Academic literature on the topic 'Calcium affinity T state'
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Journal articles on the topic "Calcium affinity T state"
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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.
Dissertations / Theses on the topic "Calcium affinity T state"
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Ding, Weixuan. "Syntheses of ternary oxyhydrates and oxides in the calcium-uranium system : stoichiometric influences on their structural affinity, precipitation mechanisms, and solid-state transformations." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/19431/.
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Calcium uranyl(VI) oxyhydrates and uranates are structurally related U(VI)-phases featuring uranium oxo-polyhedral sheets, with calcium ions occupying the interlayer. Both coordination environments appear throughout the nuclear fuel-cycle as alteration products, colloids, and sorption complexes. However, concerted studies spanning the aqueous precipitation mechanisms of uranyl(VI) oxyhydrates, their solid-state transformations, and structural relationships with uranates, have hitherto remained largely unexplored. A series of calcium-based uranyl(VI) oxyhydrates were precipitated via alkalisation of aqueous precursor solutions in titration and batch reactions. The bulk stoichiometric ratio of calcium to uranium (Ca/U) of precipitates was varied by modifying precursor stoichiometry, reaction temperature, or extraction pH. The rate of precipitation and its dependency on temperature was quantified in-situ using a quartz crystal microbalance. Novel insight was revealed on the mechanisms influencing nucleation and growth, by determining associated kinetic barriers as a function of precursor-Ca/U. Remarkably, as the bulk precipitate Ca/U increased from ~⅛ to unity, there was a transition from crystalline Becquerelite to primary or secondary amorphous phases, with uranate-like coordination environments. Formation of the latter was driven by solution alkalinity, and comprises a poorly-ordered matrix with occlusions of Ca2+-rich nano-clusters. A congruency limit lies Ca/U of ~1.5 Ca/U, whereupon discrete Portlandite crystallises. Solid-state transformation of all Ca2+-U(VI)-phases studied involved dehydration, dehydroxylation-decarbonation, and desorption processes. Associated kinetic barriers were catalysed by higher Ca2+-contents, and was reflected by reaction enthalpies for dehydration and desorption. Crystalline Becquerelite (~⅛ Ca/U) underwent amorphisation-crystallisation via partial egress of interlayer calcium, followed by reduction of β-UO3 to form a novel intercalation compound Ca0.18.α-U3O8. The endmember uranates Ca3U11O36, CaU2O7, Ca2U3O11, and CaUO4 crystallised from amorphous precursors with higher bulk Ca/U (~⅓, ~½, ~⅔, ~1), where Ca3U11O36 is a novel compound that is isostructural to (Pb/Sr)3U11O36. Nucleation and growth became predominant in the presence of Ca2+-rich occlusions. A higher Ca2+-loading facilitated the progressive ingress of interlayer-Ca2+, inducing a concerted axial compression in uranyl(VI) oxo-polyhedra towards the uranate-like coordination environment.
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McCormick, Patrick Neil. "Pre-clinical evaluation of [carbon-11]-(+)-PHNO as an agonist positron emission tomography (PET) radiotracer for imaging of the high-affinity state of the dopamine D2 receptor." 2006. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=442230&T=F.
Full textBooks on the topic "Calcium affinity T state"
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de Villiers, Rick. Eliot and Beckett's Low Modernism. Edinburgh University Press, 2021. http://dx.doi.org/10.3366/edinburgh/9781474479035.001.0001.
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Humility and humiliation have an awkward, often unacknowledged intimacy. Humility may be a queenly, cardinal, or monkish virtue, while humiliation points to an affective state at the extreme end of shame. Yet a shared etymology links the words to lowliness and, further down, to the earth. In ascetic traditions painfully aware of humanity’s quintessence – ‘dust thou art, and unto dust shalt thou return’ – humiliation cultivates humility. Like the terms in question, T. S. Eliot and Samuel Beckett share an imperfect likeness. Between them is a common interest in states of abjection, shame and suffering – and possible responses to such states. Tracing the relation between affect, ethics, and aesthetics, Low Modernism demonstrates how these two major modernists recuperate the affinity between humility and humiliation.
Book chapters on the topic "Calcium affinity T state"
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Teubl, Fabian, Katrin Schwank, Uli Ohmayer, Joachim Griesenbeck, Herbert Tschochner, and Philipp Milkereit. "Tethered MNase Structure Probing as Versatile Technique for Analyzing RNPs Using Tagging Cassettes for Homologous Recombination in Saccharomyces cerevisiae." In Ribosome Biogenesis, 127–45. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2501-9_8.
