolism and salicylic acid beta-oxidation inhibition.

PubMed

Choi, Mun Hwan; Xu, Ju; Rho, Jong Kook; Zhao, Xu Ping; Yoon, Sung Chul

2010-06-01

The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) omega-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL omega-aromatic fatty acid in the presence of salicylic acid that is known as a beta-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to approximately 10 mol%. An advantage of salicylic acid inhibition technique is that salicylic acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL-PHA with improved physicochemical properties in P. fluorescens BM07. (c) 2010 Elsevier Ltd. All rights reserved.

Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1.

PubMed

Kida, Hiroshi; Sugano, Yuri; Iizuka, Ryo; Fujihashi, Masahiro; Yohda, Masafumi; Miki, Kunio

2008-11-14

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of alpha and beta subunits and forms a "jellyfish-like" structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two alpha and two beta subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of alpha (alpha1 and alpha2) and beta subunits (beta1 and beta2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD beta1 subunit at 1.9 A resolution and its functional analysis. TsPFD beta1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The beta hairpin linkers of beta1 subunits assemble to form a beta barrel "body" around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the beta1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric beta1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD beta1 subunits act as molecular chaperones in living cells of some archaea.

Rigidity and pH dependent Morphology of Beta-Lactoglobulin Spherulites

NASA Astrophysics Data System (ADS)

Gayetsky, Lisa; Armstead, Douglas

2008-03-01

Beta-Lactoglobulin is a milk protein that will denature in acidic solution (less than 2.0 pH) and if heated for extended periods (greater than 18 hours) it will form radial structures called Spherulites. Spherulites, along with the amyloid fibrils that compose them, are of practical importance because they form in the human body and cause the amyloidosis diseases. Different amyloidosis are caused by different types of denatured proteins occurring in different parts of the body. Since it is believed that Spherulite formation is a generic protein characteristic, Beta-Lactoglobulin is a legitimate and easy to use protein to study these structures. In this study we are quantifying the shape of Beta-Lactoglobulin Spherulites to determine if the pH of the protein solution has an impact on the morphology due to side chain interactions or other causes. We are also testing the rigidity of these structures to determine the relevance of small shape changes.

Beta-and gamma-turns in proteins revisited: a new set of amino acid turn-type dependent positional preferences and potentials.

PubMed

Guruprasad, K; Rajkumar, S

2000-06-01

The number of beta-turns in a representative set of 426 protein three-dimensional crystal structures selected from the recent Protein Data Bank has nearly doubled and the number of gamma-turns in a representative set of 320 proteins has increased over seven times since the previous analysis. Beta-turns (7153) and gamma-turns (911) extracted from these proteins were used to derive a revised set of type-dependent amino acid positional preferences and potentials. Compared with previous results, the preference for proline, methionine and tryptophan has increased and the preference for glutamine, valine, glutamic acid and alanine has decreased for beta-turns. Certain new amino acid preferences were observed for both turn types and individual amino acids showed turn-type dependent positional preferences. The rationale for new amino acid preferences are discussed in the light of hydrogen bonds and other interactions involving the turns. Where main-chain hydrogen bonds of the type NH(i + 3) --> CO(i) were not observed for some beta-turns, other main-chain hydrogen bonds or solvent interactions were observed that possibly stabilize such beta-turns. A number of unexpected isolated beta-turns with proline at i + 2 position were also observed. The NH(i + 2) --> CO(i) hydrogen bond was observed for almost all gamma-turns. Nearly 20% classic gamma-turns and 43% inverse gamma-turns are isolated turns.

Cleavage of beta,beta-carotene to flavor compounds by fungi.

PubMed

Zorn, H; Langhoff, S; Scheibner, M; Berger, R G

2003-09-01

More than 50 filamentous fungi and yeasts, known for de novo synthesis or biotransformation of mono-, sesqui-, tri-, or tetraterpenes, were screened for their ability to cleave beta,beta-carotene to flavor compounds. Ten strains discolored a beta,beta-carotene-containing growth agar, indicating efficient degradation of beta,beta-carotene. Dihydroactinidiolide was formed as the sole conversion product of beta,beta-carotene in submerged cultures of Ganoderma applanatum, Hypomyces odoratus, Kuehneromyces mutabilis, and Trametes suaveolens. When mycelium-free culture supernatants from five species were applied for the conversions, nearly complete degradation of beta,beta-carotene was observed after 12 h. Carotenoid-derived volatile products were detected in the media of Ischnoderma benzoinum, Marasmius scorodonius, and Trametes versicolor. beta-Ionone proved to be the main metabolite in each case, whereas beta-cyclocitral, dihydroactinidiolide, and 2-hydroxy-2,6,6-trimethylcyclohexanone were formed in minor quantities. Using a photometric bleaching test, the beta,beta-carotene cleaving enzyme activities of M. scorodonius were partially characterized.

The effect of TGF-beta2 on MMP-2 production and activity in highly metastatic human bladder carcinoma cell line 5637.

PubMed

Dehnavi, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ataei, Zahra; Aryan, Hajar

2009-06-01

Transforming growth factor-beta (TGF-beta) superfamily regulates matrix metalloproteinases (MMP), which intrinsically regulate various cell behaviors leading to metastasis. We investigated the effect of TGF-beta(2) on MMP-2 regulation in human bladder carcinoma cell line 5637. Zymography, ELISA, and real-time polymerase chain reaction revealed that TGF-beta(2) stimulated MMP-2 production, but the transcription of its gene remained unchanged. Wortmannin could not inhibit MMP-2 secretion and activity and conversely the amount of the protein and its enzymatic activity were increased. These data suggest that TGF-beta(2) increased MMP-2 at the posttranscriptional level and this upregulation was independent of phosphatidylinositol 3-kinase signaling pathway.

BCL11B enhances TCR/CD28-triggered NF-kappaB activation through up-regulation of Cot kinase gene expression in T-lymphocytes.

PubMed

Cismasiu, Valeriu B; Duque, Javier; Paskaleva, Elena; Califano, Danielle; Ghanta, Sailaja; Young, Howard A; Avram, Dorina

2009-01-15

BCL11B is a transcriptional regulator with an important role in T-cell development and leukaemogenesis. We demonstrated recently that BCL11B controls expression from the IL (interleukin)-2 promoter through direct binding to the US1 (upstream site 1). In the present study, we provide evidence that BCL11B also participates in the activation of IL-2 gene expression by enhancing NF-kappaB (nuclear factor kappaB) activity in the context of TCR (T-cell receptor)/CD28-triggered T-cell activation. Enhanced NF-kappaB activation is not a consequence of BCL11B binding to the NF-kappaB response elements or association with the NF-kappaB-DNA complexes, but rather the result of higher translocation of NF-kappaB to the nucleus caused by enhanced degradation of IkappaB (inhibitor of NF-kappaB). The enhanced IkappaB degradation in cells with increased levels of BCL11B was specific for T-cells activated through the TCR, but not for cells activated through TNFalpha (tumour necrosis factor alpha) or UV light, and was caused by increased activity of IkappaB kinase, as indicated by its increase in phosphorylation. As BCL11B is a transcription factor, we investigated whether the expression of genes upstream of IkappaB kinase in the TCR/CD28 signalling pathway was affected by increased BCL11B expression, and found that Cot (cancer Osaka thyroid oncogene) kinase mRNA levels were elevated. Cot kinase is known to promote enhanced IkappaB kinase activity, which results in the phosphorylation and degradation of IkappaB and activation of NF-kappaB. The implied involvement of Cot kinase in BCL11B-mediated NF-kappaB activation in response to TCR activation is supported by the fact that a Cot kinase dominant-negative mutant or Cot kinase siRNA (small interfering RNA) knockdown blocked BCL11B-mediated NF-kappaB activation. In support of our observations, in the present study we report that BCL11B enhances the expression of several other NF-kappaB target genes, in addition to IL-2. In addition, we

beta-Hexachlorocyclohexane (beta-HCH)

Integrated Risk Information System (IRIS)

beta - Hexachlorocyclohexane ( beta - HCH ) ; CASRN 319 - 85 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Asses

Beta-hairpin formation in aqueous solution and in the presence of trifluoroethanol: a (1)H and (13)C nuclear magnetic resonance conformational study of designed peptides.

PubMed

Santiveri, Clara M; Pantoja-Uceda, David; Rico, Manuel; Jiménez, M Angeles

2005-10-15

In order to check our current knowledge on the principles involved in beta-hairpin formation, we have modified the sequence of a 3:5 beta-hairpin forming peptide with two different purposes, first to increase the stability of the formed 3:5 beta-hairpin, and second to convert the 3:5 beta-hairpin into a 2:2 beta-hairpin. The conformational behavior of the designed peptides was investigated in aqueous solution and in 30% trifluoroethanol (TFE) by analysis of the following nuclear magnetic resonance (NMR) parameters: nuclear Overhauser effect (NOE) data, and C(alpha)H, (13)C(alpha), and (13)C(beta) conformational shifts. From the differences in the ability to adopt beta-hairpin structures in these peptides, we have arrived to the following conclusions: (i) beta-Hairpin population increases with the statistical propensity of residues to occupy each turn position. (ii) The loop length, and in turn, the beta-hairpin type, can be modified as a function of the type of turn favored by the loop sequence. These two conclusions reinforce previous results about the importance of beta-turn sequence in beta-hairpin folding. (iii) Side-chain packing on each face of the beta-sheet may play a major role in beta-hairpin stability; hence simplified analysis in terms of isolated pair interactions and intrinsic beta-sheet propensities is insufficient. (iv) Contributions to beta-hairpin stability of turn and strand sequences are not completely independent. (v) The burial of hydrophobic surface upon beta-hairpin formation that, in turn, depends on side-chain packing also contributes to beta-hairpin stability. (vi) As previously observed, TFE stabilizes beta-hairpin structures, but the extent of the contribution of different factors to beta-hairpin formation is sometimes different in aqueous solution and in 30% TFE. (c) 2005 Wiley Periodicals, Inc. Biopolymers 79: 150-162, 2005.

