Journal articles on the topic 'Calcium affinity T state'
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1
Aguilar, Alicia Virginia, Bryan Benson, Joseph Rathkey, Luis Correa, Lucy Li, Jay Myers, Jeffrey Tomalka, et al. "PIEZO1 forms an adhesive-mechanosensitive complex with activated LFA-1 on T lymphocytes." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 51.21. http://dx.doi.org/10.4049/jimmunol.202.supp.51.21.
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Abstract In order to respond to infection, injury and stress, T lymphocytes must successfully migrate from the bloodstream into inflamed tissue in a process called the leukocyte adhesion cascade. Of these steps, blockade of adhesion has shown clear clinical benefit in autoimmunity, and the step of intraluminal crawling is of great potential clinical importance. In order to coordinate adhesion and crawling while in circulation, leukocytes must respond appropriately to complex hemodynamic forces, including shear stresses and erythrocyte driven margination against the venule walls. Therefore, we hypothesized that the mechanosensitive calcium channel PIEZO1, which is preferentially expressed in T lymphocytes, mediates T lymphocyte responses to force during adhesion and crawling. To test this hypothesis, we genetically ablated PIEZO1 in primary T lymphocytes from healthy human donors using CRISPR/Cas9, and observed crawling ability in vitro. In this assay, PIEZO1 knockout cells exhibited decreased crawling and disrupted morphology. Moreover, we performed immunofluorescence of PIEZO1 on chemokine-activated crawling T lymphocytes, which demonstrated that PIEZO1 redistributes to the contact zone of crawling cells in a pattern reminiscent of a high-affinity LFA-1 focal zone. Additionally, PIEZO1 colocalizes specifically with high affinity LFA-1. Using co-immunoprecipitation indicated that PIEZO1 preferentially associates with the alpha integrin subunit of LFA-1 in its active form, through a conserved amphipathic eight amino acid motif. Taken together, our data suggest that PIEZO1 contributes to coordination of T lymphocyte crawling via interaction with integrins, modulating the affinity state and turnover of LFA-1.
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Mirit, Eynan, Chaya Gross, Yonathan Hasin, Aharon Palmon, and Michal Horowitz. "Changes in cardiac mechanics with heat acclimation: adrenergic signaling and SR-Ca regulatory proteins." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 279, no. 1 (July 1, 2000): R77—R85. http://dx.doi.org/10.1152/ajpregu.2000.279.1.r77.
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The involvement of adrenergic signaling and sarcoplasmic calcium regulatory proteins in the development of heat acclimation-induced adaptations in cardiac mechanics was studied in heat-acclimated (34°C) rats for 2, 5, and 30 days (AC2, AC5, and AC30, respectively). Control (C) rats were held at 24 ± 1°C. Systolic pressure (LVP) and velocities of contraction (dP/d t/P) and relaxation (−dP/d t/P) were measured using a Langendorff system. For adrenergic signaling, β-adrenoreceptor (AR) density and affinity (Scatchard plots) and cardiac inotropic response to norepinephrine (10− 7 mM, ± 10− 6 mM propranolol) were measured. For the regulatory proteins, steady-state levels of Ca2+-ATPase and phospholamban (PLB) mRNAs and the encoded proteins Ca2+-ATPase [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)] and PLB were measured using semiquantitative RT-PCR and Western immunoblotting, respectively. Both short (STHA; AC2 and AC5)- and long-term heat acclimation (LTHA; AC30) enhanced LVP. However, dP/d t ⋅ P and −dP/d t ⋅ P in STHA hearts resembled that of the controls, whereas on LTHA, both parameters decreased ( P< 0.05), implying decreased velocity of contraction and relaxation. β-AR density remained unchanged with their affinity markedly decreased ( P < 0.05). AR responsiveness, however, diminished in AC2 but was markedly enhanced on LTHA. During STHA, PLB and sarcoplasmic reticulum Ca2+-ATPase transcripts were upregulated with no change in the encoded proteins except for SERCA downregulation on AC5, leading to an increased PLB/SERCA ratio ( P < 0.05). This mismatched preacclimation lusitropic state on STHA and increased PLB/SERCA ratio was evident ( P < 0.05) due to downregulation of SERCA and upregulation of PLB. Our data fit a biphasic acclimation model in which desensitized adrenergic signaling is dominant during STHA, whereas on LTHA, the contractile machinery is influenced by altered expression of the calcium regulatory proteins leading to both augmented adrenergic inotropic response (via PLB elevation) and decreased velocity of relaxation. The sustained low thyroxin measured on LTHA causally associates with this response.
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Wang, J. M., D. W. McVicar, J. J. Oppenheim, and D. J. Kelvin. "Identification of RANTES receptors on human monocytic cells: competition for binding and desensitization by homologous chemotactic cytokines." Journal of Experimental Medicine 177, no. 3 (March 1, 1993): 699–705. http://dx.doi.org/10.1084/jem.177.3.699.
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RANTES (regulated on activation, normal T expressed and secreted) is a member of the chemotactic cytokine (chemokine) beta subfamily. High affinity receptors for RANTES have been identified on a human monocytic leukemia cell line THP-1, which responded to RANTES in chemotaxis and calcium mobilization assays. Steady-state binding data analyses revealed approximately 700 binding sites/cell on THP-1 cells with a Kd value of 400 pM, comparable to that expressed on human peripheral blood monocytes. The RANTES binding to monocytic cells was competed for by monocyte chemotactic and activating factor (MCAF) and macrophage inflammatory protein 1 (MIP-1) alpha, two other chemokine beta cytokines. Although MCAF and MIP-1 alpha competed for RANTES binding to monocytes with apparent lower affinity (with estimated Kd of 6 and 1.6, nM respectively) both of these cytokines effectively desensitized the calcium mobilization induced by RANTES. The chemotactic response of THP-1 cells to RANTES was also markedly inhibited by preincubation with MCAF or MIP-1 alpha. In contrast, RANTES did not desensitize the THP-1 calcium mobilization and chemotaxis in response to MCAF or MIP-1 alpha. These results, together with our previous observations that RANTES did not compete for MCAF or MIP-1 alpha binding on monocytic cells, indicate the expression of promiscuous receptors on monocytes that recognize one or more cytokines within the chemokine beta family.
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Freedman, Bruce D., Qing-Hua Liu, Selin Somersan, Michael I. Kotlikoff, and Jennifer A. Punt. "Receptor Avidity and Costimulation Specify the Intracellular Ca2+ Signaling Pattern in Cd4+Cd8+ Thymocytes." Journal of Experimental Medicine 190, no. 7 (October 4, 1999): 943–52. http://dx.doi.org/10.1084/jem.190.7.943.
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Thymocyte maturation is governed by antigen–T cell receptor (TCR) affinity and the extent of TCR aggregation. Signals provided by coactivating molecules such as CD4 and CD28 also influence the fate of immature thymocytes. The mechanism by which differences in antigen–TCR avidity encode unique maturational responses of lymphocytes and the influence of coactivating molecules on these signaling processes is not fully understood. To better understand the role of a key second messenger, calcium, in governing thymocyte maturation, we measured the intracellular free calcium concentration ([Ca2+]i) response to changes in TCR avidity and costimulation. We found that TCR stimulation initiates either amplitude- or frequency-encoded [Ca2+]i changes depending on (a) the maturation state of stimulated thymocytes, (b) the avidity of TCR interactions, and (c) the participation of specific coactivating molecules. Calcium signaling within immature but not mature thymocytes could be modulated by the avidity of CD3/CD4 engagement. Low avidity interactions induced biphasic calcium responses, whereas high avidity engagement initiated oscillatory calcium changes. Notably, CD28 participation converted the calcium response to low avidity receptor engagement from a biphasic to oscillatory pattern. These data suggest that calcium plays a central role in encoding the nature of the TCR signal received by thymocytes and, consequently, a role in thymic selection.
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Lüttgau, H. C., G. Gottschalk, and Dorothea Berwe. "The effect of calcium and Ca antagonists upon excitation–contraction coupling." Canadian Journal of Physiology and Pharmacology 65, no. 4 (April 1, 1987): 717–23. http://dx.doi.org/10.1139/y87-118.
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The effect of a Ca2+ -free tetraethylammonium sulfate solution on force development in short skeletal muscle fibres of the frog was investigated under voltage clamp control. Maximum force could still be reached under this condition. The removal of external Ca2+, however, caused an acceleration of force inactivation leading to a shift of the steady-state potential dependence of force inactivation to more negative potentials. With reference to the "modulated-receptor hypothesis" this result was explained by assuming a potential-dependent binding of Ca2+ to a force-controlling system in the T-tubular membrane, with a low affinity in the depolarized-inactivated state. A dissociation of Ca2+ is assumed to turn the system into a secondary inactivated state (paralysis) from which it only slowly recovers after repolarization. Ca antagonists like D600 and diltiazem accelerated the shift into paralysis, probably by an allosteric displacement of Ca2+ from its binding site. The application of 1–2 μM of the Ca antagonist nifedipine blocked the inward Ca2+ current and caused a prolongation of the transient force development following a depolarization. A similar retardation of force inactivation and a threshold shift to more negative potentials occurred when the Ca2+ chelator ethyleneglycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) was injected into the fibre and when in Ca2+-free solutions sodium ions entered the cell through Ca2+ channels.
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McCarthy, R. T., and C. J. Cohen. "Nimodipine block of calcium channels in rat vascular smooth muscle cell lines. Exceptionally high-affinity binding in A7r5 and A10 cells." Journal of General Physiology 94, no. 4 (October 1, 1989): 669–92. http://dx.doi.org/10.1085/jgp.94.4.669.