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AbstractMicrococcal nuclease (MNase) originating from Staphylococcus aureus is a calcium dependent ribo- and desoxyribonuclease which has endo- and exonucleolytic activity of low sequence preference. MNase is widely used to analyze nucleosome positions in chromatin by probing the enzyme’s DNA accessibility in limited digestion reactions. Probing reactions can be performed in a global way by addition of exogenous MNase, or locally by “chromatin endogenous cleavage” (ChEC) reactions using MNasefusion proteins. The latter approach has recently been adopted for the analysis of local RNA environments of MNasefusion proteins which are incorporated in vivo at specific sites of ribonucleoprotein (RNP) complexes. In this case, ex vivo activation of MNase by addition of calcium leads to RNA cleavages in proximity to the tethered anchor protein thus providing information about the folding state of its RNA environment.Here, we describe a set of plasmids that can be used as template for PCR-based MNase tagging of genes by homologous recombination in S. cerevisiae. The templates enable both N- and C-terminal tagging with MNase in combination with linker regions of different lengths and properties. In addition, an affinity tag is included in the recombination cassettes which can be used for purification of the particle of interest before or after induction of MNase cleavages in the surrounding RNA or DNA. A step-by-step protocol is provided for tagging of a gene of interest, followed by affinity purification of the resulting fusion protein together with associated RNA and subsequent induction of local MNase cleavages.
Conference papers on the topic "Calcium affinity T state"
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Ersdel, E., M. Andersson, and S. Rosen. "DETERMINATION OF SOLUBLE FIBRIN IN PLASMA WITH A CHR0M0GENIC KIT METHOD UTILIZING THE HIGH AFFINITY PLASMIN SUBSTRATE S-2390." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643058.
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A sensitive and quantitative assay of soluble fibrin is of clinically diagnostic relevance in an early thrombotic state where there is a risk for development of DIC. Recently Wiman and Ranby (Thromb. Haemostas 55, 189-193 (1986)) published a spectro-photometric assay which met these criterions. The single-stage assay procedure is based upon activation of Glu-Plasminogen to Plasmin by t-PA in the presence of soluble fibrin and hydrolysis of the chromogenic plasmin substrate S-2390, H-D-Val-Phe-Lys-pNA, which has a high affinity for plasmin. The rate of plasmin generation is correlated to the amount of soluble fibrin monomers present in the sample.A complete kit containing optimized, stable reagents has now been developed which allows a quantitative determination of soluble fibrin in the range 30-200 nmol/1 within 30 min. at room temperature (20-25°C). The assay procedure is straightforward involving addition of 200 pi diluted plasma sample to 200 pi Glu-Plasminogen and 100 ul of a t-PA/S-2390-reagent.The results show a high resolution of the standard curve as illustrated by a AA405 amounting to about one absorbance unit between a 200 nmol/1 sample of soluble fibrin and the reagent blank, some variation, ±0.1 absorbance unit, being caused mainly by differences in temperature. In combination with an intra-assay variation coefficient = 6.3% and 5.0% at 150 and 50 nmol/1, respectively, this will allow safe and reliable differentiation of pathological levels of soluble fibrin from levels found in healthy subjects (below 10 nmol/1). A similar precision is also obtained when the assay is performed in microplates.In the original procedure fresh frozen human plasma was utilized as a dilution medium for soluble fibrin. Comparisons with carefully collected bovine plasma proved this source to be a convenient substitute. Furthermore, lyophilization of the bovine plasma did not produce any significant degradation of fibrinogen which otherwise might interfere in the assay. This simple kit procedure should make it a suitable tool in early determinations of soluble fibrin in a number of pathological states which may result in severe haemostatic disturbances.
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Belin, D., D. Baccino, A. Wohlwend, A. Estreicher, J. Hurate, and J.-D. Vassalli. "A CELLULAR RECEPTOR FOR UROKINASE-TYPE PLASMINOGEN ACTIVATOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642957.