Folding cooperativity in a three-stranded beta-sheet model.

PubMed

Roe, Daniel R; Hornak, Viktor; Simmerling, Carlos

2005-09-16

The thermodynamic behavior of a previously designed three-stranded beta-sheet was studied via several microseconds of standard and replica exchange molecular dynamics simulations. The system is shown to populate at least four thermodynamic minima, including two partially folded states in which only a single hairpin is formed. Simulated melting curves show different profiles for the C and N-terminal hairpins, consistent with differences in secondary structure content in published NMR and CD/FTIR measurements, which probed different regions of the chain. Individual beta-hairpins that comprise the three-stranded beta-sheet are observed to form cooperatively. Partial folding cooperativity between the component hairpins is observed, and good agreement between calculated and experimental values quantifying this cooperativity is obtained when similar analysis techniques are used. However, the structural detail in the ensemble of conformations sampled in the simulations permits a more direct analysis of this cooperativity than has been performed on the basis of experimental data. The results indicate the actual folding cooperativity perpendicular to strand direction is significantly larger than the lower bound obtained previously.

Structure-activity relationship studies of (+/-)-terbutaline and (+/-)-fenoterol on beta3-adrenoceptors in the guinea pig gastric fundus.

PubMed

Horinouchi, T; Nakagawa, Y; Wakabayashi, M; Koike, K

2001-08-01

(+/-)-Terbutaline and (+/-)-fenoterol are both arylethanolamine analogs that have tertbutyl and aryliso-propyl substituents respectively at the a position on the nitrogen of the ethanolamine side chain. In the present study, we have investigated the structure-activity relationships of (+/-)-terbutaline and (+/-)-fenoterol as beta3-adrenoceptor agonists in the guinea pig gastric fundus. (+/-)-Terbutaline and (+/-)-fenoterol induced concentration-dependent relaxation of the precontracted gastric fundus with pD2 values of 4.45+/-0.10 and 5.90+/-0.09, and intrinsic activities of 1.00+/-0.03 and 0.99+/-0.01 respectively. The combination of the selective beta1-adrenoceptor antagonist (+/-)-atenolol (100 microM), and the selective beta2-adrenoceptor antagonist (+/-)-butoxamine (100 microM), produced a 2 and 6 fold rightward shift of the concentration-response curves for (+/-)-terbutaline and (+/-)-fenoterol respectively, without depressing the maximal responses. The order of potency of these agonists was (pD2 value): (+/-)-fenoterol (5.09+/-0.10) > (+/-)-terbutaline (4.13+/-0.08). In the presence of (+/-)-atenolol and (+/-)-butoxamine, however, the non-selective beta1, beta2- and beta3-adrenoceptor antagonist (+/-)-bupranolol caused a concentration-dependent rightward shift of the concentration-response curves for (+/-)-terbutaline and (+/-)-fenoterol. Schild plot analyses of the effects of (+/-)-bupranolol against these agonists gave pA2 values of 6.21+/-0.07 ((+/-)-terbutaline) and 6.37+/-0.06 ((+/-)-fenoterol) respectively, and the slopes of the Schild plot were not significantly different from unity (p>0.05). These results suggest that the relaxant responses to (+/-)-terbutaline and (+/-)-fenoterol are mainly mediated through beta3-adrenoceptors in the guinea pig gastric fundus. The beta3-adrenoceptor agonist potencies of arylethanolamine analogs depend on the size of the end of the alkylamine side chain.

Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

PubMed Central

1995-01-01

Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells. PMID:7721945

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Extended 3{beta}-alkyl steranes and 3-alkyl triaromatic steroids in crude oils and rock extracts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahl, J.; Moldowan, J.M.; Summons, R.E.

1995-09-01

In oils and Precambian- to Miocene-age source rocks from varying depositional environments, we have conclusively identified several novel 3-alkyl sterane and triaromatic steroid series, including (1) 3{beta}-n-pentyl steranes, (2) 3{beta}-isopentyl steranes, (3) 3{beta}-n-hexyl steranes, (4) 3{beta}-n-hepatyl steranes, (5) 3,4-dimethyl steranes, (6) 3{beta}-butyl,4-methyl steranes, (7) triaromatic 3-n-pentyl steroids, and (8) triaromatic 3-isopentyl steroids. We have also tentatively identified additional homologs with 3-alkyl substituents as large as C{sub 11}. The relative abundances of these compounds vary substantially between samples, as indicated by (1) the ratio of 3{beta}-n-pentyl steranes to 3{beta}-isopentyl steranes and (2) the ratio of 3-n-pentyl triaromatic steroids to 3-isopentyl triaromaticmore » steroids. These data suggest possible utility for these parameters as tools for oil-source rock correlations and reconstruction of depositional environments. Although no 3-alkyl steroid natural products are currently known, several lines of evidence suggest that 3{beta}-alkyl steroids result from bacterial side-chain additions to diagenetic {delta}{sup 2}-sterenes.« less

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor.

PubMed

Kim, Jaewon; Lee, Jihun; Brych, Stephen R; Logan, Timothy M; Blaber, Michael

2005-02-01

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

A theoretical case study of type I and type II beta-turns.

PubMed

Czinki, Eszter; Császár, Attila G; Perczel, András

2003-03-03

NMR chemical shielding anisotropy tensors have been computed by employing a medium size basis set and the GIAO-DFT(B3LYP) formalism of electronic structure theory for all of the atoms of type I and type II beta-turn models. The models contain all possible combinations of the amino acid residues Gly, Ala, Val, and Ser, with all possible side-chain orientations where applicable in a dipeptide. The several hundred structures investigated contain either constrained or optimized phi, psi, and chi dihedral angles. A statistical analysis of the resulting large database was performed and multidimensional (2D and 3D) chemical-shift/chemical-shift plots were generated. The (1)H(alpha-13)C(alpha), (13)C(alpha-1)H(alpha-13)C(beta), and (13)C(alpha-1)H(alpha-13)C' 2D and 3D plots have the notable feature that the conformers clearly cluster in distinct regions. This allows straightforward identification of the backbone and side-chain conformations of the residues forming beta-turns. Chemical shift calculations on larger For-(L-Ala)(n)-NH(2) (n=4, 6, 8) models, containing a single type I or type II beta-turn, prove that the simple models employed are adequate. A limited number of chemical shift calculations performed at the highly correlated CCSD(T) level prove the adequacy of the computational method chosen. For all nuclei, statistically averaged theoretical and experimental shifts taken from the BioMagnetic Resonance Bank (BMRB) exhibit good correlation. These results confirm and extend our previous findings that chemical shift information from selected multiple-pulse NMR experiments could be employed directly to extract folding information for polypeptides and proteins.

Contributions of arginines-43 and -94 of human choriogonadotropin. beta. to receptor binding and activation as determined by oligonucleotide-based mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang Chen; Puett, D.

1991-10-22

Members of the glycoprotein hormone family contain a common {alpha} subunit and a hormone-specific {beta} subunit. Human choriogonadotropin (hCG) {beta} is a 145 amino acid residue protein glycosylated at 6 positions (2 N-linked and 4 O-linked oligosaccharides). In an effort to elucidate receptor determinants on hCG{beta}, the authors have used site-directed mutagenesis to prepare and express several mutant cDNAs with replacements at arginines-43 and -94. Arg-43 is invariant in all known mammalian CG/lutropin {beta} amino acid sequences, and Arg-94 is conserved in 10 of the 12 sequences. Moreover, various studies involving synthetic peptides and enzymatic digestions of intact {beta} chainsmore » suggest that these residues may be important in hCG receptor binding. Point mutants were made in which these two arginines were replaced with the corresponding residues in human follitropin {beta}, Leu-43 and Asp-94. The wild-type and mutant {beta} chains were expressed in CHO cells containing a stably integrated gene for bovine {alpha}, and heterodimer formation occurred. These heterologous gonadotropins were active in assays using transformed Leydig cells, competitive binding with standard {sup 125}I-hCG, and cAMP and progesterone production, but the potency was considerably less than that associated with the hCG{beta} wild-type-containing gonadotropin. The double-mutant protein Arg-43 to Leu/Arg-94 to Asp also associated with bovine {alpha}, but the resultant heterodimer exhibited only low activity. Replacement but the Lys-43-containing {beta} chain appeared to exhibit a low degree of subunit association or reduced stability relative to the expressed hCG{beta} wild type. These results demonstrate that arginines-43 and -94 contribute to receptor binding through a positive charge.« less

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

PubMed

Bidmon, Nicole; Kind, Sonja; Welters, Marij J P; Joseph-Pietras, Deborah; Laske, Karoline; Maurer, Dominik; Hadrup, Sine Reker; Schreibelt, Gerty; Rae, Richard; Sahin, Ugur; Gouttefangeas, Cécile; Britten, Cedrik M; van der Burg, Sjoerd H

2018-07-01

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Dynamics of beta-cell turnover: evidence for beta-cell turnover and regeneration from sources of beta-cells other than beta-cell replication in the HIP rat.

PubMed

Manesso, Erica; Toffolo, Gianna M; Saisho, Yoshifumi; Butler, Alexandra E; Matveyenko, Aleksey V; Cobelli, Claudio; Butler, Peter C

2009-08-01

Type 2 diabetes is characterized by hyperglycemia, a deficit in beta-cells, increased beta-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). These characteristics are recapitulated in the human IAPP transgenic (HIP) rat. We developed a mathematical model to quantify beta-cell turnover and applied it to nondiabetic wild type (WT) vs. HIP rats from age 2 days to 10 mo to establish 1) whether beta-cell formation is derived exclusively from beta-cell replication, or whether other sources of beta-cells (OSB) are present, and 2) to what extent, if any, there is attempted beta-cell regeneration in the HIP rat and if this is through beta-cell replication or OSB. We conclude that formation and maintenance of adult beta-cells depends largely ( approximately 80%) on formation of beta-cells independent from beta-cell duplication. Moreover, this source adaptively increases in the HIP rat, implying attempted beta-cell regeneration that substantially slows loss of beta-cell mass.