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Calcium channel currents were studied in the A10 and A7r5 cell lines derived from rat thoracic aorta muscle cells. The whole-cell variation of the patch voltage clamp technique was used. Results with each cell line were nearly identical. Two types of Ca channels were found in each cell line that are similar to the L-type and T-type Ca channels found in excitable cells. Nimodipine block of the L-type Ca channels in both cell lines is more potent than in previously studied tissues. The kinetics of nimodipine block are accounted for by a model that postulates 1:1 drug binding to open Ca channels with an apparent dissociation constant (KO) of 16-45 pM. In A7r5 cells, the rate of onset of nimodipine block increases with the test potential, in quantitative agreement with the model of open channel block. The apparent association rate (f) is 1.4 x 10(9) M-1 s-1; the dissociation rate (b) is about 0.024 s-1. In anterior pituitary cells (GH4C1 cells), KO is 30 times larger; b is only twice as fast, but f is 15 times slower. The comparative kinetic analysis indicates that the high-affinity binding site for nimodipine is similar in both GH4C1 and A7r5 cells, but nimodipine diffuses much faster or has a larger partition coefficient into the plasmalemma of A7r5 cells than for GH4C1 cells. Unusually high-affinity binding was not observed in earlier 45Ca flux studies with A10 and A7r5 cells. The model of open channel block accounts for the discrepancy; only a small fraction of the Ca channels are in the high affinity open state under the conditions used in 45Ca flux studies, so an effective binding constant is measured that is much greater than the dissociation constant for high-affinity binding.
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Waldmann, TA, JD White, CK Goldman, L. Top, A. Grant, R. Bamford, E. Roessler, ID Horak, S. Zaknoen, and C. Kasten-Sportes. "The interleukin-2 receptor: a target for monoclonal antibody treatment of human T-cell lymphotrophic virus I-induced adult T-cell leukemia." Blood 82, no. 6 (September 15, 1993): 1701–12. http://dx.doi.org/10.1182/blood.v82.6.1701.1701.
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Abstract Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotrophic virus-I (HTLV-I). It is an aggressive leukemia with an overall mortality rate of 50% within 5 months; no conventional chemotherapy regimen appears successful in inducing long-term disease-free survival in ATL patients. However, ATL cells constitutively express high-affinity interleukin-2 receptors (IL-2Rs) identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference in receptor expression, we administered anti-Tac intravenously (IV) to 19 patients with ATL. In general the patients did not suffer untoward reactions, and in 18 of 19 cases did not have a reduction in normal formed elements of the blood. Seven patients developed remissions that were mixed (1 patient), partial (4 patients), or complete (2 patients), with partial and complete remissions lasting from 9 weeks to more than 3 years as assessed by routine hematologic tests, immunofluorescence analysis, and molecular genetic analysis of T-cell receptor gene rearrangements and of HTLV-I proviral integration. Furthermore, remission was associated with a return to normal serum calcium levels and an improvement of liver function tests. Remission was also associated in some cases with an amelioration of the profound immunodeficiency state that characterizes ATL. Thus the use of a monoclonal antibody that blocks the interaction of IL-2 with its receptor expressed on ATL cells provides a rational approach for treatment of this aggressive malignancy.
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Waldmann, TA, JD White, CK Goldman, L. Top, A. Grant, R. Bamford, E. Roessler, ID Horak, S. Zaknoen, and C. Kasten-Sportes. "The interleukin-2 receptor: a target for monoclonal antibody treatment of human T-cell lymphotrophic virus I-induced adult T-cell leukemia." Blood 82, no. 6 (September 15, 1993): 1701–12. http://dx.doi.org/10.1182/blood.v82.6.1701.bloodjournal8261701.
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Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotrophic virus-I (HTLV-I). It is an aggressive leukemia with an overall mortality rate of 50% within 5 months; no conventional chemotherapy regimen appears successful in inducing long-term disease-free survival in ATL patients. However, ATL cells constitutively express high-affinity interleukin-2 receptors (IL-2Rs) identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference in receptor expression, we administered anti-Tac intravenously (IV) to 19 patients with ATL. In general the patients did not suffer untoward reactions, and in 18 of 19 cases did not have a reduction in normal formed elements of the blood. Seven patients developed remissions that were mixed (1 patient), partial (4 patients), or complete (2 patients), with partial and complete remissions lasting from 9 weeks to more than 3 years as assessed by routine hematologic tests, immunofluorescence analysis, and molecular genetic analysis of T-cell receptor gene rearrangements and of HTLV-I proviral integration. Furthermore, remission was associated with a return to normal serum calcium levels and an improvement of liver function tests. Remission was also associated in some cases with an amelioration of the profound immunodeficiency state that characterizes ATL. Thus the use of a monoclonal antibody that blocks the interaction of IL-2 with its receptor expressed on ATL cells provides a rational approach for treatment of this aggressive malignancy.
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Rogawski, Michael A. "New Evidence Supporting a Role for T-Type Ca2+ Channels in Absence Epilepsy and in the Action of Ethosuximide." Epilepsy Currents 2, no. 2 (March 2002): 57. http://dx.doi.org/10.1111/j.1535-7597.2002.00020.x.
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Lack of the Burst Firing of Thalamocortical Relay Neurons and Resistance to Absence Seizures in Mice Lacking α1G T-Type Ca2+ Channels Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, Shin HS Neuron 2001;31:35–45 T-type Ca2+ currents have been proposed to be involved in the genesis of spike-and-wave discharges, a sign of absence seizures, but direct evidence in vivo to support this hypothesis has been lacking. To address this question, we generated a null mutation of the α1G subunit of T-type Ca2+ channels. The thalamocortical relay neurons of the α1G-deficient mice lacked the burst mode firing of action potentials, whereas they showed the normal pattern of tonic mode firing. The α1G-deficient thalamus was specifically resistant to the generation of spike-and-wave discharges in response to GABAB receptor activation. Thus, the modulation of the intrinsic firing pattern mediated by α1G T-type Ca2+ channels plays a critical role in the genesis of absence seizures in the thalamocortical pathway. Block of Cloned Human T-Type Calcium Channels by Succinimide Antiepileptic Drugs Gomora JC, Daud AN, Weiergraber M, Perez-Reyes E Mol Pharmacol 2001;60:1121–1132 Inhibition of T-type Ca2+ channels has been proposed to play a role in the therapeutic action of succinimide antiepileptic drugs. Despite the widespread acceptance of this hypothesis, recent studies using rat and cat neurons have failed to confirm inhibition of T-type currents at therapeutically relevant concentrations. The present study re-examines this issue using the three cloned human channels that constitute the T-type family: α1G, α1H, and α1I. The cloned cDNAs were stably transfected and expressed into mammalian cells, leading to the appearance of typical T-type currents. The results demonstrate that both ethosuximide and the active metabolite of methsuximide, α-methyl-α-phenylsuccinimide (MPS), block human T-type channels in a state-dependent manner, with higher affinity for inactivated channels. In contrast, succinimide analogs that are not anticonvulsive were relatively poor blockers. The apparent affinity of MPS for inactivated states of the three channels was estimated using two independent measures: K1 for α1G and α1I was 0.3 to 0.5 mM and for α1H was 0.6 to 1.2 mM. T-type channels display current at the end of long pulses (persistent current), and this current was especially sensitive to block (ethosuximide IC50 = 0.6 mM). These drugs also reduced both the size of the T-type window current region and the currents elicited by a mock low threshold spike. We conclude that succinimide antiepileptic drugs are capable of blocking human T-type channels at therapeutically relevant concentrations.
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Alam, M. Faiyaz, M. Azmat Rana, and M. Shamshad Alam. "Osteocalcin, a promising marker of osteoporosis: evaluation in post-menopausal females with osteoporosis." International Journal of Advances in Medicine 6, no. 6 (November 25, 2019): 1746. http://dx.doi.org/10.18203/2349-3933.ijam20194639.
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Background: Osteocalcin, has high affinity for calcium. In osteoporotic women, deficiency of calcium may lead to lowering of the formation of hydroxyapatite crystals. Thus, in the state of hypo mineralization, free osteocalcin available in the circulation. Therefore, present study was designed to evaluate significance of serum osteocalcin in diagnosis of osteoporosis, and relationship between Serum Osteocalcin and BMD (Bone mineral Density) in post-menopausal females with osteoporosis and without osteoporosis.Methods: One hundred and forty seven post-menopausal women between age 45 to 80 years attending the hospital OPD were studied. To be eligible for the study they had to have been postmenopausal for at least one year. The diagnosis of osteoporosis was made based on T-Scores (BMD) at the lumber spine (L1 to L4 and femaral neck) by DEXA (GE lunar Densitometer). Serum osteocalcin level was estimated by LIAISON osteocalcin assay. Patients with chronic conditions affecting skeletal health and patients on drugs affecting the skeleton were excluded from the study.Results: Serum osteocalcin level in post-menopausal female without osteoporosis was 9.87±1.04ng/ml, while post-menopausal female with osteoporosis had 22.62±2.25ng/ml suggesting significant increase in bone marker level in osteoporotic females (p<0.05.) Correlation study between BMD and osteocalcin showed strong Negative Correlation (r=-0.77, p<0.05).Conclusions: Serum osteocalcin can be considered as a specific marker of osteoblast function as its levels have been shown to correlate with bone formation rates. Thus, serum osteocalcin can be used for diagnosis and monitoring of response to therapy and this may be the better predictor than BMD.
