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Cox Terhorst - One of the best experts on this subject based on the ideXlab platform.
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expression of a cd3 epsilon transgene in cd3 epsilon null mice does not restore cd3 gamma and delta expression but efficiently rescues t cell development from a subpopulation of prothymocytes
International Immunology, 1998Co-Authors: Ninghai Wang, Baoping Wang, Mariolina Salio, Deborah Allen, Jian She, Cox TerhorstAbstract:The TCR-associated CD3 complex consists of four subunits, i.e. CD3 gamma, delta, epsilon and zeta, which are expressed very early in T cell development prior to the expression of the TCR and the pre-TCR alpha chain. It is unclear whether the expression of each CD3 protein is independent of, or is influenced by, other CD3 subunits. To study whether CD3 epsilon regulates expression of CD3 gamma and delta genes, we generated a strain of CD3 epsilon-deficient mice termed CD3 epsilon(delta P/delta P) (epsilon(delta P)), in which the promoter of CD3E was disrupted, and subsequently reconstituted these mice with a CD3 epsilon transgene. In the epsilon(delta P) mice, T cell development is arrested at the double-negative stage and targeting the CD3 epsilon gene caused severe inhibition of CD3 gamma and delta gene expression. Introduction of the CD3 epsilon transgene did not restore CD3 gamma and delta expression. However, a very small fraction of prothymocytes that expressed CD3 gamma and delta was rescued upon reconstitution of the CD3 epsilon transgene. Remarkably, this rescue led to a very efficient differentiation and maturation of thymocytes, resulting in a significant T cell population in the periphery. These results demonstrate that CD3 epsilon does not regulate expression of CD3 gamma and delta genes, and underscore the capacity of each prothymocyte to give rise to a large number of mature peripheral T cells.
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the t cell receptor associated cd3 epsilon protein is phosphorylated upon t cell activation in the two tyrosine residues of a conserved signal transduction motif
European Journal of Immunology, 1993Co-Authors: Jaime Sancho, Rafael Franco, Talal A Chatila, C Hall, Cox TerhorstAbstract:Signal transduction through the T cell receptor for antigen, the TcR/CD3 complex, involves phosphorylation of tyrosine residues in the CD3-zeta chain. Since both CD3-epsilon and the zeta chain contain a tyrosine-based signaling motif, we examine phosphorylation of CD3-epsilon in human T cells. Engagement of the TcR/CD3 complex induced tyrosine phosphorylation of CD3-epsilon in vivo. Induction of CD3-epsilon phosphorylation followed similar kinetics to that of the zeta chain phosphorylation. In contrast to zeta, CD3-epsilon phosphorylation was strictly dependent upon cell surface expression of this member of the TcR/CD3 complex. Chemical and proteolytic cleavage combined with peptide-specific Western blotting established that CD3-epsilon phosphorylation occurred in the two tyrosine residues located in the signal transduction motif in the C-terminal portion of the molecule. Taken together, these data indicated that phosphorylation of CD3-epsilon by tyrosine protein kinases may serve to couple the TcR/CD3 complex to other effector molecules in the signaling cascade.
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cd3 zeta surface expression is required for cd4 p56lck mediated upregulation of t cell antigen receptor cd3 signaling in t cells
Journal of Biological Chemistry, 1992Co-Authors: J Sancho, Jeffrey A Ledbetter, Myung Sik Choi, Steven B Kanner, Julie P Deans, Cox TerhorstAbstract:It has been proposed that during T cell receptor antigen recognition, CD4- or CD8-p56lck molecules interact with the T cell antigen receptor-CD3 complex (TCR-CD3) to phosphorylate various undefined substrates, which then initiate signal transduction through the TCR-CD3 complex. The ability of CD4 to modulate the TCR-CD3-induced increase in intracellular Ca2+, [Ca2+]i, and substrate tyrosine phosphorylation was studied in mutants of the human leukemic T cell line HPB-ALL characterized by their low expression of the TCR-CD3 complex on the cell surface. In TCR-CD3low cells, in which CD3-zeta was found to be associated with the TCR-CD3 complex, cross-linking CD3 with CD4 resulted in a profile of calcium mobilization, CD3-zeta, and phospholipase C-gamma 1 tyrosine phosphorylation similar to that observed in HPB-ALL cells, although the magnitude of generalized substrate tyrosine phosphorylation appeared to be smaller, as compared with wild-type cells. Responses were weak or absent when CD3 was cross-linked alone. In contrast, in a mutant in which association of CD3-zeta 2 with the TCR-CD3 was defective, cross-linking of CD3 with CD4 had a weaker effect on any of the activation parameters tested. These experiments showed that the presence of CD3-zeta 2 in the TCR-CD3 complex is of critical importance for the ability of CD4 to enhance early transducing signals inside the cell. The data also suggest that CD4-associated protein tyrosine kinase p56lck could up-regulate defective CD3-mediated induction of phospholipase C activity by increasing tyrosine phosphorylation of phospholipase C-gamma 1.