[Beta thalassemia major and pregnancy during adolescence: report of two cases].

PubMed

Trigo, Lucas Augusto Monteiro Castro; Surita, Fernanda Garanhani; Parpinelli, Mary Angela; Pereira, Belmiro Gonçalves; Fertrin, Kleber Yotsumoto; Costa, Maria Laura

2015-06-01

Beta thalassemia major is a rare hereditary blood disease in which impaired synthesis of beta globin chains causes severe anemia. Medical treatment consists of chronic blood transfusions and iron chelation. We describe two cases of adolescents with beta thalassemia major with unplanned pregnancies and late onset of prenatal care. One had worsening of anemia with increased transfusional requirement, fetal growth restriction, and placental senescence. The other was also diagnosed with hypothyroidism and low maternal weight, and was admitted twice during pregnancy due to dengue shock syndrome and influenza H1N1-associated respiratory infection. She also developed fetal growth restriction and underwent vaginal delivery at term complicated by uterine hypotonia. Both patients required blood transfusions after birth and chose medroxyprogesterone as a contraceptive method afterwards. This report highlights the importance of medical advice on contraceptive methods for these women and the role of a specialized prenatal follow-up in association with a hematologist.

TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

PubMed

Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

2010-01-01

Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

Proinsulin Expression Shapes the TCR Repertoire but Fails to Control the Development of Low-Avidity Insulin-Reactive CD8+ T Cells

PubMed Central

Pearson, James A.; Thayer, Terri C.; McLaren, James E.; Ladell, Kristin; De Leenheer, Evy; Phillips, Amy; Davies, Joanne; Kakabadse, Dimitri; Miners, Kelly; Morgan, Peter; Wen, Li; Price, David A.

2016-01-01

NOD mice, a model strain for human type 1 diabetes, express proinsulin (PI) in the thymus. However, insulin-reactive T cells escape negative selection, and subsequent activation of the CD8+ T-cell clonotype G9C8, which recognizes insulin B15-23 via an αβ T-cell receptor (TCR) incorporating TRAV8-1/TRAJ9 and TRBV19/TRBJ2-3 gene rearrangements, contributes to the development of diabetes. In this study, we used fixed TRAV8-1/TRAJ9 TCRα-chain transgenic mice to assess the impact of PI isoform expression on the insulin-reactive CD8+ T-cell repertoire. The key findings were: 1) PI2 deficiency increases the frequency of insulin B15-23–reactive TRBV19+CD8+ T cells and causes diabetes; 2) insulin B15-23–reactive TRBV19+CD8+ T cells are more abundant in the pancreatic lymph nodes of mice lacking PI1 and/or PI2; 3) overexpression of PI2 decreases TRBV19 usage in the global CD8+ T-cell compartment; 4) a biased repertoire of insulin-reactive CD8+ T cells emerges in the periphery regardless of antigen exposure; and 5) low-avidity insulin-reactive CD8+ T cells are less affected by antigen exposure in the thymus than in the periphery. These findings inform our understanding of the diabetogenic process and reveal new avenues for therapeutic exploitation in type 1 diabetes. PMID:26953160

Cytoskeleton and paclitaxel sensitivity in breast cancer: the role of beta-tubulins.

PubMed

Tommasi, Stefania; Mangia, Anita; Lacalamita, Rosanna; Bellizzi, Antonia; Fedele, Vita; Chiriatti, Annalisa; Thomssen, Christopher; Kendzierski, Nancy; Latorre, Agnese; Lorusso, Vito; Schittulli, Francesco; Zito, Francesco; Kavallaris, Maria; Paradiso, Angelo

2007-05-15

The antineoplastic effect of paclitaxel is mainly related to its ability to bind the beta subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I beta-tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel-binding site of beta-tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of beta-tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC(50). The Class I beta-tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF-7 and SK-BR-3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III beta-tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III beta-tubulin could at least partially increase paclitaxel-chemosensitivity. The hypothesis of a relationship between beta-tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel-based chemotherapy. Thirty-five percent (95% CI: 45-31) of patients with high Class III beta-tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III beta-tubulin tumor expression could be considered a predictive biomarker of paclitaxel-clinical resistance for breast cancer patients. (c) 2007 Wiley-Liss, Inc.

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

PubMed

Kimmich, Tanja; Brüning, Ansgar; Käufl, Stephanie D; Makovitzky, Josef; Kuhn, Christina; Jeschke, Udo; Friese, Klaus; Mylonas, Ioannis

2010-08-01

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in

Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands.

PubMed

Das, Dibyendu Kumar; Mallis, Robert J; Duke-Cohan, Jonathan S; Hussey, Rebecca E; Tetteh, Paul W; Hilton, Mark; Wagner, Gerhard; Lang, Matthew J; Reinherz, Ellis L

2016-12-02

The pre-T cell receptor (pre-TCR) is a pTα-β heterodimer functioning in early αβ T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αβTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cβ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαβ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vβ patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of β chains with self-reactivity but is occluded by α subunit replacement of pTα upon αβTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αβTCR repertoire tuning via the pre-TCR. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands*♦

PubMed Central

Das, Dibyendu Kumar; Mallis, Robert J.; Duke-Cohan, Jonathan S.; Hussey, Rebecca E.; Tetteh, Paul W.; Hilton, Mark; Wagner, Gerhard; Lang, Matthew J.; Reinherz, Ellis L.

2016-01-01

The pre-T cell receptor (pre-TCR) is a pTα-β heterodimer functioning in early αβ T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αβTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cβ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαβ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vβ patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of β chains with self-reactivity but is occluded by α subunit replacement of pTα upon αβTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αβTCR repertoire tuning via the pre-TCR. PMID:27707880

Modeling alternative binding registers of a minimal immunogenic peptide on two class II major histocompatibility complex (MHC II) molecules predicts polarized T-cell receptor (TCR) contact positions.

PubMed

Murray, J S; Fois, S D S; Schountz, T; Ford, S R; Tawde, M D; Brown, J C; Siahaan, T J

2002-03-01

Several major histocompatibility complex class II (MHC II) complexes with known minimal immunogenic peptides have now been solved by X-ray crystallography. Specificity pockets within the MHC II binding groove provide distinct peptide contacts that influence peptide conformation and define the binding register within different allelic MHC II molecules. Altering peptide ligands with respect to the residues that contact the T-cell receptor (TCR) can drastically change the nature of the ensuing immune response. Here, we provide an example of how MHC II (I-A) molecules may indirectly effect TCR contacts with a peptide and drive functionally distinct immune responses. We modeled the same immunogenic 12-amino acid peptide into the binding grooves of two allelic MHC II molecules linked to distinct cytokine responses against the peptide. Surprisingly, the favored conformation of the peptide in each molecule was distinct with respect to the exposure of the N- or C-terminus of the peptide above the MHC II binding groove. T-cell clones derived from each allelic MHC II genotype were found to be allele-restricted with respect to the recognition of these N- vs. C-terminal residues on the bound peptide. Taken together, these data suggest that MHC II alleles may influence T-cell functions by restricting TCR access to specific residues of the I-A-bound peptide. Thus, these data are of significance to diseases that display genetic linkage to specific MHC II alleles, e.g. type 1 diabetes and rheumatoid arthritis.

Insights into the phosphoregulation of beta-secretase sorting signal by the VHS domain of GGA1.

PubMed

Shiba, Tomoo; Kametaka, Satoshi; Kawasaki, Masato; Shibata, Masahiro; Waguri, Satoshi; Uchiyama, Yasuo; Wakatsuki, Soichi

2004-06-01

BACE (beta-site amyloid precursor protein cleaving enzyme, beta-secretase) is a type-I membrane protein which functions as an aspartic protease in the production of beta-amyloid peptide, a causative agent of Alzheimer's disease. Its cytoplasmic tail has a characteristic acidic-cluster dileucine motif recognized by the VHS domain of adaptor proteins, GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-interacting). Here we show that BACE is colocalized with GGAs in the trans-Golgi network and peripheral structures, and phosphorylation of a serine residue in the cytoplasmic tail enhances interaction with the VHS domain of GGA1 by about threefold. The X-ray crystal structure of the complex between the GGA1-VHS domain and the BACE C-terminal peptide illustrates a similar recognition mechanism as mannose 6-phosphate receptors except that a glutamine residue closes in to fill the gap created by the shorter BACE peptide. The serine and lysine of the BACE peptide point their side chains towards the solvent. However, phosphorylation of the serine affects the lysine side chain and the peptide backbone, resulting in one additional hydrogen bond and a stronger electrostatic interaction with the VHS domain, hence the reversible increase in affinity.

9-Methyl-beta-carboline has restorative effects in an animal model of Parkinson's disease.