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Liu, C. C., B. Perussia, Z. A. Cohn, and J. D. Young. "Identification and characterization of a pore-forming protein of human peripheral blood natural killer cells." Journal of Experimental Medicine 164, no. 6 (December 1, 1986): 2061–76. http://dx.doi.org/10.1084/jem.164.6.2061.
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We show here that human peripheral blood NK cells contain a pore-forming protein (PFP) with an Mr of 70,000-72,000 that assembles structural lesions (with an average internal diameter of 150-170 A) and forms functional channels. The PFP was isolated by affinity chromatography from human NK cells, using a specific anti-C9 antiserum as the immunoadsorbent. The NK cells were isolated from PBL by positive or negative selection by indirect rosetting using a panel of monoclonal antibodies directed against different NK and T cell surface antigens. PFP was identified in NK cells freshly isolated and isolated from cultured PBL, both stimulated with interleukin 2, but not in NK cell-depleted lymphocytes. In planar bilayers, the channels formed by the NK cell-derived PFP are highly voltage resistant, with most channels persisting in the open state once they have inserted into the bilayer. The unit conductances of these channels range 0.3-1 nS in 0.1 M NaCl. The channels show poor selectivity for monovalent and divalent ions. The PFP is also released from human NK cells stimulated with the calcium ionophore A23187, suggesting that this protein, like the one produced by murine CTL lines, may be similarly secreted during cell-mediated killing. Its identification in primary human NK cell cultures indicates that this protein may play an active role in NK cell-mediated killing.
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Dasgupta, Swapan Kumar, Qi Da, Anhquyen Le, Miguel A. Cruz, and Perumal Thiagarajan. "Wdr1-Mediated Actin Reorganization Is Essential for Integrin αIIbβ3 Activation in Platelets." Blood 126, no. 23 (December 3, 2015): 2231. http://dx.doi.org/10.1182/blood.v126.23.2231.2231.
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Abstract In resting platelets, the heterodimeric integrin αIIbβ3 is present in a low-affinity state. During platelet activation, the intracytoplasmic signals induce conformational changes that results in a swung-out conformation of the extracellular domain competent to bind ligands such as fibrinogen with high affinity to mediate platelet aggregation. Actin turnover is essential for this process and dynamic assembly and disassembly of actin filaments regulate it. We have identified Wdr1, a cofilin and actin binding protein containing WD40 repeats, as an essential component of the machinery that orchestrates actin fiber reorganization that leads to integrin αIIbβ3 activation. Methods: Wdr1-deficient mouse strain, Wdr1rd/rd. was obtained through an N-ethyl-N-nitrosourea mutagenesis screen in Baylor College of Medicine. The mutant mouse has a T>A transversion in the second dinucleotide of the intron 9 splice donor site and it produces a mutant transcript containing a 6-bp in-frame deletion that results in a incorrectly folded, nonfunctional protein. Normal splicing produces a small amount of Wdr1 protein (~2%) resulting in a hypomorphic allele. Wdr1-deficient mice are moderately thrombocytopenic (85± 11 x 106 ml Wdr1 deficient versus 427± 52 x 106/ml for wild-type). Platelets were isolated from Wdr1-deficient and control mice. Platelet aggregation was carried out by standard turbidometric methods. Calcium mobilization was measured by incubating Wdr1-deficient and WT (wild-type) platelets with Fura 2 AM and measuring the Fura 2 fluorescence after collagen treatment. Conformational change in αIIbβ3 was determined by flow cytometry with a conformation-specific anti-αIIbβ3 antibody JON/A. In vivo hemostasis was assessed by tail bleeding time and FeCl3-induced endothelial carotid injury/thrombosis model was used to assess the occlusion in carotid artery of mice. Results: Aggregation response of Wdr1-deficient platelets to different doses of collagen was significantly impaired compared to WT platelets. Under similar conditions, the calcium response was similar to the WT. In a parallel-plate flow chamber assay, WT platelets stably adhered to collagen surface and formed stable thrombus. On the other hand, significantly less number of Wdr1 deficient platelets were stably attach to the collagen surface and it did not form stable thrombus. As expected the tail bleeding time of Wdr1 deficient mice is significantly prolonged (> 10 minutes) compared to WT mice (<2 min). In vivo, in FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1 deficient was prolonged significantly compared wild type mice (15.8 ± 12.6 minutes versus 9.0 ± 10.5 minutes (p=0.041, Mann-Whitney non parametric comparison). To examine directly the activation of αIIbβ3, we used JON/A antibody, which selectively binds to activated αIIbβ3 integrins on mouse platelets. Binding of collagen treated Wdr1-deficient platelets to JON/A as determined by flow cytometry, is significantly less compared to WT platelets (6.1±0.3 fluorescence units (FU) versus 17.4±0.6 FU, p≤0.05) indicating impaired inside-out activation of αIIbβ3. Since, Wdr1 promotes actin disassembly, which is essential for the rearrangement of the actin fibers that occurs during platelet activation, we measured actin turn over by measuring F-actin and G-actin ratios of collagen treated platelets at various time points. Actin turnover is highly impaired in Wdr1 deficient platelets compared to WT platelets. Furthermore, integrin αIIbβ3 association with actin cytoskeleton was markedly impaired in Wdr1 deficient mice compared to their WT controls. These studies show that Wdr1 mediated actin cytoskeleton reorganization is essential for integrin αIIbβ3 activation. Disclosures No relevant conflicts of interest to declare.
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Fischer, Charlotte Froese, and Tomas Brage. "Contributions to the electron affinity of calcium and scandium." Canadian Journal of Physics 70, no. 12 (December 1, 1992): 1283–90. http://dx.doi.org/10.1139/p92-209.
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Theoretical predictions of the electron affinity of Ca vary from 0 to 130 meV. Not all calculations have included the same effects. In this paper, the different approaches are reviewed, the effect of assumptions estimated whenever possible, and some new ab initio results reported that estimate the effect of core polarization on electron affinity for both Ca and Sc. For the latter our predicted electron affinity is in good agreement with the experimental value for the lowest 4s23d4p1D state and underestimates the electron affinity for 4s23d4p3D, where the calculation of outer correlation is more demanding and the core-polarization effect is small.
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Chandrasekera, P. Charukeshi, Margaret E. Kargacin, Julie P. Deans, and Jonathan Lytton. "Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3." American Journal of Physiology-Cell Physiology 296, no. 5 (May 2009): C1105—C1114. http://dx.doi.org/10.1152/ajpcell.00650.2008.
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The sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca2+ concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca2+; however, Ca2+ affinity of native SERCA3 in an endogenous setting has not been examined. Such a measurement is complicated, because SERCA3 is always coexpressed with the housekeeping isoform SERCA2b. We used a fluorescence-based assay for monitoring continuous Ca2+ uptake into microsomes to examine the properties of endogenous human SERCA3 and SERCA2b. The kinetic parameters were derived using a cooperative two-component uptake model for Ca2+ activation, and the values assigned to SERCA3 were confirmed using the highly specific human SERCA3 inhibitory antibody PL/IM430. First, using recombinant human SERCA3 and SERCA2b proteins transiently expressed in HEK-293 cells, we confirmed the previously observed low apparent Ca2+ affinity for SERCA3 compared with SERCA2b (1.10 ± 0.04 vs. 0.26 ± 0.01 μM), and using mixtures of recombinant protein isoforms, we validated the two-component uptake model. Then we determined apparent Ca2+ affinity for SERCA proteins present endogenously in cultured Jurkat T lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca2+ affinity in these two preparations was 1.04 ± 0.07 and 1.1 ± 0.2 μM for SERCA3 and 0.27 ± 0.02 and 0.26 ± 0.01 μM for SERCA2b, respectively. Our data demonstrate, for the first time, that affinity for Ca2+ is inherently lower for SERCA3 expressed in situ than for other SERCA isoforms.
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Singh, Nathan, Noelle V. Frey, Boris Engels, David M. Barrett, Olga Shestova, Pranali Ravikumar, Amy Shyu, et al. "Single Chain Variable Fragment Linker Length Regulates CAR Biology and T Cell Efficacy." Blood 134, Supplement_1 (November 13, 2019): 247. http://dx.doi.org/10.1182/blood-2019-131024.