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Recent cell biological and biochemical studies on the urokinase-type plasminogen activator (u-PA) have revealed an unsuspected property of this protein: it binds with high affinity and specificity to the plasma membrane of a number of cell types. Hence, while the interaction of tissue-type plasminogen activator (t-PA) with fibrin suggests a preferred role for this enzyme in the maintenance of fluidity of the extracellular milieu, the cellular binding of u-PA results in the focalisation of plasmin generation to the close environment of the cell surface; this appears as an optimal configuration if u-PA is to participate in the enzymatic events required for cell migration.The available information on the cellular binding of u-PA can be summarized as follows:1. Human monocytes-macrophages, monocyte-like cell lines, fibroblasts, and a variety of other cell lines all express u-PA binding sites. The number of u-PA binding sites on a given cell type may vary as a function of the functional state of the cells. In some cases all sites are occupied by “endogenous” u-PA.2. Binding does not require u-PA activity, and prou-PA binds with the same affinity as does the active enzyme.3. The Kd for u-PA binding is between 1 and 10×10-10 M. The binding site appears to be specific for u-PA.4. Binding requires the presence of the A chain of u-PA; the growth factor module of the A chain is involved in this interaction.5. Bound enzyme does not dissociate readily, nor is it rapidly endocytosed; most importantly, it retains catalytic activity.Studies in progress are aimed at further defining the u-PA determinants responsible for binding. In this context it is noteworthy that there is a tight species specificity of binding: human and murine u-PA, for instance, bind only to cells of the homologous species. Characterization of the u-PA binding site suggests that it is an integral membrane protein that includes at least one Mf 50.000 polypeptide chain.In addition to allowing for the peri-cellular focalisation of u-PA catalysed proteolysis, expression of the u-PA binding site provides a mecanism whereby one cell type can acquire membrane-bound u-PA activity following secretion of the (pro)enzyme by another cell population. A striking example of this is the binding of u-PA, synthesized by the epithelial layer of the male genital tract, to the head region of murine spermatozoa.
Reports on the topic "Calcium affinity T state"
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Levy, Maggie, Raymond Zielinski, and Anireddy S. Reddy. IQD1 Function in Defense Responses. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7699842.bard.
Full textAbstract:
The main objective of the proposed research was to study IQD1's mechanism of action and elucidate its role in plant protection. Preliminary experiments suggest that IQD1 binds CaM in a Ca²⁺-dependent manner and functions in general defense responses. We propose to identify proteins and genes that interact with IQD1, which may provide some clues to its mechanism of action. We also plan to dissect IQD1's integration in defense pathways and to study and modulate its binding affinity to CaM in order to enhance crop resistance. Our specific objectives were: (1) Analysis of IQD1's CaM-binding properties; (2) Identification of IQD1 targets;(3) Dissection of IQD1 integration into defense signaling pathways. Analysis of IQD1's CaM-binding properties defined four potential classes of sequences that should affect CaM binding: one is predicted to raise the affinity for Ca²⁺-dependent interaction but have no effect on Ca²⁺-independent binding; a second is predicted to act like the first mutation but eliminate Ca²⁺-independent binding; a third has no predicted effect on Ca²⁺-dependent binding but eliminates Ca²⁺-independent binding; and the fourth is predicted to eliminate or greatly reduce both Ca²⁺-dependent and Ca²⁺-independent binding. Following yeast two hybrid analysis we found that IQD1 interact with AtSR1 (Arabidopsis thalianaSIGNALRESPONSIVE1), a calcium/calmodulin-binding transcription factor, which has been shown to play an important role in biotic and abiotic stresses. We tested IQD1 interaction with both N-terminal or C-terminal half of SR1. These studies have uncovered that only the N-terminal half of the SR1 interacts with the IQD1. Since IQD1 has an important role in herbivory, its interaction with SR1 suggests that it might also be involved in plant responses to insect herbivory. Since AtSR1, like IQD1, is a calmodulin-binding protein and the mutant showed increased sensitivity to a herbivore, we analyzed WT, Atsr1 and the complemented line for the levels of GS to determine if the increased susceptibility of Atsr1 plants to T. ni feeding is associated with altered GS content. In general, Atsr1 showed a significant reduction in both aliphatic and aromatic GS levels as compared to WT. In order to study IQD1's molecular basis integration into hormone-signaling pathways we tested the epistatic relationships between IQD1 and hormone-signaling mutants. For that purpose we construct double mutants between IQD1ᴼXᴾ and mutants defective in plant-hormone signaling and GS accumulation. Epitasis with SA mutant NahG and npr1-1 and JA mutant jar1-1 suggested IQD1 function is dependent on both JA and SA as indicated by B. cinerea infection assays. We also verified the glucosinolate content in the crosses siblings and found that aliphatic GSL content is reduced in the double transgenic plants NahG:IQD1ᴼXᴾ as compare to parental lines while the aliphatic GSL content in the npr1-1:IQD1ᴼXᴾ and jar1-1: IQD1ᴼXᴾ double mutants was intimidated to the parental lines. This suggests that GSL content dependency on SA is downstream to IQD1. As a whole, this project should contribute to the development of new defense strategies that will improve crop protection and reduce yield losses and the amount of pesticides required; these will genuinely benefit farmers, consumers and the environment.