PubMed

Wernicke, Catrin; Hellmann, Julian; Zieba, Barbara; Kuter, Katarzyna; Ossowska, Krystyna; Frenzel, Monika; Dencher, Norbert A; Rommelspacher, Hans

2010-01-01

In a previous study, a primary culture of midbrain cells was exposed to 9-methyl-beta-carboline for 48 h, which caused an increase in the number of tyrosine hydroxylase-positive cells. Quantitative RT-PCR revealed increased transcription of genes participating in the maturation of dopaminergic neurons. These in vitro findings prompted us to investigate the restorative actions of 9-methyl-beta-carboline in vivo. The compound was delivered for 14 days into the left cerebral ventricle of rats pretreated with the neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP+) for 28 days applying a dose which lowered dopamine by approximately 50%. Interestingly, 9-methyl-beta-carboline reversed the dopamine-lowering effect of the neurotoxin in the left striatum. Stereological counts of tyrosine hydroxylase-immunoreactive cells in the substantia nigra revealed that the neurotoxin caused a decrease in the number of those cells. However, when treated subsequently with 9-methyl-beta-carboline, the number reached normal values. In search of an explanation for the restorative activity, we analyzed the complexes that compose the respiratory chain in striatal mitochondria by 2-dimension gel electrophoresis followed by MALDI-TOF peptide mass fingerprinting.We found no changes in the overall composition of the complexes. However, the activity of complex I was increased by approximately 80% in mitochondria from rats treated with MPP+ and 9-methyl-beta-carboline compared to MPP+ and saline and to sham-operated rats, as determined by measurements of nicotinamide adenine dinucleotide dehydrogenase activity. Microarray technology and single RT-PCR revealed the induction of neurotrophins: brain-derived neurotrophic factor, conserved dopamine neurotrophic factor, cerebellin 1 precursor protein, and ciliary neurotrophic factor. Selected western blots yielded consistent results. The findings demonstrate restorative effects of 9-methyl-beta-carboline in an animal model of Parkinson's disease that

Expression of transforming growth factor-beta1, -beta2 and -beta3 in normal and diseased canine mitral valves.

PubMed

Aupperle, H; März, I; Thielebein, J; Schoon, H-A

2008-01-01

The pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, but activation and proliferation of valvular stromal cells (VSC) and their transdifferentiation into myofibroblast-like cells has been described. These alterations may be influenced by transforming growth factor-beta (TGF-beta), a cytokine involved in extracellular matrix (ECM) regulation and mesenchymal cell differentiation. The present study investigates immunohistochemically the expression of TGF-beta1, -beta2, -beta3 and smooth muscle alpha actin (alpha-SMA) in normal canine mitral valves (MVs) (n=10) and in the valves of dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal mitral valves there was no expression of alpha-SMA but VSC displayed variable expression of TGF-beta1 (10% of VSC labelled), TGF-beta2 (1-5% labelled) and TGF-beta3 (50% labelled). In mild CVD the affected atrialis contain activated and proliferating alpha-SMA-positive VSC, which strongly expressed TGF-beta1 and -beta3, but only 10% of these cells expressed TGF-beta2. In unaffected areas of the leaflet there was selective increase in expression of TGF-beta1 and -beta3. In advanced CVD the activated subendothelial VSC strongly expressed alpha-SMA, TGF-beta1 and -beta3. Inactive VSC within the centre of the nodules had much less labelling for TGF-beta1 and -beta3. TGF-beta1 labelling was strong within the ECM. These data suggest that TGF-beta plays a role in the pathogenesis of CVD by inducing myofibroblast-like differentiation of VSC and ECM secretion. Changed haemodynamic forces and expression of matrix metalloproteinases (MMPs) may in turn regulate TGF-beta expression.

Selective regulation of beta 1- and beta 2-adrenoceptors in the human heart by chronic beta-adrenoceptor antagonist treatment.

PubMed Central

Michel, M. C.; Pingsmann, A.; Beckeringh, J. J.; Zerkowski, H. R.; Doetsch, N.; Brodde, O. E.

1988-01-01

1. In 44 patients undergoing coronary artery bypass grafting, the effect of chronic administration of the beta-adrenoceptor antagonists sotalol, propranolol, pindolol, metoprolol and atenolol on beta-adrenoceptor density in right atria (containing 70% beta 1- and 30% beta 2-adrenoceptors) and in lymphocytes (having only beta 2-adrenoceptors) was studied. 2. beta-Adrenoceptor density in right atrial membranes and in intact lymphocytes was assessed by (-)-[125I]-iodocyanopindolol (ICYP) binding; the relative amount of right atrial beta 1- and beta 2-adrenoceptors was determined by inhibition of ICYP binding by the selective beta 2-adrenoceptor antagonist ICI 118,551 and analysis of the resulting competition curves by the iterative curve fitting programme LIGAND. 3. With the exception of pindolol, all beta-adrenoceptor antagonists increased right atrial beta-adrenoceptor density compared to that observed in atria from patients not treated with beta-adrenoceptor antagonists. 4. All beta-adrenoceptor antagonists increased right atrial beta 1-adrenoceptor density; on the other hand, only sotalol and propranolol also increased right atrial beta 2-adrenoceptor density, whereas metoprolol and atenolol did not affect it and pindolol decreased it. 5. Similarly, in corresponding lymphocytes, only sotalol or propranolol increased beta 2-adrenoceptor density, while metoprolol and atenolol did not affect it and pindolol decreased it. 6. It is concluded that beta-adrenoceptor antagonists subtype-selectively regulate cardiac and lymphocyte beta-adrenoceptor subtypes. The selective increase in cardiac beta 1-adrenoceptor density evoked by metoprolol and atenolol may be one of the reasons for the beneficial effects observed in patients with end-stage congestive cardiomyopathy following intermittent treatment with low doses of selective beta 1-adrenoceptor antagonists. PMID:2902891

Comparison between qualitative and real-time polymerase chain reaction to evaluate minimal residual disease in children with acute lymphoblastic leukemia.

PubMed

Paula, Francisco Danilo Ferreira; Elói-Santos, Silvana Maria; Xavier, Sandra Guerra; Ganazza, Mônica Aparecida; Jotta, Patricia Yoshioka; Yunes, José Andrés; Viana, Marcos Borato; Assumpção, Juliana Godoy

2015-01-01

Minimal residual disease is an important independent prognostic factor that can identify poor responders among patients with acute lymphoblastic leukemia. The aim of this study was to analyze minimal residual disease using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements by conventional polymerase chain reaction followed by homo-heteroduplex analysis and to compare this with real-time polymerase chain reaction at the end of the induction period in children with acute lymphoblastic leukemia. Seventy-four patients diagnosed with acute lymphoblastic leukemia were enrolled. Minimal residual disease was evaluated by qualitative polymerase chain reaction in 57 and by both tests in 44. The Kaplan-Meier and multivariate Cox methods and the log-rank test were used for statistical analysis. Nine patients (15.8%) were positive for minimal residual disease by qualitative polymerase chain reaction and 11 (25%) by real-time polymerase chain reaction considering a cut-off point of 1×10(-3) for precursor B-cell acute lymphoblastic leukemia and 1×10(-2) for T-cell acute lymphoblastic leukemia. Using the qualitative method, the 3.5-year leukemia-free survival was significantly higher in children negative for minimal residual disease compared to those with positive results (84.1%±5.6% versus 41.7%±17.3%, respectively; p-value=0.004). There was no significant association between leukemia-free survival and minimal residual disease by real-time polymerase chain reaction. Minimal residual disease by qualitative polymerase chain reaction was the only variable significantly correlated to leukemia-free survival. Given the difficulties in the implementation of minimal residual disease monitoring by real-time polymerase chain reaction in most treatment centers in Brazil, the qualitative polymerase chain reaction strategy may be a cost-effective alternative. Copyright © 2015 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier

Crystal structure of bovine Cu,Zn superoxide dismutase at 3 A resolution: chain tracing and metal ligands.

PubMed Central

Richardson, J; Thomas, K A; Rubin, B H; Richardson, D C

1975-01-01

An electron density map at 3 angstrom resolution has been calculated for Cu2+, Zn2+ superoxide dismutase from bovine erythrocytes, and the course of the main chain has been traced. The dominant structural feature is an 8-stranded barrel of antiparallel beta-pleated sheet. There is one very short helical section and two long loops of non-repetitive structure. The Cu and Zn are bound between the loops and one side of the beta barrel and are about 6 Angstrom apart, with a common histidine ligand. The Cu has four histidine ligands in a somewhat distorted square plane, and the Zn has three histidines and an aspartate in approximately tetrahedral arrangement. The two coppers of a dimer are about 34 Angstrom apart. The two subunits have essentially the same conformation and have an extensive contact area that mainly involves hydrophobic side chain interactions. The overall folding pattern of the polypeptide chain is very similar to that of an immunoglobulin domain. Images PMID:1055410

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold.

PubMed Central

Rosa, J. C.; De Oliveira, P. S.; Garratt, R.; Beltramini, L.; Resing, K.; Roque-Barreira, M. C.; Greene, L. J.

1999-01-01

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature. PMID:10210179

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

Synthesis and evaluation of N-alkyl-beta-D-glucosylamines on the growth of two wood fungi, Coriolus versicolor and Poria placenta.

PubMed

Muhizi, Théoneste; Coma, Véronique; Grelier, Stéphane

2008-09-22

Various glucosylamines were synthesized from glucose and different alkyl amine compounds. These amino compounds are beta-D-glucopyranosylamine (GPA), N-ethyl-beta-D-glucopyranosylamine (EtGPA), N-butyl-beta-D-glucopyranosylamine (BuGPA), N-hexyl-beta-D-glucopyranosylamine (HeGPA), N-octyl-beta-D-glucopyranosylamine (OcGPA), N-dodecyl-beta-D-glucopyranosylamine (DoGPA), N-(2-hydroxyethyl)-beta-D-glucopyranosylamine (HEtGPA) and N,N-di(2-hydroxyethyl)-beta-D-glucopyranosylamine (DHEtGPA). They were tested for their antifungal activity against the growth of Coriolus versicolor and Poria placenta. An improvement of the antifungal activity with the increase of alkyl chain length was observed. DoGPA exhibited the best antifungal activity against both strains. It completely inhibited the fungal growth at 0.01x10(-3)molmL(-1) and 0.0075x10(-3)molmL(-1) for C. versicolor and P. placenta, respectively. For other glucosylamines higher concentrations were needed for complete inhibition of fungi.

Decursin suppresses human androgen-independent PC3 prostate cancer cell proliferation by promoting the degradation of beta-catenin.