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We recently conducted a clinical trial of CD22-directed chimeric antigen receptor (CAR) T cells in children and adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). While we did observe some transient responses, overall outcomes were inferior to another recent trial of CD22 CAR T cells in ALL performed at the NCI (Fry, T.J. et al. Nat Med, 2018). Intriguingly, these trials used a CAR that employed the same antigen-binding and intracellular signaling domains, and differed only in the length of linker connecting the variable regions of the single chain variable fragment (scFv). Based on these clinical observations, we sought to identify how the scFv linker impacts CAR biology and regulates CAR-driven T cell activity. The University of Pennsylvania's CD22 CAR contained a long 20 amino acid scFv linker ("CAR22-L") while the NCI's CAR had a 5 amino acid linker ("CAR22-S"). We began by investigating the impact of linker length on CAR biochemistry. Both CAR22-L and CAR22-S had similar antigen-binding affinities (KD of 1.67nM and 6.05nM, respectively). Chromatography revealed that CAR22-L remained monomeric in solution while CAR22-S formed homodimers. To explore how dimerization influenced surface-membrane biology, we developed GFP-tagged versions of each CAR and performed confocal microscopy on CAR+ T cells. CAR22-L exhibited homogenous surface membrane expression, while CAR22-S appeared to self-aggregate and cluster (Fig. 1a). We investigated the impact of this clustering on receptor signaling and found that CAR22-S demonstrated high levels of signaling molecule activation (i.e. Akt, p70-S6 and STAT3) in the absence of antigen engagement. This is consistent with previous reports establishing that CAR clustering can lead to tonic signaling (Long, A.H. et al. Nat Med, 2015). Importantly, this tonic signaling did not lead to autonomous T cell proliferation. We proceeded to evaluate how clustering and tonic signaling impacted CAR function upon antigen engagement. Microscopic evaluation of CAR T cells combined with CD22+ Nalm6 cells revealed greater actin and microtubule organizing complex polarization (P = 0.02 and 0.01, respectively) in CAR22-S cells, consistent with superior immune synapse formation. This was accompanied by increased phosphorylation of PI3K, MAPK and calcium signaling proteins (Fig. 1b) after CAR engagement. RNA sequencing revealed significantly greater activation of immune response gene programs in CAR22-S cells as compared to CAR22-L after overnight exposure to Nalm6. We next investigated the impact that this enhanced receptor-driven activity had on CAR T cell anti-tumor function. CAR T cells were combined with Nalm6 in vitro and residual Nalm6 was serially quantified, revealing that CAR22-S mediated greater tumor control than CAR22-L, particularly at later time periods (P < 0.001). This was associated with greater secretion of IFNg, IL-2 and TNFa (all P < 0.001). Finally, we compared anti-tumor efficacy in xenograft models of systemic Nalm6. NOD/SCID/cg-/- mice were engrafted with Nalm6 and received 1x106 CAR T cells 7 days later. CAR22-S demonstrated greater in vivo expansion (P < 0.0001) and enhanced control of systemic disease (Fig. 1c,P = 0.017), resulting in prolongation of animal survival (Fig. 1d,P = 0.013). Based on these observations, we have designed a novel, affinity-enhanced CD22 CAR and confirmed that shorter linker length improves anti-tumor activity of this CAR. T cells expressing this CAR are currently undergoing evaluation in a phase I clinical trial (ClinicalTrials.org Identifiers NCT03620058 and NCT02650414). Thus far, 4 children and 2 adults have been infused with manageable toxicity. Early outcomes are promising, with 67% achieving complete remission at day 28, compared to 50% in our original CART22 trials. In summary, by investigating the potential mechanisms for an apparent discrepancy in outcomes between two different clinical trials, we demonstrate that reducing the length of the scFv linker results in significant changes to CAR biochemistry that directly lead to antigen-independent receptor activity. In contrast to previously published data demonstrating that tonic signaling of CD28-costimulated CARs is detrimental to T cell function (Long, A.H. et al. Nat Med, 2015), we found that tonic signaling of 4-1BB-costimulated CARs may be beneficial, possibly by priming T cells for rapid response to antigen. Disclosures Singh: University of Pennsylvania: Patents & Royalties. Frey:Novartis: Research Funding. Engels:Novartis: Employment. Zhao:Novartis: Employment. Peng:Novartis: Employment. Granda:Novartis: Employment. Ramones:Novartis: Employment. Lacey:Novartis: Research Funding; Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding. Young:novartis: Research Funding. Brogdon:Novartis: Employment. Grupp:Roche: Consultancy; GSK: Consultancy; Novartis: Consultancy, Research Funding; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Maude:Novartis: Consultancy; Kite: Consultancy. Gill:Novartis: Research Funding; Tmunity Therapeutics: Research Funding; Carisma Therapeutics: Research Funding; Amphivena: Consultancy; Aro: Consultancy; Intellia: Consultancy; Sensei Bio: Consultancy; Carisma Therapeutics: Equity Ownership. Ruella:AbClon: Membership on an entity's Board of Directors or advisory committees; Nanostring: Consultancy, Speakers Bureau; Novartis: Patents & Royalties: CART for cancer.
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Bruno, Stefano, Maria Bonaccio, Stefano Bettati, Claudio Rivetti, Cristiano Viappiani, Stefania Abbruzzetti, and Andrea Mozzarelli. "High and low oxygen affinity conformations of T state hemoglobin." Protein Science 10, no. 11 (December 31, 2008): 2401–7. http://dx.doi.org/10.1110/ps.20501.
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Martinez, Ryan J., Rakieb Andargachew, Hunter Martinez, and Brian D. Evavold. "Absence of preferential high-affinity TCR expansion during CD4 T cell responses." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 133.27. http://dx.doi.org/10.4049/jimmunol.196.supp.133.27.
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Abstract Current views assume higher-affinity T cells, as identified by pMHCII tetramers, possess enhanced capabilities while lower-affinity T cells are inconsequential to the immune response. We set out to formally address the contribution of low-affinity T cells to the expanded repertoire. To perform this study we employed a micropipette based assay and T cell receptor signaling functional reporter to quantify the total number of antigen-specific CD4 T cells from naïve precursors through their expansion to antigen. In the naïve state, an in vivo limiting dilution assay revealed hundreds more precursor T cells than defined by pMHCII tetramers, finding high-affinity T cells comprise only 5–30% of the total naïve repertoire. Upon immunization, low-affinity T cells expand similarly to higher-affinity counterparts, demonstrating no affinity maturation or preferential expansion of the highest-affinity T cells as the immune response progressed. These findings demonstrate affinity diversity of CD4 T cells is maintained without a selective shift favoring the highest-affinity cells.
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Sundholm, Dage, and Jeppe Olsen. "Core—valence correlation effects on the ground state electron affinity of calcium." Chemical Physics Letters 217, no. 4 (January 1994): 451–55. http://dx.doi.org/10.1016/0009-2614(93)e141s-z.
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NYITRAI, Miklós, Andrew G. SZENT-GYÖRGYI, and Michael A. GEEVES. "A kinetic model of the co-operative binding of calcium and ADP to scallop (Argopecten irradians) heavy meromyosin." Biochemical Journal 365, no. 1 (July 1, 2002): 19–30. http://dx.doi.org/10.1042/bj20020099.
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Analysis of the kinetics of ATP and ADP binding to scallop (Argopecten irradians) heavy meromyosin (HMM) showed that the only calcium-dependent process is the rate of ADP release. At physiological ionic strength calcium accelerated ADP release about 20-fold. Notably in the absence of calcium only one ADP bound HMM, with an affinity of 0.5–1μM. The second nucleotide site remained unoccupied at up to 50μM ADP yet could bind ATP rapidly. The calcium dependence of ADP-release rates showed that calcium binds co-operatively to scallop HMM with an affinity of 0.78μM and a Hill coefficient of 1.9. Detailed interpretation of the data suggests that HMM exists in equilibrium between the on and off states and that calcium and ADP modulate the equilibrium between the two states. The on state is favoured in the presence of calcium and in the absence of both calcium and nucleotide. The off state is favoured by ADP (or ADP·Pi) in the absence of calcium. A detailed co-operative model of the interaction of ADP and calcium with HMM is presented.
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Batters, Christopher, Dario Brack, Heike Ellrich, Beate Averbeck, and Claudia Veigel. "Calcium can mobilize and activate myosin-VI." Proceedings of the National Academy of Sciences 113, no. 9 (January 25, 2016): E1162—E1169. http://dx.doi.org/10.1073/pnas.1519435113.
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The ability to coordinate the timing of motor protein activation lies at the center of a wide range of cellular motile processes including endocytosis, cell division, and cancer cell migration. We show that calcium dramatically alters the conformation and activity of the myosin-VI motor implicated in pivotal steps of these processes. We resolved the change in motor conformation and in structural flexibility using single particle analysis of electron microscopic data and identified interacting domains using fluorescence spectroscopy. We discovered that calcium binding to calmodulin increases the binding affinity by a factor of 2,500 for a bipartite binding site on myosin-VI. The ability of calcium-calmodulin to seek out and bridge between binding site components directs a major rearrangement of the motor from a compact dormant state into a cargo binding primed state that is nonmotile. The lack of motility at high calcium is due to calmodulin switching to a higher affinity binding site, which leaves the original IQ-motif exposed, thereby destabilizing the lever arm. The return to low calcium can either restabilize the lever arm, required for translocating the cargo-bound motors toward the center of the cell, or refold the cargo-free motors into an inactive state ready for the next cellular calcium flux.
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Goenka, Radhika, Andrew H. Matthews, Bochao Zhang, Patrick J. O’Neill, Jean L. Scholz, Thi-Sau Migone, Warren J. Leonard, William Stohl, Uri Hershberg, and Michael P. Cancro. "Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation." Journal of Experimental Medicine 211, no. 1 (December 23, 2013): 45–56. http://dx.doi.org/10.1084/jem.20130505.
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We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21–mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (TFH cells) provide a local source of BLyS. Whereas T cell–derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on TFH-derived BLyS for their persistence.
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Lu, You, and Ming Li. "A New Computer Model for Evaluating the Selective Binding Affinity of Phenylalkylamines to T-Type Ca2+ Channels." Pharmaceuticals 14, no. 2 (February 10, 2021): 141. http://dx.doi.org/10.3390/ph14020141.
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To establish a computer model for evaluating the binding affinity of phenylalkylamines (PAAs) to T-type Ca2+ channels (TCCs), we created new homology models for both TCCs and a L-type calcium channel (LCC). We found that PAAs have a high affinity for domains I and IV of TCCs and a low affinity for domains III and IV of the LCC. Therefore, they should be considered as favorable candidates for TCC blockers. The new homology models were validated with some commonly recognized TCC blockers that are well characterized. Additionally, examples of the TCC blockers created were also evaluated using these models.