Carsten Geisler - One of the best experts on this subject based on the ideXlab platform.
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distinct domains of the cd3 gamma chain are involved in surface expression and function of the t cell antigen receptor
Journal of Biological Chemistry, 1995Co-Authors: A M K Wegener, Xiaohong Hou, Jes Dietrich, Carsten GeislerAbstract:Abstract The T cell antigen receptor (TcR) is a multisubunit complex that consists of at least six different polypeptides. We have recently demonstrated that the CD3-δ subunit cannot substitute for the CD3-γ subunit in TcR cell surface expression, in spite of significant amino acid homology between these two subunits. To identify CD3-γ-specific domains that are required for assembly of the complete TcR and for surface expression and function of the TcR, chimeric CD3-γ/CD3-δ molecules were constructed and expressed in T cells devoid of endogenous CD3-γ. Substitution of the extracellular domain of CD3-γ with that of CD3-δ did not allow cell surface expression of the TcR. In contrast, substitution of the transmembrane and/or the intracellular domains of CD3-γ with those of CD3-δ did allow TcR cell surface expression. These results conclusively demonstrate that the extracellular domain of CD3-γ plays a unique role in TcR assembly. Functional analyses of the transfectants demonstrated that the intracellular domain of CD3-γ is required for protein kinase C-mediated down-regulation of TcR but is dispensable for the pattern of tyrosine phosphorylation observed following activation through TcR.
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failure to synthesize the cd3 gamma chain consequences for t cell antigen receptor assembly processing and expression
Journal of Immunology, 1992Co-Authors: Carsten GeislerAbstract:The TCR consists of the Ti alpha beta heterodimer and the associated CD3 chains, CD3 gamma delta epsilon zeta 2 or zeta eta. The structural relationships between the subunits of the Ti/CD3 complex are still not fully understood. To explore the roles of the individual CD3 chains for the assembly, intracellular processing, and expression of the TCR, mutants of the T cell line Jurkat were isolated. One variant, JGN, was found to produce all the Ti/CD3 components with the exception of CD3-gamma. The results indicate that: 1) the tetrameric form (Ti alpha beta-CD3 delta epsilon) of the Ti/CD3 complex is produced in the endoplasmic reticulum in the absence of CD3-gamma; 2) CD3-zeta does not associate with the Ti alpha beta-CD3 delta epsilon complex; 3) the Ti alpha beta-CD3 delta epsilon complex is not exported from the endoplasmic reticulum to the Golgi apparatus; and 4) CD3-gamma is required for cell surface expression of the Ti/CD3 complex. Transfection of the wild-type CD3-gamma gene into JGN reconstituted expression of functional Ti/CD3 complexes, and analysis of T cell lines producing different amounts of CD3-gamma indicated that CD3-gamma and CD3-delta competed for the binding to CD3-epsilon.
Wei Li - One of the best experts on this subject based on the ideXlab platform.
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protective effects of novel derivatives of vitamin d3 and lumisterol against uvb induced damage in human keratinocytes involve activation of nrf2 and p53 defense mechanisms
Redox biology, 2019Co-Authors: Edith K Y Tang, Robert C Tuckey, Anyamanee Chaiprasongsuk, Zorica Janjetovic, Stuart G Jarrett, John A Dorazio, Michael F Holick, Uraiwan Panich, Wei LiAbstract:Abstract We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50–200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.