PubMed

Song, Gyu-Yong; Lee, Jee-Hyun; Cho, Munju; Park, Byeoung-Soo; Kim, Dong-Eun; Oh, Sangtaek

2007-12-01

Alterations in the Wnt/beta-catenin pathway are associated with the development and progression of human prostate cancer. Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, inhibits the growth of androgen-independent human prostate cancer cells, but little is known about its mechanism of action. Using a cell-based screen, we found that decursin attenuates the Wnt/beta-catenin pathway. Decursin antagonized beta-catenin response transcription (CRT), which was induced with Wnt3a-conditioned medium and LiCl, by promoting the degradation of beta-catenin. Furthermore, decursin suppressed the expression of cyclin D1 and c-myc, which are downstream target genes of beta-catenin and thus inhibited the growth of PC3 prostate cancer cells. In contrast, decursinol, in which the (CH3)2-C=CH-COO- side chain of decursin is replaced with -OH, had no effect on CRT, the level of intracellular beta-catenin, or PC3 cell proliferation. Our findings suggest that decursin exerts its anticancer activity in prostate cancer cells via inhibition of the Wnt/beta-catenin pathway.

Relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-beta1 levels.

PubMed

Hueso, Miguel; Navarro, Estanis; Moreso, Francesc; Beltrán-Sastre, Violeta; Ventura, Francesc; Grinyó, Josep M; Serón, Daniel

2006-05-27

Transforming growth factor (TGF)-beta(1) is increased in allograft rejection and its production is associated with single nucleotide polymorphisms (SNPs). The contribution of SNPs at codons 10 and 25 of the TGF-beta(1) gene to renal allograft damage was assessed in 6-month protocol biopsies and their association with TGF-beta(1) production. TGF-beta(1) genotypes were evaluated by polymerase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-beta(1) messenger RNA (mRNA) was measured by real-time PCR and TGF-beta(1) plasma levels were assessed by enzyme-linked immunosorbent assay. Eighty consecutive patients were included. Allele T at codon 10 (risk ratio, 6.7; P = 0.02) and an episode of acute rejection before protocol biopsy (risk ratio, 6.2; P = 0.01) were independent predictors of subclinical rejection (SCR). TGF-beta(1) plasma levels, but not those of TGF-beta(1) mRNA, were increased in patients with SCR (2.59 ng/mL +/- 0.91 [n = 22] vs. 2.05 ng/mL +/- 0.76 [n = 43]; P = 0.01). There was no association between allele T and TGF-beta(1) plasma or intragraft levels. Allele T at codon 10 of the TGF-beta(1) gene is associated with a higher incidence of SCR.

Enzymatic preparation of. cap alpha. - and. beta. -deuterated or tritiated amino acids with l-methionine. gamma. -lyase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Esaki, N.; Sawada, S.; Tanaka, H.

L-Methionine ..gamma..-lyase catalyzes the exchange of ..cap alpha..- and ..beta..-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium or tritium of solvents. The rate of ..cap alpha..-hydrogen exchange with deuterium was about 40 times faster than that of the elimination reactions. The deuterium and tritium were exchanged also with the ..cap alpha..- and ..beta..-hydrogens of the straight-chain amino acids which do not undergo the elimination: L-alanine, L-..cap alpha..-aminobutyrate, L-norvaline, and L-norleucine. No exchange occurs for the D-isomers, acidic L-amino acids, basic L-amino acids, and branched-chain L-amino acids, although ..cap alpha..-hydrogen of glycine, L-trypotophan, and L-phenylalanine is exchanged slowly. These enzymatic hydrogen-exchange reactionsmore » facilitate specific labeling of the L-amino acids with deuterium and tritium.« less

IFN-γ regulates CD8+ memory T cell differentiation and survival in response to weak, but not strong, TCR signals.

PubMed

Stoycheva, Diana; Deiser, Katrin; Stärck, Lilian; Nishanth, Gopala; Schlüter, Dirk; Uckert, Wolfgang; Schüler, Thomas

2015-01-15

In response to primary Ag contact, naive mouse CD8(+) T cells undergo clonal expansion and differentiate into effector T cells. After pathogen clearance, most effector T cells die, and only a small number of memory T cell precursors (TMPs) survive to form a pool of long-lived memory T cells (TMs). Although high- and low-affinity CD8(+) T cell clones are recruited into the primary response, the TM pool consists mainly of high-affinity clones. It remains unclear whether the more efficient expansion of high-affinity clones and/or cell-intrinsic processes exclude low-affinity T cells from the TM pool. In this article, we show that the lack of IFN-γR signaling in CD8(+) T cells promotes TM formation in response to weak, but not strong, TCR agonists. The IFN-γ-sensitive accumulation of TMs correlates with reduced mammalian target of rapamycin activation and the accumulation of long-lived CD62L(hi)Bcl-2(hi)Eomes(hi) TMPs. Reconstitution of mammalian target of rapamycin or IFN-γR signaling is sufficient to block this process. Hence, our data suggest that IFN-γR signaling actively blocks the formation of TMPs responding to weak TCR agonists, thereby promoting the accumulation of high-affinity T cells finally dominating the TM pool. Copyright © 2015 by The American Association of Immunologists, Inc.

Insulin-producing cells could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells

PubMed Central

2013-01-01

Objective The aim of this study was to compare the difference between insulin-producing cells (IPCs) and normal human pancreatic beta cells both in physiological function and morphological features in cellular level. Methods The levels of insulin secretion were measured by enzyme-linked immunosorbent assay. The insulin gene expression was determined by real-time quantitative polymerase chain reaction. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy. Results IPCs and normal human pancreatic beta cells were similar to each other under the observation in AFM with the porous structure features in the cytoplasm. Both number of membrane particle size and average roughness of normal human beta cells were higher than those of IPCs. Conclusions Our results firstly revealed that the cellular ultrastructure of IPCs was closer to that of normal human pancreatic beta cells, but they still could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells. PMID:23421382

Discovery of nonsteroidal 17beta-hydroxysteroid dehydrogenase 1 inhibitors by pharmacophore-based screening of virtual compound libraries.

PubMed

Schuster, Daniela; Nashev, Lyubomir G; Kirchmair, Johannes; Laggner, Christian; Wolber, Gerhard; Langer, Thierry; Odermatt, Alex

2008-07-24

17Beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) plays a pivotal role in the local synthesis of the most potent estrogen estradiol. Its expression is a prognostic marker for the outcome of patients with breast cancer and inhibition of 17beta-HSD1 is currently under consideration for breast cancer prevention and treatment. We aimed to identify nonsteroidal 17beta-HSD1 inhibitor scaffolds by virtual screening with pharmacophore models built from crystal structures containing steroidal compounds. The most promising model was validated by comparing predicted and experimentally determined inhibitory activities of several flavonoids. Subsequently, a virtual library of nonsteroidal compounds was screened against the 3D pharmacophore. Analysis of 14 selected compounds yielded four that inhibited the activity of human 17beta-HSD1 (IC 50 below 50 microM). Specificity assessment of identified 17beta-HSD1 inhibitors emphasized the importance of including related short-chain dehydrogenase/reductase (SDR) members to analyze off-target effects. Compound 29 displayed at least 10-fold selectivity over the related SDR enzymes tested.

A new common mutation in the cardiac beta-myosin heavy chain gene in Finnish patients with hypertrophic cardiomyopathy.

PubMed

Jääskeläinen, Pertti; Heliö, Tiina; Aalto-Setälä, Katriina; Kaartinen, Maija; Ilveskoski, Erkki; Hämäläinen, Liisa; Melin, John; Kärkkäinen, Satu; Peuhkurinen, Keijo; Nieminen, Markku S; Laakso, Markku; Kuusisto, Johanna

2014-09-01

In the nationwide FinHCM Study including 306 Finnish patients with hypertrophic cardiomyopathy (HCM), we have previously identified two founder mutations in the alpha-tropomyosin (TPM1-D175N) and myosin-binding protein C (MYBPC3-Q1061X) genes, accounting for 18% of all cases. Objective. To screen additional mutations, previously identified in eastern Finnish cohorts with HCM, in the FinHCM Study population. Ten mutations in the beta-myosin heavy chain gene (MYH7), TPM1, and MYBPC3 were screened. MYH7-R1053Q was found in 17 of 306 patients (5.6%). No carriers of MYH7-R719W or N696S were found. A novel TPM1-D175G mutation was found in a single patient. MYBPC3 mutations were found in 14 patients: IVS5-2A-C in two, IVS14-13G-A in two, K811del in six, and A851insT in four patients. Altogether, a HCM-causing mutation was identified in 32 patients, accounting for 10.5% of all cases. In addition, two MYBPC3 variants R326Q and V896M with uncertain pathogenicity were found in eight and in 10 patients, respectively. Combining the present findings with our previous results, a causative mutation was identified in 28% of the FinHCM cohort. MYH7-R1053Q was the third most common mutation, and should be screened in all new cases of HCM in Finland.

ENU mutagenesis identifies mice with cardiac fibrosis and hepatic steatosis caused by a mutation in the mitochondrial trifunctional protein beta-subunit.

PubMed

Kao, Hsiao-Jung; Cheng, Ching-Feng; Chen, Yen-Hui; Hung, Shuen-Iu; Huang, Cheng-Chih; Millington, David; Kikuchi, Tateki; Wu, Jer-Yuarn; Chen, Yuan-Tsong

2006-12-15

Using the metabolomics-guided screening coupled to N-ethyl-N-nitrosourea-mediated mutagenesis, we identified mice that exhibited elevated levels of long-chain acylcarnitines. Whole genome homozygosity mapping with 262 SNP markers mapped the disease gene to chromosome 5 where candidate genes Hadha and Hadhb, encoding the mitochondria trifunctional protein (MTP) alpha- and beta-subunits, respectively, are located. Direct sequencing revealed a normal alpha-subunit, but detected a nucleotide T-to-A transversion in exon 14 (c.1210T>A) of beta-subunit (Hadhb) which resulted in a missense mutation of methionine to lysine (M404K). Western blot analysis showed a significant reduction of both the alpha- and beta-subunits, consistent with reduced enzyme activity in both the long-chain 3-hydroxyacyl-CoA dehydrogenase and the long-chain 3-ketoacyl-CoA thiolase activities. These mice had a decreased weight gain and cardiac arrhythmias which manifested from a prolonged PR interval to a complete atrio-ventricular dissociation, and died suddenly between 9 and 16 months of age. Histopathological studies showed multifocal cardiac fibrosis and hepatic steatosis. This mouse model will be useful to further investigate the mechanisms underlying arrhythmogenesis relating to lipotoxic cardiomyopathy and to investigate pathophysiology and treatment strategies for human MTP deficiency.