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Ashwell, J. D., R. J. Robb, and T. R. Malek. "Proliferation of T lymphocytes in response to interleukin 2 varies with their state of activation." Journal of Immunology 137, no. 8 (October 15, 1986): 2572–78. http://dx.doi.org/10.4049/jimmunol.137.8.2572.
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Abstract The interleukin 2 (IL 2) receptor on T lymphocytes can be upregulated by a variety of stimuli including antigen, lectin, and IL 2 itself. In this report, the direct binding of radiolabeled IL 2 and a quantitative bioassay of T cell responsiveness to IL 2 were used to determine the biological significance of upregulation of the murine IL 2 receptor. Antigen and lectin, and to a lesser extent IL 2, were found to cause an increase in the expression of the high affinity form of the IL 2 receptor on both a T cell clone and concanavalin A-induced T cell blasts. A 2-day stimulation with antigen resulted in an increase in the sensitivity of the T cell clone to IL 2, whereas activation with IL 2 caused a decrease in the sensitivity of these cells to subsequent stimulation with IL 2. Comparison of the direct binding and the functional data revealed that IL 2-preactivated T cells required a greater number of occupied high affinity IL 2 receptors to achieve a given fractional response than did unactivated T cells. These observations suggest that the sensitivity with which a T cell responds to IL 2 is not determined solely by the number of high affinity IL 2 receptors it bears.
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Bouvard, D., A. Molla, and M. R. Block. "Calcium/calmodulin-dependent protein kinase II controls alpha5beta1 integrin-mediated inside-out signaling." Journal of Cell Science 111, no. 5 (March 1, 1998): 657–65. http://dx.doi.org/10.1242/jcs.111.5.657.
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Fibronectin binding on alpha5beta1 integrin is strictly dependent on intracellular calcium. Using an in vitro assay, we previously found that either calcineurin inhibitors or a blocking calcineurin monoclonal antibody added to cell lysates completely abolished the fibronectin/integrin interaction, which suggested that the activity of calcineurin, a calcium/calmodulin-dependent phosphatase, was required to counteract some kinase activity and maintain the high affinity state of alpha5beta1. In this paper, we show that blocking of the calcium/calmodulin kinase II (CaMKII) activity with the specific inhibitor KN-62 or with its pseudosubtrate Autocamtide-2 preserved the high affinity state of the integrin even under experimental conditions that inhibit calcineurin. Conversely, the addition of purified CaMKII to the cell lysate inhibited alpha5beta1 binding to fibronectin in vitro. Consistent with these results, cell adhesion on fibronectin was stimulated by KN-62. Moreover, Scatchard analysis of fibronectin binding on CHO cells revealed that KN-62 decreased the Kd value from 0.3 to 0.05 microM. Finally the expression of exogenous constitutively active CaMKII resulted in a dramatic defect in cell adhesion with no significant modification in alpha5beta1 cell surface expression. In summary our results demonstrate that CaMKII controls the affinity state of the integrin alpha5beta1 in vitro and in living cells.
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Serrano, Jose R., Edward Perez-Reyes, and Stephen W. Jones. "State-Dependent Inactivation of the α1g T-Type Calcium Channel." Journal of General Physiology 114, no. 2 (August 1, 1999): 185–202. http://dx.doi.org/10.1085/jgp.114.2.185.
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We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the α1G channel, with symmetrical Na+i and Na+o and 2 mM Ca2+o. After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential −100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from −120 to −70 mV \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{e-fold\;for}}\;31\;{\mathrm{mV}};\;{\mathrm{{\tau}}}\;=\;2.5\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{\mathrm{{\tau}}}\;=\;12{\raisebox{1mm}{\line(1,0){6}}}17\;{\mathrm{ms\;from}}-40\;{\mathrm{to}}\;+60\;{\mathrm{mV}}\end{equation*}\end{document}. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100–300 ms, inactivation was strong but incomplete (∼98%). Inactivation was also produced by long, weak depolarizations \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{{\tau}}}\;=\;220\;{\mathrm{ms\;at}}\;-80\;{\mathrm{mV}};\;{\mathrm{V}}_{1/2}\;=\;-82\;{\mathrm{mV}})\end{equation*}\end{document}, which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{{\tau}}}\;=\;85\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but weakly voltage dependent. Recovery was similar after 60-ms steps to −20 mV or 600-ms steps to −70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at −100 mV during recovery from inactivation, consistent with ≤8% of the channels recovering through the open state. The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate. One consequence of state-dependent inactivation is that α1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.
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Wong, Wing Ki, Claire Marks, Jinwoo Leem, Alan P. Lewis, Jiye Shi, and Charlotte M. Deane. "TCRBuilder: multi-state T-cell receptor structure prediction." Bioinformatics 36, no. 11 (March 17, 2020): 3580–81. http://dx.doi.org/10.1093/bioinformatics/btaa194.
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Abstract Motivation T-cell receptors (TCRs) are immune proteins that primarily target peptide antigens presented by the major histocompatibility complex. They tend to have lower specificity and affinity than their antibody counterparts, and their binding sites have been shown to adopt multiple conformations, which is potentially an important factor for their polyspecificity. None of the current TCR-modelling tools predict this variability which limits our ability to accurately predict TCR binding. Results We present TCRBuilder, a multi-state TCR structure prediction tool. Given a paired αβTCR sequence, TCRBuilder returns a model or an ensemble of models covering the potential conformations of the binding site. This enables the analysis of structurally driven polyspecificity in TCRs, which is not possible with existing tools. Availability and implementation http://opig.stats.ox.ac.uk/resources. Contact deane@stats.ox.ac.uk Supplementary information Supplementary data are available at Bioinformatics online.
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Shakiba, Mojdeh, Mary Philip, Ellen Horste, Steven Camara, and Andrea Schietinger. "Impact of antigen affinity on T cell dysfunction in solid tumors." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 141.1. http://dx.doi.org/10.4049/jimmunol.198.supp.141.1.
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Abstract Although tumor-specific T cells (TST) are found in human solid tumors, cancers progress, indicating that these T cells are dysfunctional. TSTs express high levels of inhibitory receptors and fail to execute effector functions; however, the regulatory mechanisms underlying TST dysfunction remain poorly defined. T cell mediated immune response is triggered by T cell receptor (TCR) binding to peptide-major histocompatibility (pMHC) complex on the surface of cells. The affinity of TCR:pMHC interaction is a critical determinant of T cell expansion and effector function in acute infections, where T cells with high affinity interactions generally show superior function. However, little is known about how TCR:pMHC affinity impacts T cells activation, induction of dysfunction and susceptibility to immunotherapy in progressing tumors. To elucidate this, we generated tumor cell lines expressing altered peptide ligands derived from SV40 large T antigen epitope I (Tag) and recognized by Tag-specific transgenic CD8 T cells (TCRTag) with varying functional avidity. We found that tumor-infiltrating TCRTag encountering low and high affinity tumor antigens were equally activated and expressed similar levels of inhibitory receptors, suggesting that even very weak TCR ligations can induce a typical “exhaustion” phenotype. Strikingly, while high affinity TCR:pMHC interactions led to complete loss of cytokine production, T cells with low affinity interactions remained functional, suggesting that TSTs with high affinity TCR:pMHC interactions enter a profound state of dysfunction. Future experiments will test the impact of tumor antigen affinity on the efficacy of therapeutic reprogramming, with important implications for immunotherapy.
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NYITRAI, Miklos, Andrew G. SZENT-GYÖRGYI, and Michael A. GEEVES. "Interactions of the two heads of scallop (Argopecten irradians) heavy meromyosin with actin: influence of calcium and nucleotides." Biochemical Journal 370, no. 3 (March 15, 2003): 839–48. http://dx.doi.org/10.1042/bj20021519.
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We recently proposed a co-operative model for the influence of calcium and ADP on scallop (Argopecten irradians) muscle heavy meromyosin (scHMM), in which scHMM exists in two conformations (designated ‘off’ and ‘on'), and calcium and ADP are allosteric effectors of the equilibrium between the off and on conformations [Nyitrai, Szent-Gyorgyi and Geeves (2002) Biochem. J. 365, 19—30]. Here we examine the influence of actin on scHMM. In the absence of nucleotide, both heads of scHMM bind very tightly to actin, independent of the presence of calcium. In the absence of calcium, ADP dissociates scHMM from actin completely, and little evidence of ternary complex formation can be found (actin affinity >20μM). The off state of scHMM therefore does not interact with actin. In the presence of calcium, ADP and actin lower each other's affinity for scHMM by 30—50-fold, although both heads remain strongly attached to actin (actin affinity 0.17μM). Detailed analysis suggests that the second head contributes far more to the overall binding energy than is the case for mammalian skeletal muscle HMM. This is consistent with a different stereochemical relationship between the two heads in scallop and mammalian HMM molecules.
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Strege, Peter R., Cheryl E. Bernard, Yijun Ou, Simon J. Gibbons, and Gianrico Farrugia. "Effect of mibefradil on sodium and calcium currents." American Journal of Physiology-Gastrointestinal and Liver Physiology 289, no. 2 (August 2005): G249—G253. http://dx.doi.org/10.1152/ajpgi.00022.2005.