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detection of novel cyp11a1 derived secosteroids in the human epidermis and serum and pig adrenal gland
Scientific Reports, 2015Co-Authors: Andrzej Slominski, Edith K Y Tang, Elaine W Tieu, Arnold E. Postlethwaite, Wei Li, Robert C TuckeyAbstract:To investigate whether novel pathways of vitamin D3 (D3) and 7-dehydrocholesterol (7DHC) metabolism initiated by CYP11A1 and previously characterized in vitro, occur in vivo, we analyzed samples of human serum and epidermis, and pig adrenals for the presence of intermediates and products of these pathways. We extracted human epidermis from 13 individuals and sera from 13 individuals and analyzed them by LC/qTOF-MS alongside the corresponding standards. Pig adrenal glands were also analyzed for these steroids and secosteroids. Epidermal, serum and adrenal samples showed the presence of D3 hydroxy-derivatives corresponding to 20(OH)D3, 22(OH)D3, 25(OH)D3, 1,25(OH)2D3, 20,22(OH)2D3, 20,23(OH)2D3, 20,24(OH)2D3, 20,25(OH)2D3, 20,26(OH)2D3, 1,20,23(OH)3D3 and 17,20,23(OH)3D3, plus 1,20(OH)2D3 which was detectable only in the epidermis. Serum concentrations of 20(OH)D3 and 22(OH)D3 were only 30- and 15-fold lower than 25(OH)D3, respectively, and at levels above those required for biological activity as measured in vitro. We also detected 1,20,24(OH)3D3, 1,20,25(OH)3D3 and 1,20,26(OH)3D3 in the adrenals. Products of CYP11A1 action on 7DHC, namely 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone were also detected in serum, epidermis and the adrenal. Thus, we have detected novel CYP11A1-derived secosteroids in the skin, serum and adrenal gland and based on their concentrations and biological activity suggest that they act as hormones in vivo.
Anyamanee Chaiprasongsuk - One of the best experts on this subject based on the ideXlab platform.
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protective effects of novel derivatives of vitamin d3 and lumisterol against uvb induced damage in human keratinocytes involve activation of nrf2 and p53 defense mechanisms
Redox biology, 2019Co-Authors: Edith K Y Tang, Robert C Tuckey, Anyamanee Chaiprasongsuk, Zorica Janjetovic, Stuart G Jarrett, John A Dorazio, Michael F Holick, Uraiwan Panich, Wei LiAbstract:Abstract We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50–200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.
Noboru Kubodera - One of the best experts on this subject based on the ideXlab platform.
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c 3 epimerization of vitamin d3 metabolites and further metabolism of c 3 epimers 25 hydroxyvitamin d3 is metabolized to 3 epi 25 hydroxyvitamin d3 and subsequently metabolized through c 1α or c 24 hydroxylation
Journal of Biological Chemistry, 2004Co-Authors: Maya Kamao, Toshiyuki Sakaki, Noboru Kubodera, Satyanarayana G Reddy, Syuichiro Tatematsu, Susumi Hatakeyama, Natsumi Sawada, Kuniyo Inouye, Keiichi Ozono, Toshio OkanoAbstract:Recently, it was revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and 24R,25-dihydroxyvitamin D3 (24,25(OH)2D3) were metabolized to their respective epimers of the hydroxyl group at C-3 of the A-ring. We now report the isolation and structural assignment of 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3 as a major metabolite of 25-hydroxyvitamin D3 (25(OH)D3) and the further metabolism of C-3 epimers of vitamin D3 metabolites. When 25(OH)D3 was incubated with various cultured cells including osteosarcoma, colon adenocarcinoma, and hepatoblastoma cell lines, 3-epi-25(OH)D3 and 24,25 (OH)2D3 were commonly observed as a major and minor metabolite of 25(OH)D3, respectively. 25(OH)D3 was at least as sensitive to C-3 epimerization as 1α, 25(OH)2D3 which has been reported as a substrate for the C-3 epimerization reaction. Unlike these cultured cells, LLC-PK1 cells, a porcine kidney cell line, preferentially produced 24,25(OH)2D3 rather than 3-epi-25(OH)D3. We also confirmed the existence of 3-epi-25(OH)D3 in the serum of rats intravenously given pharmacological doses of 25(OH)D3. The cultured cells metabolized 3-epi-25(OH)D3 and 3-epi-1α,25(OH)2D3 to 3-epi-24,25(OH)2D3 and 3-epi-1α,24,25(OH)3D3, respectively. In addition, we demonstrated that 3-epi-25(OH)D3 was metabolized to 3-epi-1α,25(OH)2D3 by CYP27B1 and to 3-epi-24,25(OH)2D3 by CYP24 using recombinant Escherichia coli cell systems. 3-Epi-25(OH)D3, 3-epi-1α,25(OH)2D3, and 3-epi-24,25(OH)2D3 were biologically less active than 25(OH)D3, 1α,25(OH)2D3, and 24,25(OH)2D3, but 3-epi-1α,25(OH)2D3 showed to some extent transcriptional activity toward target genes and anti-proliferative/differentiation-inducing activity against human myeloid leukemia cells (HL-60). These results indicate that C-3 epimerization may be a common metabolic pathway for the major metabolites of vitamin D3.