Polymorphism in 'L' shaped lipids: structure of N-, O-diacylethanolamines with mixed acyl chains.

PubMed

Tarafdar, Pradip K; Swamy, Musti J

2009-11-01

Although solid state polymorphism in lipids has been established by spectroscopic and calorimetric studies long ago, only in a few cases crystal structures of different polymorphs of the same compound have been reported, possibly due to difficulties in obtaining high quality single crystals of individual polymorphs. Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of the stress-related lipids, the N-acylethanolamines under physiological conditions. In this study, two DAEs with mixed acyl chains, namely N-palmitoyl, O-octanoylethanolamine and N-palmitoyl, O-decanoylethanolamine have been synthesized and their three-dimensional structures were determined. Both the compounds were found to adopt 'L' shaped structures and exist in two polymorphic forms, alpha and beta. In the alpha form a mixed-type chain packing has been observed whereas in the beta form the chain packing is symmetric. Similar polymorphic forms are likely to exist in other 'L' shaped lipids such as 1,3-diacylglycerols and ceramides, where polymorphism has been detected earlier, but three-dimensional structures - which can give precise information about the packing at atomic resolution - have not been reported.

Molecular cloning of a novel widely expressed human 80 kDa 17 beta-hydroxysteroid dehydrogenase IV.

PubMed Central

Adamski, J; Normand, T; Leenders, F; Monté, D; Begue, A; Stéhelin, D; Jungblut, P W; de Launoit, Y

1995-01-01

Reactions of oestrogens and androgens at position C-17 are catalysed by 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs). Cloning of the cDNA of a novel human 17 beta-HSD IV and expression of its mRNA are described. A probe derived from the recently discovered porcine 17 beta-oestradiol dehydrogenase (17 beta-EDH) was used to isolate a 2.6 kb human cDNA encoding a continuous protein of 736 amino acids of high (84%) similarity to the porcine 17 beta-EDH. The calculated molecular mass of the human enzyme is 79,595 Da. Other sequence similarities shared by the two enzymes are: an N-terminal sequence which is similar to that of members of the short-chain alcohol dehydrogenase family; amino acids 343-607 which are similar to the C-terminal domains of a trifunctional Candida tropicalis enzyme and the FOX2 gene product of Saccharomyces cerevisiae; amino acids 596-736 which are similar to human sterol carrier protein 2. The previously cloned human 17 beta-HSD I, II and III are less than 25% identical with 17 beta-HSD IV. mRNA for HSD IV is a single species of 3.0 kb, present in many tissues with highest concentrations in liver, heart, prostate and testes. When over-expressed in mammalian cells, the human 17 beta-HSD IV enzyme displays a specific unidirectional oxidative 17 beta-HSD activity. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7487879

Risk Factors for and the Clinical Impact of Cytomegalovirus and Epstein-Barr Virus Infections in Pediatric Recipients of TCR-α/β- and CD19-Depleted Grafts.

PubMed

Laberko, Alexandra; Bogoyavlenskaya, Anna; Shelikhova, Larisa; Shekhovtsova, Zhanna; Balashov, Dmitriy; Voronin, Kirill; Kurnikova, Elena; Boyakova, Elena; Raykina, Elena; Brilliantova, Varvara; Pirumova, Valentina; Novichkova, Galina; Maschan, Alexei; Maschan, Michael

2017-03-01

Alpha/beta T cell and CD19 depletion are used to improve the outcomes of hematopoietic stem cell transplantation (HSCT). We evaluated the burden of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in pediatric patients after this HSCT type. A cohort of 182 patients with malignant (n = 114) or nonmalignant (n = 68) disorders was transplanted from either matched unrelated (n = 124) or haploidentical (n = 58) donors. The cumulative incidence of CMV and EBV viremia were 51% and 33%, respectively. Acute graft-versus-host disease (GVHD) grades II to IV, D-/R+ serology, and malignant HSCT indications were associated with increased risk of CMV viremia. CMV disease developed in 10 patients (6%). The occurrence of CMV viremia was not associated with inferior outcomes. Acute GVHD grade ≥ II was the only factor significantly associated with an increased risk of EBV viremia. Rituximab significantly decreased the rate of EBV reactivation in a subgroup that received a higher B cell dose in the graft. The rate of EBV-associated disease was .5%, and EBV viremia did not affect survival. TCR-α/β and CD19 depletion are associated with a significant rate of CMV viremia that does not affect survival. The hazard of EBV post-transplant lymphoproliferative disease (PTLD) is eliminated by the combination of CD19 depletion and rituximab. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

PubMed

Wu, A M; Lin, S R; Chin, L K; Chow, L P; Lin, J Y

1992-09-25

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.

I. The design, synthesis, and structure of antiparallel beta-sheet and beta-strand mimics. II. The design of a scripted chemistry outreach program to high schools

NASA Astrophysics Data System (ADS)

Waldman, Amy Sue

I. Protein structure is not easily predicted from the linear sequence of amino acids. An increased ability to create protein structures would allow researchers to develop new peptide-based therapeutics and materials, and would provide insights into the mechanisms of protein folding. Toward this end, we have designed and synthesized two-stranded antiparallel beta-sheet mimics containing conformationally biased scaffolds and semicarbazide, urea, and hydrazide linker groups that attach peptide chains to the scaffold. The mimics exhibited populations of intramolecularly hydrogen-bonded beta-sheet-like conformers as determined by spectroscopic techniques such as FTIR, sp1H NMR, and ROESY studies. During our studies, we determined that a urea-hydrazide beta-strand mimic was able to tightly hydrogen bond to peptides in an antiparallel beta-sheet-like configuration. Several derivatives of the urea-hydrazide beta-strand mimic were synthesized. Preliminary data by electron microscopy indicate that the beta-strand mimics have an effect on the folding of Alzheimer's Abeta peptide. These data suggest that the urea-hydrazide beta-strand mimics and related compounds may be developed into therapeutics which effect the folding of the Abeta peptide into neurotoxic aggregates. II. In recent years, there has been concern about the low level of science literacy and science interest among Americans. A declining interest in science impacts the abilities of people to make informed decisions about technology. To increase the interest in science among secondary students, we have developed the UCI Chemistry Outreach Program to High Schools. The Program features demonstration shows and discussions about chemistry in everyday life. The development and use of show scripts has enabled large numbers of graduate and undergraduate student volunteers to demonstrate chemistry to more than 12,000 local high school students. Teachers, students, and volunteers have expressed their enjoyment of The UCI

Conformational analysis of the ΜΒΡ83-99 (Phe91) and ΜΒΡ83-99 (Tyr91) peptide analogues and study of their interactions with the HLA-DR2 and human TCR receptors by using Molecular Dynamics

NASA Astrophysics Data System (ADS)

Potamitis, C.; Matsoukas, M.-T.; Tselios, T.; Mavromoustakos, T.; Golič Grdadolnik, S.

2011-09-01

The two new synthetic analogues of the MBP83-99 epitope substituted at Lys91 (primary TCR contact) with Phe [MBP83-99 (Phe91)] or Tyr [MBP83-99 (Tyr91)], have been structurally elucidated using 1D and 2D high resolution NMR studies. The conformational analysis of the two altered peptide ligands (APLs) has been performed and showed that they adopt a linear and extended conformation which is in agreement with the structural requirements of the peptides that interact with the HLA-DR2 and TCR receptors. In addition, Molecular Dynamics (MD) simulations of the two analogues in complex with HLA-DR2 (DRA, DRB1*1501) and TCR were performed. Similarities and differences of the binding motif of the two analogues were observed which provide a possible explanation of their biological activity. Their differences in the binding mode in comparison with the MBP83-99 epitope may also explain their antagonistic versus agonistic activity. The obtained results clearly indicate that substitutions in crucial amino acids (TCR contacts) in combination with the specific conformational characteristics of the MBP83-99 immunodominant epitope lead to an alteration of their biological activity. These results make the rational drug design intriguing since the biological activity is very sensitive to the substitution and conformation of the mutated MBP epitopes.

Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

PubMed

Klein, B; Pawlowski, K; Höricke-Grandpierre, C; Schell, J; Töpfer, R

1992-05-01

A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata. This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity. Hybridization studies revealed that in C. lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver.

PubMed Central

Kawashima, Y; Uy-Yu, N; Kozuka, H

1989-01-01

Inductions by perfluoro-octanoic acid (PFOA) of hepatomegaly, peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase were compared in liver between male and female rats. Marked inductions of these four parameters were seen concurrently in liver of male rats, whereas the inductions in liver of female rats were far less pronounced. The sex-related difference in the response of rat liver to PFOA was much more marked than that seen with p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). Hormonal manipulations revealed that this sex-related difference in the inductions is strongly dependent on sex hormones, namely that testosterone is necessary for the inductions, whereas oestradiol prevented the inductions by PFOA. PMID:2570571

5Beta,6beta-epoxy-17-oxoandrostan-3beta-yl acetate and 5beta,6beta-epoxy-20-oxopregnan-3beta-yl acetate.