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Interstitial cells of Cajal (ICC) generate the electrical slow wave. The ionic conductances that contribute to the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant Na+ current (NaV1.5) encoded by SCN5A contributes to the rising phase of the slow wave, whereas T-type Ca2+ currents have been reported from cultured mouse intestine ICC and also from canine colonic ICC. Mibefradil has a higher affinity for T-type over L-type Ca2+ channels, and the drug has been used in the gastrointestinal tract to identify T-type currents. However, the selectivity of mibefradil for T-type Ca2+ channels over ICC and smooth muscle Na+ channels has not been clearly demonstrated. The aim of this study was to determine the effect of mibefradil on T-type and L-type Ca2+ and Na+ currents. Whole cell currents were recorded from HEK-293 cells coexpressing green fluorescent protein with either the rat brain T-type Ca2+ channel α13.3b + β2, the human intestinal L-type Ca2+ channel subunits α1C + β2, or NaV1.5. Mibefradil significantly reduced expressed T-type Ca2+ current at concentrations ≥ 0.1 μM (IC50 = 0.29 μM), L-type Ca2+ current at > 1 μM (IC50 = 2.7 μM), and Na+ current at ≥ 0.3 μM (IC50 = 0.98 μM). In conclusion, mibefradil inhibits the human intestinal tetrodotoxin-resistant Na+ channel at submicromolar concentrations. Caution must be used in the interpretation of the effects of mibefradil when several ion channel classes are coexpressed.
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George, J., S. J. Penner, J. Weber, J. Berry, and J. L. Claflin. "Influence of membrane Ig receptor density and affinity on B cell signaling by antigen. Implications for affinity maturation." Journal of Immunology 151, no. 11 (December 1, 1993): 5955–65. http://dx.doi.org/10.4049/jimmunol.151.11.5955.
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Abstract We have initiated a series of experiments to explore the effect of changes in density of the surface Ig receptor (mIg) on Ag:mIg interactions. We transfected into the surface Ig-negative B cell line M12.4 H and L chain constructs known to effect a 10-fold change in antibody binding affinity for the naturally occurring hapten phosphocholine (PC). Two sets of stable transfectants were generated and those expressing levels of mIg comparable to the range normally seen on splenic B cells were studied. One set expressed an unmutated VH and an unmutated VL. The second set expressed the same pair of V regions except for a single somatic change in CDR3 of VH; this substitution increases the affinity of antibody for PC from 3 x 10(4) M-1 to 3 x 10(5) M-1. Ag:mIg interactions were assessed in the transfected cell lines by measuring calcium mobilization induced by stimulation with soluble PC Ag. As expected, the mutation that increased affinity for PC increased the sensitivity of transfectants to PC Ag. Relatively small changes in receptor number had a dramatic effect in the quantity and quality of a calcium response. Significantly, we found that Ag-specific signaling could occur with only a few thousand receptors per cell. Signaling differences were most noticeable with PC protein Ag (T-dependent form) compared with PC polysaccharide Ag (T-independent form). These results suggest that the down-regulation of mIg that follows B cell activation may have evolved to assist in the selection of B cell clones with higher affinity for Ag. Furthermore, the results also provide an explanation for why selection of higher affinity clones can occur with protein Ag but only poorly so with polymeric Ag.
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Faull, R. J., N. L. Kovach, J. M. Harlan, and M. H. Ginsberg. "Stimulation of integrin-mediated adhesion of T lymphocytes and monocytes: two mechanisms with divergent biological consequences." Journal of Experimental Medicine 179, no. 4 (April 1, 1994): 1307–16. http://dx.doi.org/10.1084/jem.179.4.1307.
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We show that the adhesion of T lymphoid cells to immobilized fibronectin can be increased by two distinct mechanisms. The first is by increasing the affinity of the fibronectin receptor/ligand interaction using the anti-beta 1 integrin monoclonal antibody 8A2. The second is by treating the cells with phorbol 12-myristate 13-acetate (PMA), which alters events that occur after receptor occupancy (e.g., cell spreading) without affecting receptor affinity. The effects of these two mechanisms on adhesion in the presence of physiological concentrations of soluble fibronectin suggest that they have different biological consequences. Under these conditions, the net effect of increasing the affinity of the fibronectin receptors is to decrease cell adhesion, whereas the increase in adhesion induced by PMA is unaffected. This suggests that the high affinity receptors are not primarily available for cell adhesion under these circumstances, and that they have an alternative function. We further show that high affinity binding of soluble fibronectin can be induced by either differentiation of the monocytic cell line THP-1 or by cross-linking the T cell receptor complexes on the T lymphoid cell line HUT-78. The differentiated monocytic cells express two populations of fibronectin receptors: a minority in a high affinity state, and the majority in a low affinity state. Thus they will both continue to adhere in the presence of physiological concentrations of soluble fibronectin and bind significant amounts of soluble fibronectin at the cell surface.
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Shibayama, Naoya, and Satoshi Saigo. "Direct observation of two distinct affinity conformations in the T state human deoxyhemoglobin." FEBS Letters 492, no. 1-2 (March 7, 2001): 50–53. http://dx.doi.org/10.1016/s0014-5793(01)02225-6.
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Balasubramanian, Moovarkumudalvan, Ponnuraj Sathya Moorthy, Kamariah Neelagandan, Ramya Ramadoss, Prasanna R. Kolatkar, and M. N. Ponnuswamy. "Structure of liganded T-state haemoglobin from cat (Felis silvestris catus), a low oxygen-affinity species, in two different crystal forms." Acta Crystallographica Section D Biological Crystallography 70, no. 7 (June 29, 2014): 1898–906. http://dx.doi.org/10.1107/s139900471400916x.
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Haemoglobin (Hb) is an iron-containing metalloprotein which plays a major role in the transportation of oxygen from the lungs to tissues and of carbon dioxide back to the lungs. Hb is in equilibrium between low-affinity tense (T) and high-affinity relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express `high' or `low' oxygen-affinity Hbs. Although Hbs from the former group have been studied extensively, a more limited number of structural studies have been performed for low oxygen-affinity Hbs. Here, the crystal structure of low oxygen-affinity cat methaemoglobin (metHb) has been solved at 2.0 and 2.4 Å resolution in two different crystal forms. Even though both structures are fully liganded, they unusually adopt a T-state-like quaternary conformation but with several localized R-like tertiary-structural and quaternary-structural features. The study provides atomic-level insights into the ligand-binding properties of this Hb, including its low cooperativity, blunt response to allosteric effectors and low affinity for oxygen, as well as further contributing to the mechanism underlying Hb allostery.
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Yokomizo, Takehiko, Kazuhiko Kato, Hiroshi Hagiya, Takashi Izumi, and Takao Shimizu. "Hydroxyeicosanoids Bind to and Activate the Low Affinity Leukotriene B4Receptor, BLT2." Journal of Biological Chemistry 276, no. 15 (January 18, 2001): 12454–59. http://dx.doi.org/10.1074/jbc.m011361200.
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Leukotriene B4, an arachidonate metabolite, is a potent chemoattractant of leukocytes involved in various inflammatory diseases. Two G-protein-coupled receptors for leukotriene B4have been cloned and characterized. BLT1 (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997)Nature387, 620–624) is a high affinity receptor exclusively expressed in leukocytes, and BLT2 (Yokomizo, T., Kato, K., Terawaki, K., Izumi, T., and Shimizu, T. (2000)J. Exp. Med.192, 421–432) is a low affinity receptor expressed more ubiquitously. Here we report the binding profiles of various BLT antagonists and eicosanoids to either BLT1 or BLT2 using the membrane fractions of Chinese hamster ovary cells stably expressing the receptor. BLT antagonists are grouped into three classes: BLT1-specific U-75302, BLT2-specific LY255283, and BLT1/BLT2 dual-specific ZK 158252 and CP 195543. We also show that 12(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroperxyeicosatetraenoic acid, and 15(S)-hydroxyeicosatetraenoic acid competed with [3H]LTB4binding to BLT2, but not BLT1, dose dependently. These eicosanoids also cause calcium mobilization and chemotaxis through BLT2, again in contrast to BLT1. These findings suggest that BLT2 functions as a low affinity receptor, with broader ligand specificity for various eicosanoids, and mediates distinct biological and pathophysiological roles from BLT1.
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COLOMER-PALLAS, Anne, Yannick PEREIRA, Marie-Françoise PETIT-GLATRON, and Régis CHAMBERT. "Calcium triggers the refolding of Bacillus subtilis chitosanase." Biochemical Journal 369, no. 3 (February 1, 2003): 731–38. http://dx.doi.org/10.1042/bj20021459.
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We characterized the reversible folding—unfolding transition of Bacillus subtilis exocellular chitosanase from either thermal or urea denaturation of the protein. The transitions were monitored in each case by intrinsic fluorescence changes and resistance to proteolysis. Unfolding and refolding kinetics and differential scanning calorimetry analysis suggested a two-state equilibrium. The equilibrium between the folded and unfolded states was rapidly displaced towards the folded state in the presence of a low concentration of calcium (2—20mM). The binding titration curve indicated that chitosanase possesses one weak Ca2+-binding site (with an equilibrium affinity constant, KA, of 0.3×103M-1). These results support the hypothesis that this metal ion, which is accumulated in the cell wall environment of B. subtilis, is an effector that influences folding and stability of newly translocated proteins.
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Johnson, D. E., and A. Hudmon. "Activation State-Dependent Substrate Gating in Ca2+/Calmodulin-Dependent Protein Kinase II." Neural Plasticity 2017 (2017): 1–13. http://dx.doi.org/10.1155/2017/9601046.