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differential catabolism of 22 oxacalcitriol and 1 25 dihydroxyvitamin d3 by normal human peripheral monocytes
Endocrinology, 1993Co-Authors: S Kamimura, Noboru Kubodera, Eduardo Slatopolsky, Alex J Brown, Y Nishii, Maurizio Gallieni, Adriana DussoAbstract:Oxacalcitriol (1,25-(OH)Z-220xa-D3) mimics the action of 1,25- dihydroxyvitamin D3 (1,25-(OH),D,) in a variety of target tissues, including the systemic control of calcitriol metabolism. Similar to 1,25- (OH)1D3, 1,25-(OH)Z-220xa-D3 decreases the rate of 1,25-(OH)2D3 syn- thesis and accelerates its metabolic clearance rate. We have previously shown that in normal human monocytes, physiological concentrations of 1,25-(OH)*D3 and 1,25-(OH)2-220xa-D3 determine identical suppres- sion of 1,25-(OH)*D3 synthesis. Moreover, both sterols have a similar potency to induce vitamin D degradation through stimulation of the C24-hydroxylation pathway. In this study, we examined the ability of normal human monocytes to metabolize 1,25-(OH)2-220xa-D3 and whether the enzymes involved are the same as those that catabolize 1,25-(OH)2D3. Time-course experiments demonstrated no detectable basal catabolic activity. However, exogenous 1,25-(OH)QDa at physio- logical concentrations induced 1,25-(OH)2-220xa-D3 degradation by normal human monocytes. Competition experiments showed that a lo- fold molar excess of unlabeled 1,25-(OH)*D3 inhibited tritiated-1,25- (OH)2-220xa-D3 catabolism by 85%, whereas a lo-fold excess of unla- beled 1,25-(OH)2-220xa-D3 reduced tritiated-1,25-(OH)2-220xa-D3 ca- tabolism by 33%. In contrast, although a lo-fold excess of unlabeled 1,25-(OH)2D3 reduced tritiat=ed 1,25-(OH)2D3 catabolism by 60%, a 1000-fold excess of 1,25-(OH)2-220xa-D3 was required to reduce triti- ated 1,25-(OH)*D3 catabolism to this degree. The apparent K, for 1,25- (OH)2-220xa-D3 was significantly higher than that of 1,25-(OH)*D3 (2.0 rt 0.8 us. 0.9 -t 0.2 nM, respectively; P < 0.001) for the catabolic pathway induced by physiological concentrations of 1,25-(OH)2D3. Moreover, the presence of 0.65 nM lr25-(OH)ZD3 caused an additional increase in the K, for 1,25-(OH)Z-220xa-D3 (3.2 rf: 0.8 nM). These data suggest that 1,25-(OH)z-220xa-D3 may be less accessible than 1,25-(OH)zD3 to the hydroxylases involved in vitamin D catabolism. The resulting prolonged biological half-life of the analog in certain target tissues may be involved in its selectivity. (Endocrinology 133: 2719-2723, 1993)