PubMed

Pinto, Rui M A; Salvador, Jorge A R; Paixão, José A

2008-05-01

In the title compounds, C(21)H(30)O(4), (I), and C(23)H(34)O(4), (II), respectively, which are valuable intermediates in the synthesis of important steroid derivatives, rings A and B are cis-(5beta,10beta)-fused. The two molecules have similar conformations of rings A, B and C. The presence of the 5beta,6beta-epoxide group induces a significant twist of the steroid nucleus and a strong flattening of the B ring. The different C17 substituents result in different conformations for ring D. Cohesion of the molecular packing is achieved in both compounds only by weak intermolecular interactions. The geometries of the molecules in the crystalline environment are compared with those of the free molecules as given by ab initio Roothan Hartree-Fock calculations. We show in this work that quantum mechanical ab initio methods reproduce well the details of the conformation of these molecules, including a large twist of the steroid nucleus. The calculated twist values are comparable, but are larger than the observed values, indicating a possible small effect of the crystal packing on the twist angles.

Characterization of beta-turn and Asx-turns mimicry in a model peptide: stabilization via C--H . . . O interaction.

PubMed

Thakur, A K; Kishore, R

2006-04-15

The chemical synthesis and single-crystal X-ray diffraction analysis of a model peptide, Boc-Thr-Thr-NH2 (1) comprised of proteinogenic residues bearing an amphiphilic Cbeta -stereogenic center, has been described. Interestingly, the analysis of its molecular structure revealed the existence of a distinct conformation that mimics a typical beta-turn and Asx-turns, i.e., the two Thr residues occupy the left- and right-corner positions. The main-chain torsion angles of the N- and C-terminal residues i.e., semiextended: phi = -68.9 degrees , psi = 128.6 degrees ; semifolded: phi = -138.1 degrees , psi = 2.5 degrees conformations, respectively, in conjunction with a gauche- disposition of the obligatory C-terminus Thr CgammaH3 group, characterize the occurrence of the newly described beta-turn- and Asx-turns-like topology. The preferred molecular structure is suggested to be stabilized by an effective nonconventional main-chain to side-chain Ci=O . . . H--Cgamma(i+2)-type intraturn hydrogen bond. Noteworthy, the observed topology of the resulting 10-membered hydrogen-bonded ring is essentially similar to the one perceived for a classical beta-turn and the Asx-turns, stabilized by a conventional intraturn hydrogen bond. Considering the signs as well as magnitudes of the backbone torsion angles and the orientation of the central peptide bond, the overall mimicked topology resembles the type II beta-turn or type II Asx-turns. An analysis of Xaa-Thr sequences in high-resolution X-ray elucidated protein structures revealed the novel topology prevalence in functional proteins (unpublished). In view of indubitable structural as well as functional importance of nonconventional interactions in bioorganic and biomacromolecules, we intend to highlight the participation of Thr CgammaH in the creation of a short-range C=O . . . H--Cgamma -type interaction in peptides and proteins. Copyright 2006 Wiley Periodicals, Inc.

Specific reaction of alpha,beta-unsaturated carbonyl compounds such as 6-shogaol with sulfhydryl groups in tubulin leading to microtubule damage.

PubMed

Ishiguro, Kazuhiro; Ando, Takafumi; Watanabe, Osamu; Goto, Hidemi

2008-10-15

6-Shogaol and 6-gingerol are ginger components with similar chemical structures. However, while 6-shogaol damages microtubules, 6-gingerol does not. We have investigated the molecular mechanism of 6-shogaol-induced microtubule damage and found that the action of 6-shogaol results from the structure of alpha,beta-unsaturated carbonyl compounds. alpha,beta-Unsaturated carbonyl compounds such as 6-shogaol react with sulfhydryl groups of cysteine residues in tubulin, and impair tubulin polymerization. The reaction with sulfhydryl groups depends on the chain length of alpha,beta-unsaturated carbonyl compounds. In addition, alpha,beta-unsaturated carbonyl compounds are more reactive with sulfhydryl groups in tubulin than in 2-mercaptoethanol, dithiothreitol, glutathione and papain, a cysteine protease.

Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain sophorolipid production.

PubMed

Van Bogaert, Inge N A; Sabirova, Julia; Develter, Dirk; Soetaert, Wim; Vandamme, Erick J

2009-06-01

The nonpathogenic yeast Candida bombicola synthesizes sophorolipids. These biosurfactants are composed of the disaccharide sophorose linked to a long-chain hydroxy fatty acid and have potential applications in the food, pharmaceutical, cosmetic and cleaning industries. In order to expand the range of application, a shift of the fatty acid moiety towards medium-chain lengths would be recommendable. However, the synthesis of medium-chain sophorolipids by C. bombicola is a challenging objective. First of all, these sophorolipids can only be obtained by fermentations on unconventional carbon sources, which often have a toxic effect on the cells. Furthermore, medium-chain substrates are partially metabolized in the beta-oxidation pathway. In order to redirect unconventional substrates towards sophorolipid synthesis, the beta-oxidation pathway was blocked on the genome level by knocking out the multifunctional enzyme type 2 (MFE-2) gene. The total gene sequence of the C. bombicola MFE-2 (6033 bp) was cloned (GenBank accession number EU371724), and the obtained nucleotide sequence was used to construct a knock-out cassette. Several knock-out mutants with the correct geno- and phenotype were evaluated in a fermentation on 1-dodecanol. All mutants showed a 1.7-2.9 times higher production of sophorolipids, indicating that in those strains the substrate is redirected towards the sophorolipid synthesis.

Laboratory modeling of multiple zonal jets on the polar beta-plane

NASA Astrophysics Data System (ADS)

Afanasyev, Y.

2011-12-01

Zonal jets observed in the oceans and atmospheres of planets are studied in a laboratory rotating tank. The fluid layer in the rotating tank has parabolic free surface and dynamically simulates the polar beta-plane where the Coriolis parameter varies quadratically with distance from the pole. Velocity and surface elevation fields are measured with an optical altimetry method (Afanasyev et al., Exps Fluids 2009). The flows are induced by a localized buoyancy source along radial direction. The baroclinic flow consisting of a field of eddies propagates away from the source due West and forms zonal jets (Fig. 1). Barotropic jets ahead of the baroclinic flow are formed by radiation of beta plumes. Inside the baroclinic flow the jets flow between the chains of eddies. Experimental evidence of so-called noodles (baroclinic instability mode with motions in the radial, North-South direction) theoretically predicted by Berloff et al. (JFM, JPO 2009) was found in our experiments. Beta plume radiation mechanism and the mechanism associated with the instability of noodles are likely to contribute to formation of jets in the baroclinic flow.

Are genetic variants in the platelet-derived growth factor [beta] gene associated with chronic pancreatitis?

PubMed

Muddana, Venkata; Park, James; Lamb, Janette; Yadav, Dhiraj; Papachristou, Georgios I; Hawes, Robert H; Brand, Randall; Slivka, Adam; Whitcomb, David C

2010-11-01

Platelet-derived growth factor [beta] (PDGF-[beta]) is a major signal in proliferation and matrix synthesis through activated pancreatic stellate cells, leading to fibrosis of the pancreas. Recurrent acute pancreatitis (RAP) seems to predispose to chronic pancreatitis (CP) in some patients but not others. We tested the hypothesis that 2 known PDGF-[beta] polymorphisms are associated with progression from RAP to CP. We also tested the hypothesis that PDGF-[beta] polymorphisms in combination with environmental risk factors such as alcohol and smoking are associated with CP. Three hundred eighty-two patients with CP (n = 176) and RAP (n = 206) and 251 controls were evaluated. Platelet-derived growth factor [beta] polymorphisms +286 A/G (rs#1800818) seen in 5'-UTR and +1135 A/C (rs#1800817) in first intron were genotyped using single-nucleotide polymorphism polymerase chain reaction approach and confirmed by DNA sequencing. The genotypic frequencies for PDGF-[beta] polymorphisms in positions +286 and +1135 were found to be similar in controls and patients with RAP and CP. There was no difference in genotypic frequencies among RAP, CP, and controls in subjects in the alcohol and smoking subgroups. Known variations in the PDGF-[beta] gene do not have a significant effect on promoting or preventing fibrogenesis in pancreatitis. Further evaluation of this important pathway is warranted.

Highly restricted diversity of TCR delta chains of the amphibian Mexican axolotl (Ambystoma mexicanum) in peripheral tissues.

PubMed

André, Sébastien; Kerfourn, Fabienne; Affaticati, Pierre; Guerci, Aline; Ravassard, Philippe; Fellah, Julien S

2007-06-01

Gammadelta T cells localize at mammalian epithelial surfaces to exert both protective and regulatory roles in response to infections. We have previously characterized the Mexican axolotl (Ambystoma mexicanum) T cell receptor delta (TRD) chain. In this study, TRD repertoires in spleen, liver, intestine and skin from larvae, pre-adult and adult axolotls were examined and compared to the thymic TRD repertoire. A TRDV transcript without N/D diversity, TRDV1S1-TRDJ1, dominates the TRD repertoires until sexual maturation. In adult tissues, this canonical transcript is replaced by another dominant TRDV1S1-TRDJ1 transcript. In the thymus, these two transcripts are detected early in development. Our results suggest that gammadelta T cells that express the canonical TRDV1S1-TRDJ1 transcript emerge from the thymus and colonize the peripheral tissues, where they are selectively expanded by recurrent ligands. This particular situation is probably related to the neotenic state and the slow development of the axolotl. In thymectomized axolotls, TRD repertoires appear different from those of normal axolotls, suggesting that extrathymic gammadelta T cell differentiation could occur. Gene expression analysis showed the importance of the gut in T cell development.

T-cell clonality analysis in biopsy specimens from two different skin sites shows high specificity in the diagnosis of patients with suggested mycosis fungoides.