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Calcium/calmodulin-dependent protein kinase II (CaMKII) is highly concentrated in the brain where its activation by the Ca2+sensor CaM, multivalent structure, and complex autoregulatory features make it an ideal translator of Ca2+signals created by different patterns of neuronal activity. We provide direct evidence that graded levels of kinase activity and extent of T287(T286αisoform) autophosphorylation drive changes in catalytic output and substrate selectivity. The catalytic domains of CaMKII phosphorylate purified PSDs much more effectively when tethered together in the holoenzyme versus individual subunits. Using multisubstrate SPOT arrays, high-affinity substrates are preferentially phosphorylated with limited subunit activity per holoenzyme, whereas multiple subunits or maximal subunit activation is required for intermediate- and low-affinity, weak substrates, respectively. Using a monomeric form of CaMKII to control T287autophosphorylation, we demonstrate that increased Ca2+/CaM-dependent activity for all substrates tested, with the extent of weak, low-affinity substrate phosphorylation governed by the extent of T287autophosphorylation. Our data suggest T287autophosphorylation regulates substrate gating, an intrinsic property of the catalytic domain, which is amplified within the multivalent architecture of the CaMKII holoenzyme.
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Bugge, Jon, and Roy E. Weber. "Oxygen binding and its allosteric control in hemoglobin of the pulmonate snail, Biomphalaria glabrata." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 276, no. 2 (February 1, 1999): R347—R356. http://dx.doi.org/10.1152/ajpregu.1999.276.2.r347.
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Pulmonate snails that experience extreme variations in gas tensions and temperatures possess extracellular, high-molecular mass (∼1.7 × 106 Da) hemoglobins (Hbs) that are little known as regards oxygenation and allosteric characteristics. Biomphalaria glabrata hemolymph exhibits a high O2 affinity (half-saturation O2 tension = 6.1 mmHg; pH 7.7, 25°C), pronounced Bohr effect (Bohr factor = −0.5), and pH-dependent cooperativity (Hill’s cooperativity coefficient at half-saturation = 1.1–2.0). Divalent cations increase O2 affinity, Ca2+ exerting greater effect than Mg2+. Analyses in terms of the Monod-Wyman-Changeux model indicate novel O2 affinity control mechanisms. In contrast to vertebrate Hb, where organic phosphates and protons lower affinity via decreased O2association equilibrium constant of Hb in low-affinity state ( K T), and to extracellular annelid Hbs, where protons and cations primarily modulate O2 association equilibrium constant of Hb in high-affinity state ( K R), in B. glabrata Hb, the Bohr effect is mediated predominantly via K R and the cation effect via K T, reflecting preferential, oxygenation-linked proton binding to oxygenated Hb and cation binding to deoxygenated Hb. CO2 has no specific (pH independent) effect. Nonlinear van’t Hoff plots show temperature dependence of the overall heats of oxygenation, indicating oxy-deoxy heat capacity differences. The findings are related to possible physiological significance in pond habitats.
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Kang, Ho-Won, Iuliia Vitko, Sang-Soo Lee, Edward Perez-Reyes, and Jung-Ha Lee. "Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels." Journal of Biological Chemistry 285, no. 5 (November 23, 2009): 3271–81. http://dx.doi.org/10.1074/jbc.m109.067660.
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Mikolajewicz, Nicholas, Delaney Smith, Svetlana V. Komarova, and Anmar Khadra. "High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts." PLOS Computational Biology 17, no. 6 (June 21, 2021): e1008872. http://dx.doi.org/10.1371/journal.pcbi.1008872.
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The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10−4 and 10−2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.
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D’Souza-Schorey, Crislyn, Benjamin Boettner, and Linda Van Aelst. "Rac Regulates Integrin-Mediated Spreading and Increased Adhesion of T Lymphocytes." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 3936–46. http://dx.doi.org/10.1128/mcb.18.7.3936.
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ABSTRACT Leukocyte adhesion to the extracellular matrix (ECM) is tightly controlled and is vital for the immune response. Circulating lymphocytes leave the bloodstream and adhere to ECM components at sites of inflammation and lymphoid tissues. Mechanisms for regulating T-lymphocyte–ECM adhesion include (i) an alteration in the affinity of cell surface integrin receptors for their extracellular ligands and (ii) an alteration of events following postreceptor occupancy (e.g., cell spreading). Whereas H-Ras and R-Ras were previously shown to affect T-cell adhesion by altering the affinity state of the integrin receptors, no signaling molecule has been identified for the second mechanism. In this study, we demonstrated that expression of an activated mutant of Rac triggered dramatic spreading of T cells and their increased adhesion on immobilized fibronectin in an integrin-dependent manner. This effect was not mimicked by expression of activated mutant forms of Rho, Cdc42, H-Ras, or ARF6, indicating the unique role of Rac in this event. The Rac-induced spreading was accompanied by specific cytoskeletal rearrangements. Also, a clustering of integrins at sites of cell adhesion and at the peripheral edges of spread cells was observed. We demonstrate that expression of RacV12 did not alter the level of expression of cell surface integrins or the affinity state of the integrin receptors. Moreover, our results indicate that Rac plays a role in the regulation of T-cell adhesion by a mechanism involving cell spreading, rather than by altering the level of expression or the affinity of the integrin receptors. Furthermore, we show that the Rac-mediated signaling pathway leading to spreading of T lymphocytes did not require activation of c-Jun kinase, serum response factor, or pp70S6 kinase but appeared to involve a phospholipid kinase.
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Wang, Xiaohua, Xiuxu Chen, Lance Rodenkirch, William Simonson, Sarah Wernimont, Rachel M. Ndonye, Natacha Veerapen, et al. "Natural killer T-cell autoreactivity leads to a specialized activation state." Blood 112, no. 10 (November 15, 2008): 4128–38. http://dx.doi.org/10.1182/blood-2008-05-157529.
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Abstract Natural killer T (NKT) cells are innate-like T cells that recognize specific microbial antigens and also display autoreactivity to self-antigens. The nature of NKT-cell autoreactive activation remains poorly understood. We show here that the mitogen-activated protein kinase (MAPK) pathway is operative during human NKT-cell autoreactive activation, but calcium signaling is severely impaired. This results in a response that is biased toward granulocyte macrophage colony-stimulating factor (GM-CSF) secretion because this cytokine requires extracellular signal-regulated kinase (ERK) signaling but is not highly calcium dependent, whereas interferon-γ (IFN-γ), interleukin (IL)–4, and IL-2 production are minimal. Autoreactive activation was associated with reduced migration velocity but did not induce arrest; thus, NKT cells retained the ability to survey antigen presenting cells (APCs). IL-12 and IL-18 stimulated autoreactively activated NKT cells to secrete IFN-γ, and this was mediated by Janus kinase-signal transducers and activators of transcription (JAK-STAT)–dependent signaling without induction of calcium flux. This pathway did not require concurrent contact with CD1d+ APCs but was strictly dependent on preceding autoreactive stimulation that induced ERK activation. In contrast, NKT-cell responses to the glycolipid antigen α-galactosyl ceramide (α-GalCer) were dampened by prior autoreactive activation. These results show that NKT-cell autoreactivity induces restricted cytokine secretion and leads to altered basal activation that potentiates innate responsiveness to costimulatory cytokines while modulating sensitivity to foreign antigens.
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Goldman, D. W., L. A. Gifford, D. M. Olson, and E. J. Goetzl. "Transduction by leukotriene B4 receptors of increases in cytosolic calcium in human polymorphonuclear leukocytes." Journal of Immunology 135, no. 1 (July 1, 1985): 525–30. http://dx.doi.org/10.4049/jimmunol.135.1.525.
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Abstract The uptake of Quin-2 by human polymorphonuclear (PMN) leukocytes permitted accurate fluorimetric quantification of the cytosolic concentration of intracellular calcium [( Ca+2]in), without altering the expression of the two subsets of leukotriene B4 (LTB4) receptors, as assessed by the binding of [3H]LTB4. Chemotactic concentrations of LTB4 elicited a rapid increase in [Ca+2]in, which reached a peak within 0.6 to 1 min and then decayed back to baseline levels by 6 to 10 min. The maximal increase and the half-maximal increase in [Ca+2]in were achieved by LTB4 at mean concentrations of 5 X 10(-10) M and 2 X 10(-10) M, respectively, where the binding of LTB4 to high-affinity receptors predominates. A rank order of potency of LTB4 greater than 5(S),12(S)-6-trans-LTB4 greater than 12(S)-LTB4 was established for the elicitation of increases in [Ca+2]in, which reflects the binding of the isomers to low-affinity receptors. PMN leukocytes were preincubated with 10(-8) M LTB4 to induce chemotactic deactivation, which eliminates the expression of high-affinity receptors without altering the expression of the low-affinity receptors for LTB4. LTB4 elicited an increase in [Ca+2]in in the deactivated PMN leukocytes with an EC50 of 3 X 10(-8) M, which is similar to the Kd for LTB4 binding to the low-affinity receptors. Two lines of cultured human leukemic cells, IM-9 and HL-60, did not bind LTB4 specifically and did not show any change in [Ca+2]in upon the addition of 3 X 10(-8) M LTB4. The HL-60 human promyelocytic leukemia cell line was induced to differentiate in 1% dimethyl sulfoxide to leukocytes with more mature myelocytic characteristics. Differentiated HL-60 cells expressed an average of 54,000 low-affinity receptors for LTB4 per cell with an average dissociation constant of 7.3 X 10(-8) M and concurrently developed the capacity to respond to LTB4 with an increase in [Ca+2]in. The binding of LTB4 to either high-affinity or low-affinity receptors appears to be sufficient to initiate an increase in [Ca+2]in in human PMN leukocytes and differentiated HL-60 cells. The specificity of LTB4 receptors in transducing maximum increases in [Ca+2]in is determined by the subset of receptors that predominate as a result of the concentration of LTB4 and the state of the responding cells.