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Horwitz, Benjamin A., and Barbara Gillian Turgeon. Fungal Iron Acquisition, Oxidative Stress and Virulence in the Cochliobolus-maize Interaction. United States Department of Agriculture, March 2012. http://dx.doi.org/10.32747/2012.7709885.bard.
Full textAbstract:
Our project focused on genes for high affinity iron acquisition in Cochliobolus heterostrophus, a necrotrophic pathogen of maize, and their intertwined relationship to oxidative stress status and virulence of the fungus on the host. An intriguing question was why mutants lacking the nonribosomal peptide synthetase (NRPS) gene (NPS6) responsible for synthesis of the extracellular siderophore, coprogen, are sensitive to oxidative stress. Our overall objective was to understand the mechanistic connection between iron stress and oxidative stress as related to virulence of a plant pathogen to its host. The first objective was to examine the interface where small molecule peptide and reactive oxygen species (ROS) mechanisms overlap. The second objective was to determine if the molecular explanation for common function is common signal transduction pathways. These pathways, built around sensor kinases, response regulators, and transcription factors may link sequestering of iron, production of antioxidants, resistance to oxidative stress, and virulence. We tested these hypotheses by genetic manipulation of the pathogen, virulence assays on the host plant, and by following the expression of key fungal genes. An addition to the original program, made in the first year, was to develop, for fungi, a genetically encoded indicator of redox state based on the commercially available Gfp-based probe pHyper, designed for animal cell biology. We implemented several tools including a genetically encoded indicator of redox state, a procedure to grow iron-depleted plants, and constructed a number of new mutants in regulatory genes. Lack of the major Fe acquisition pathways results in an almost completely avirulent phenotype, showing how critical Fe acquisition is for the pathogen to cause disease. Mutants in conserved signaling pathways have normal ability to regulate NPS6 in response to Fe levels, as do mutants in Lae1 and Vel1, two master regulators of gene expression. Vel1 mutants are sensitive to oxidative stress, and the reason may be underexpression of a catalase gene. In nps6 mutants, CAT3 is also underexpressed, perhaps explaining the sensitivity to oxidative stress. We constructed a deletion mutant for the Fe sensor-regulator SreA and found that it is required for down regulation of NPS6 under Fe-replete conditions. Lack of SreA, though, did not make the fungus over-sensitive to ROS, though the mutant had a slow growth rate. This suggests that overproduction of siderophore under Fe-replete conditions is not very damaging. On the other hand, increasing Fe levels protected nps6 mutants from inhibition by ROS, implying that Fe-catalyzed Fenton reactions are not the main factor in its sensitivity to ROS. We have made some progress in understanding why siderophore mutants are sensitive to oxidative stress, and in doing so, defined some novel regulatory relationships. Catalase genes, which are not directly related to siderophore biosynthesis, are underexpressed in nps6 mutants, suggesting that the siderophore product (with or without bound Fe) may act as a signal. Siderophores, therefore, could be a target for intervention in the field, either by supplying an incorrect signal or blocking a signal normally provided during infection. We already know that nps6 mutants cause smaller lesions and have difficulty establishing invasive growth in the host. Lae1 and Vel1 are the first factors shown to regulate both super virulence conferred by T-toxin, and basic pathogenicity, due to unknown factors. The mutants are also altered in oxidative stress responses, key to success in the infection court, asexual and sexual development, essential for fungal dissemination in the field, aerial hyphal growth, and pigment biosynthesis, essential for survival in the field. Mutants in genes encoding NADPH oxidase (Nox) are compromised in development and virulence. Indeed the triple mutant, which should lack all Nox activity, was nearly avirulent. Again, gene expression experiments provided us with initial evidence that superoxide produced by the fungus may be most important as a signal. Blocking oxidant production by the pathogen may be a way to protect the plant host, in interactions with necrotrophs such as C. heterostrophus which seem to thrive in an oxidant environment.