PubMed

Thurber, Stacy E; Zhang, Bing; Kim, Youn H; Schrijver, Iris; Zehnder, James; Kohler, Sabine

2007-11-01

The diagnosis of mycosis fungoides (MF) is often difficult because of significant clinical and histopathologic overlap with inflammatory dermatoses. T-cell receptor (TCR)gamma chain rearrangement by polymerase chain reaction (PCR) (TCR-PCR) is a helpful adjuvant tool in this setting, but several of the inflammatory dermatoses in the differential diagnosis of MF may contain a clonal T-cell proliferation. We examined whether analysis for T-cell clonality and comparison of the clones with the standardized BIOMED-2 PCR multiplex primers for the TCRgamma chain from two anatomically distinct skin sites improves diagnostic accuracy. We examined two biopsy specimens each from 10 patients with unequivocal MF, from 18 patients with inflammatory dermatoses, and from 18 patients who could initially not be definitively given a diagnosis based on clinical and histopathologic criteria. Eight of 10 patients with unequivocal MF had an identical clone in both biopsy specimens. Two of 18 patients with inflammatory dermatoses were found to have a clone in one of the biopsy specimens. On further follow-up of the 18 patients with morphologically nondiagnostic biopsy specimens, 13 of 18 were later confirmed to have MF and 5 of 18 had inflammatory dermatoses. Eleven of 13 patients with MF had an identical clone in both biopsy specimens; two of 13 had a polyclonal amplification pattern in both biopsy specimens. Four of 5 patients with inflammatory dermatoses had no clone in either biopsy specimen. One patient with an inflammatory dermatosis had an identical clone in both specimens. The sensitivity of TCR-PCR analysis to evaluate for an identical clone at different anatomic skin sites (dual TCR-PCR) is 82.6% and the specificity is 95.7%. The number of patients in the study group was limited. These data suggest that dual TCR-PCR is a very promising technique with high specificity in distinguishing MF from inflammatory dermatoses.

Computational study of the fibril organization of polyglutamine repeats reveals a common motif identified in beta-helices.

PubMed

Zanuy, David; Gunasekaran, Kannan; Lesk, Arthur M; Nussinov, Ruth

2006-04-21

The formation of fibril aggregates by long polyglutamine sequences is assumed to play a major role in neurodegenerative diseases such as Huntington. Here, we model peptides rich in glutamine, through a series of molecular dynamics simulations. Starting from a rigid nanotube-like conformation, we have obtained a new conformational template that shares structural features of a tubular helix and of a beta-helix conformational organization. Our new model can be described as a super-helical arrangement of flat beta-sheet segments linked by planar turns or bends. Interestingly, our comprehensive analysis of the Protein Data Bank reveals that this is a common motif in beta-helices (termed beta-bend), although it has not been identified so far. The motif is based on the alternation of beta-sheet and helical conformation as the protein sequence is followed from the N to the C termini (beta-alpha(R)-beta-polyPro-beta). We further identify this motif in the ssNMR structure of the protofibril of the amyloidogenic peptide Abeta(1-40). The recurrence of the beta-bend suggests a general mode of connecting long parallel beta-sheet segments that would allow the growth of partially ordered fibril structures. The design allows the peptide backbone to change direction with a minimal loss of main chain hydrogen bonds. The identification of a coherent organization beyond that of the beta-sheet segments in different folds rich in parallel beta-sheets suggests a higher degree of ordered structure in protein fibrils, in agreement with their low solubility and dense molecular packing.

Chain of Custody Item Monitor Message Viewer v.1.0 Beta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwartz, Steven Robert; Fielder, Laura; Hymel, Ross W.

The CoCIM Message Viewer software allows users to connect to and download messages from a Chain of Custody Item Monitor (CoCIM) connected to a serial port on the user’s computer. The downloaded messages are authenticated and displayed in a Graphical User Interface that allows the user a limited degree of sorting and filtering of the downloaded messages as well as the ability to save downloaded files or to open previously downloaded message history files.

Role for transforming growth factor-beta1 in alport renal disease progression.

PubMed

Sayers, R; Kalluri, R; Rodgers, K D; Shield, C F; Meehan, D T; Cosgrove, D

1999-11-01

Alport syndrome results from mutations in either the alpha3(IV), alpha4(IV), or alpha5(IV) collagen genes. The disease is characterized by a progressive glomerulonephritis usually associated with a high-frequency sensorineural hearing loss. A mouse model for an autosomal form of Alport syndrome [collagen alpha3(IV) knockout] was produced and characterized. In this study, the model was exploited to demonstrate a potential role for transforming growth factor-beta1 (TGF-beta1) in Alport renal disease pathogenesis. Kidneys from normal and Alport mice, taken at different stages during the course of renal disease progression, were analyzed by Northern blot, in situ hybridization, and immunohistology for expression of TGF-beta1 and components of the extracellular matrix. Normal and Alport human kidney was examined for TGF-beta1 expression using RNase protection. The mRNAs encoding TGF-beta1 (in both mouse and human), entactin, fibronectin, and the collagen alpha1(IV) and alpha2(IV) chains were significantly induced in total kidney as a function of Alport renal disease progression. The induction of these specific mRNAs was observed in the glomerular podocytes of animals with advanced disease. Type IV collagen, laminin-1, and fibronectin were markedly elevated in the tubulointerstitium at 10 weeks, but not at 6 weeks, suggesting that elevated expression of specific mRNAs on Northern blots reflects events associated with tubulointerstitial fibrosis. The concomitant accumulation of mRNAs encoding TGF-beta1 and extracellular matrix components in the podocytes of diseased kidneys may reflect key events in Alport renal disease progression. These data suggest a role for TGF-beta1 in both glomerular and tubulointerstitial damage associated with Alport syndrome.

The tyrosine phosphatase PTPN22 discriminates weak self peptides from strong agonist TCR signals.

PubMed

Salmond, Robert J; Brownlie, Rebecca J; Morrison, Vicky L; Zamoyska, Rose

2014-09-01

T cells must be tolerant of self antigens to avoid autoimmunity but responsive to foreign antigens to provide protection against infection. We found that in both naive T cells and effector T cells, the tyrosine phosphatase PTPN22 limited signaling via the T cell antigen receptor (TCR) by weak agonists and self antigens while not impeding responses to strong agonist antigens. T cells lacking PTPN22 showed enhanced formation of conjugates with antigen-presenting cells pulsed with weak peptides, which led to activation of the T cells and their production of inflammatory cytokines. This effect was exacerbated under conditions of lymphopenia, with the formation of potent memory T cells in the absence of PTPN22. Our data address how loss-of-function PTPN22 alleles can lead to the population expansion of effector and/or memory T cells and a predisposition to human autoimmunity.

Reactivity of long chain alkylamines to lignin moieties: implications on hydrophobicity of lignocellulose materials.

PubMed

Kudanga, Tukayi; Prasetyo, Endry Nugroho; Sipilä, Jussi; Guebitz, Georg M; Nyanhongo, Gibson S

2010-08-20

Enzymatic processes provide new perspectives for modification of lignocellulose materials. In the current study, laccase catalyzed coupling of long chain alkylamines to lignin model molecules and lignocellulose was investigated. Up to two molecules of dodecylamine (DA) and dihexylamine (DHA) were successfully coupled with lignin monomers (guaiacol, catechol and ferulic acid) while coupling onto complex lignin model compounds (syringylglycerol beta-guaiacyl ether, guaiacylglycerol beta-guaiacyl ether and dibenzodioxocin) yielded 1:1 coupling products. Surface analysis of beech veneers enzymatically grafted with DA showed an increase in nitrogen content of 3.18% compared to 0.71% in laccase only treated controls while the O/C ratio decreased from 0.52 to 0.46. Concomitantly the grafting of DHA or DA onto beech veneers resulted in a 53.8% and 84.2% increase in hydrophobicity, respectively when compared to simple adsorption. Therefore, laccase-mediated grafting of long chain alkylamines onto lignocellulose materials can be potentially exploited for improving their hydrophobicity. Copyright 2010 Elsevier B.V. All rights reserved.

Beta/gamma and alpha backgrounds in CRESST-II Phase 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, R.; Angloher, G.; Ferreiro Iachellini, N.

2015-06-01

The experiment CRESST-II aims at the detection of dark matter with scintillating CaWO{sub 4} crystals operated as cryogenic detectors. Recent results on spin-independent WIMP-nucleon scattering from the CRESST-II Phase 2 allowed to probe a new region of parameter space for WIMP masses below 3 GeV/c{sup 2}. This sensitivity was achieved after background levels were reduced significantly. We present extensive background studies of a CaWO{sub 4} crystal, called TUM40, grown at the Technische Universität München. The average beta/gamma rate of 3.51/[kg keV day] (1-40 keV) and the total intrinsic alpha activity from natural decay chains of 3.08±0.04 mBq/kg are the lowestmore » reported for CaWO{sub 4} detectors. Contributions from cosmogenic activation, surface-alpha decays, external radiation and intrinsic alpha/beta emitters are investigated in detail. A Monte-Carlo based background decomposition allows to identify the origin of the majority of beta/gamma events in the energy region relevant for dark matter search.« less

Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

PubMed Central

Jazirehi, Ali R.; Economou, James S.

2012-01-01

Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

Effect of interaction range on phonon relaxation in Fermi-Pasta-Ulam beta chain.

PubMed

Santhosh, G; Kumar, Deepak

2007-08-01

We study the effect of increasing the range of interactions on phonon relaxation in a chain of atoms with quartic anharmonicity. The study is motivated by recent numerical studies, showing that the value of the exponent alpha characterizing the divergence of conductivity with system size apparently depends on the presence of second neighbor couplings. We perform a quantum calculation of the wave-vector (q) dependent relaxation rate gamma(q) in the second order perturbation theory. The nonanalytic dependence of gamma(q) arises due to small-q singularity of the collision integral. We find that gamma(q) proportional to Aq(5/3) + Bq2. This gives rise to an asymptotic value alpha = 0.4, but the q2 terms lead to a higher apparent value of alpha at small sizes of the chain.

Synthetic alleles at position 121 define a functional domain of human interleukin-1 beta.