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Altiok, S., and E. Bermek. "Effects of mitogenic agents upon glucocorticoid action in human tonsillar T-lymphocytes." Bioscience Reports 10, no. 1 (February 1, 1990): 69–72. http://dx.doi.org/10.1007/bf01116853.
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The treatment of human tonsillar T-lymphocytes with 4-phorbol 12-myristate 13-acetate (PMA), resulted in about two fold increase in glucocorticoid receptor (GR) number, without any significant change in the receptor affinity. This increase disappeared in the presence of cycloheximide.Alone, PMA and calcium inophore A23187 did not affect, but together stimulated, like phytohaemagglutinin (PHA), leucine and, in particular, thymidine incorporation. PMA enhanced slightly the stimulatory effect of PHA. Alone, these agents failed to alter the suppressive effect of dexamethasone on thymidine and leucine incorporation; however, PMA-A23187 and PMA-PHA combinations appeared to antagonize the supression by dexamethasone.
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Stewart, M. P., C. Cabanas, and N. Hogg. "T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1." Journal of Immunology 156, no. 5 (March 1, 1996): 1810–17. http://dx.doi.org/10.4049/jimmunol.156.5.1810.
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Abstract Many leukocyte integrins require activation before they can adhere to their ligands. For example, stimulation of T cells enables the integrin LFA-1 to bind to ligand. This study compares two well known protocols for inducing T cell LFA-1 mediated adhesion to intercellular adhesion molecule-1 (ICAM)-1. We how that treatment with high concentrations of the divalent cation Mg2+ induces a high affinity state of LFA-1, which is reflected in the binding of soluble ICAM-1 and correlates with the expression of the epitope recognized by mAb 24. The second stimulation protocol with the phorbol ester phorbol-12,13-dibutyrate (PDBu) does not induce a high affinity state of LFA-1, and in this situation, adhesion is dependent on cell spreading and intracellular events involving protein kinase C, [Ca2+]i, and actin polymerization. These low affinity LFA-1 receptors are responsible for the initial contact with immobilized ligand because, unlike the Mg2+-stimulated receptors, adhesion is not blocked by soluble ICAM-1. Finally, we used a third method of inducing LFA-1-mediated adhesion by stimulation of T cells through the TCR/CD3 complex. This procedure, which is considered to be a more physiologic trigger for LFA-1 activation, resembles the phorbol ester protocol in that high affinity LFA-1 receptors are not induced and cell adhesion depends on involvement of the cytoskeleton and cell spreading.
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Timin, E. N., and S. Hering. "A method for estimation of drug affinity constants to the open conformational state of calcium channels." Biophysical Journal 63, no. 3 (September 1992): 808–14. http://dx.doi.org/10.1016/s0006-3495(92)81636-3.
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Loh, C., J. A. Carew, J. Kim, P. G. Hogan, and A. Rao. "T-cell receptor stimulation elicits an early phase of activation and a later phase of deactivation of the transcription factor NFAT1." Molecular and Cellular Biology 16, no. 7 (July 1996): 3945–54. http://dx.doi.org/10.1128/mcb.16.7.3945.
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We show here that NFAT1 is rapidly activated, then slowly deactivated, by stimulation of T cells through their antigen receptor. Within minutes of T-cell receptor stimulation, NFAT1 is dephosphorylated, translocates from the cytoplasm into the nucleus, and shows an increase in its ability to bind to DNA. These changes are dependent on calcium mobilization and calcineurin activation, since they are also elicited by ionomycin and are blocked by the immunosuppressive drug cyclosporin A. After several hours of T-cell receptor stimulation, the majority of the NFAT1 in the cell reverts to its original phosphorylated form, reappears in the cytoplasm, and again displays a low affinity for DNA. Deactivation of NFAT1 is facilitated by phorbol 12-myristate 13-acetate and inhibitors of capacitative calcium entry and most likely reflects the slow return of intracellular free calcium concentrations towards resting levels. Our results suggest that calcineurin-dependent signalling pathways mediate the early activation of NFAT1, while phorbol 12-myristate 13-acetate-dependent feedback pathways contribute to the late deactivation. Persistent NFAT-dependent cytokine gene transcription in activated T cells may be mediated by other NFAT family proteins in addition to NFAT1 during the immune response.
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Nelson, M. T., J. Woo, H. W. Kang, I. Vitko, P. Q. Barrett, E. Perez-Reyes, J. H. Lee, H. S. Shin, and S. M. Todorovic. "Reducing Agents Sensitize C-Type Nociceptors by Relieving High-Affinity Zinc Inhibition of T-Type Calcium Channels." Journal of Neuroscience 27, no. 31 (August 1, 2007): 8250–60. http://dx.doi.org/10.1523/jneurosci.1800-07.2007.
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Peelen, E., A. Muehler, D. Vitt, and H. Kohlhof. "P064 IMU-838, a Small Molecule DHODH Inhibitor in Phase 2 Clinical Trial for Ulcerative Colitis, Shows Potent Anti-inflammatory Activity in Cell-Culture-Based and In Vivo Systems." Journal of Crohn's and Colitis 16, Supplement_1 (January 1, 2022): i172—i173. http://dx.doi.org/10.1093/ecco-jcc/jjab232.193.
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Abstract Background IMU-838 (vidofludimus calcium) is a well-tolerated and orally available small molecule inhibitor of dihydroorotate dehydrogenase (DHODH) and is currently in phase 2 clinical development for ulcerative colitis (UC). Here, we investigated the effect of IMU-838 on different immune cell subsets and analysed the mechanism of action in more detail in T cells. Methods The effect of IMU-838 on regulatory macrophages (Mregs) was investigated by adding IMU-838 or anti-TNFα antibodies (infliximab, adalimumab), alone or in combination, in a mixed lymphocyte reaction assay, determining Mregs (CD206+CD14+) by FACS analysis. To investigate the effect of IMU-838 on human T and B cells, PBMC were stimulated with PHA (48h) or Oligonucleotide 2006-PTO (70h), respectively. An affinity-dependent effect of IMU-838 was assessed on CD8 T cells from OT-I and OT-III mice, containing a high and a low affinity T cell receptor (TCR), respectively, for OVA257-264 peptide. These cells were stimulated with ovalbumin (OVA) peptide loaded splenocytes or anti-CD3/anti-CD28 for 3 days. Oxidative phosphorylation (OXPHOS) and glycolysis in murine T cells was investigated by the Seahorse/Agilent technique in murine CD4 and CD8 T cells activated with anti-CD3/anti-CD28 with or without IMU-838. Results IMU-838 slightly induced Mregs, but strongly and dose-dependently decreased TNFα and IL-6 secretion. Anti-TNFα antibodies alone strongly induced Mregs, but also increased IL-6 levels. When IMU-838 was added, Mregs induction was even higher, and IL-6 was reduced. Besides reducing proinflammatory cytokines secreted by macrophages, IMU-838 also inhibits proliferation of activated B and T cells, as well as inflammatory T cell cytokine secretion, IL-17A, IL-17F and IFNγ, and increased apoptosis up to 3-fold compared to vehicle control. By investigating IMU-838’s role on affinity related activity status of T cells, it was shown that IMU-838 strongly inhibited cell proliferation in the peptide stimulated high affinity, but not in the low affinity, CD8 T cells. Peptide stimulated high affinity T cells show an upregulation of OXPHOS and glycolysis pathways, which are involved in the generation of the cell’s energy supply, compared to low affinity cells. IMU-838 strongly inhibited OXPHOS and glycolysis in both CD4 and CD8 T cells. Conclusion IMU-838 reduces the proinflammatory immune cell response by inducing Mregs, reducing pro-inflammatory cytokine secretion and reducing immune cell proliferation. DHODH is only important in cells that received a strong stimulus and are highly metabolically active. Therefore, IMU-838 will only specifically target these cells and will still allow for a normal immune response, representing a huge safety advantage for treatment of IBD patients.
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Otter, M., Th J. C. Van Berkel, and D. C. Rijken. "Binding and Degradation of Tissue-Type Plasminogen Activator by the Human Hepatoma Cell Line Hep G2." Thrombosis and Haemostasis 62, no. 02 (1989): 667–72. http://dx.doi.org/10.1055/s-0038-1646880.
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SummaryIn this study, binding and degradation of tissue-type plasminogen activator (t-PA) by the human hepatoma cell line Hep G2 was investigated. Binding at 4° C was time-dependent and reached a maximum after ca. 2 hours. Scatchard analysis of saturation experiments showed about 170,000 high affinity binding sites for t-PA per cell with an apparent Kd of 90 nM. These binding sites were calcium-dependent. Part of the binding to the hepatoma cells was non-saturable, owing to a large amount of low affinity binding sites which are at least partially located on the extracellular matrix of the cells. Competition with mannose- and galactose-terminated glycoproteins had no effect on total binding of 125I-t-PA. Degradation products of 125I-t-PA were found in the supernatant after a short lag phase and then increased linearly for at least 5 hours at 37° C. Degradation could be inhibited by chloroquine, NH4Cl and NaN3. We conclude that the human hepatoma cell line Hep G2 has a specific binding mechanism for t-PA which is not mediated by known carbohydrate receptor systems. Binding is followed by cellular uptake and degradation in the lysosomes.
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Bettati, Stefano, Laura D. Kwiatkowski, Jeffrey S. Kavanaugh, Andrea Mozzarelli, Arthur Arnone, Gian Luigi Rossi, and Robert W. Noble. "Structure and Oxygen Affinity of Crystalline des-His-146β Human Hemoglobin in the T State." Journal of Biological Chemistry 272, no. 52 (December 26, 1997): 33077–84. http://dx.doi.org/10.1074/jbc.272.52.33077.
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