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1
Saedi, M. S., W. G. Conroy, and J. Lindstrom. "Assembly of Torpedo acetylcholine receptors in Xenopus oocytes." Journal of Cell Biology 112, no. 5 (March 1, 1991): 1007–15. http://dx.doi.org/10.1083/jcb.112.5.1007.
Повний текст джерелаАнотація:
To study pathways by which acetylcholine receptor (AChR) subunits might assemble, Torpedo alpha subunits were expressed in Xenopus oocytes alone or in combination with beta, gamma, or delta subunits. The maturation of the conformation of the main immunogenic region (MIR) on alpha subunits was measured by binding of mAbs and the maturation of the conformation of the AChR binding site on alpha subunits was measured by binding of alpha-bungarotoxin (alpha Bgt) and cholinergic ligands. The size of subunits and subunit complexes was assayed by sedimentation on sucrose gradients. It is generally accepted that native AChRs have the subunit composition alpha 2 beta gamma delta. Torpedo alpha subunits expressed alone resulted in an amorphous range of complexes with little affinity for alpha Bgt or mAbs to the MIR, rather than in a unique 5S monomeric assembly intermediate species. A previously recognized temperature-dependent failure in alpha subunit maturation may cause instability of the monomeric assembly intermediate and accumulation of aggregated denatured alpha subunits. Coexpression of alpha with beta subunits also resulted in an amorphous range of complexes. However, coexpression of alpha subunits with gamma or delta subunits resulted in the efficient formation of 6.5S alpha gamma or alpha delta complexes with high affinity for mAbs to the MIR, alpha Bgt, and small cholinergic ligands. These alpha gamma and alpha delta subunit pairs may represent normal assembly intermediates in which Torpedo alpha is stabilized and matured in conformation. Coexpression of alpha, gamma, and delta efficiently formed 8.8S complexes, whereas complexes containing alpha beta and gamma or alpha beta and delta subunits are formed less efficiently. Assembly of beta subunits with complexes containing alpha gamma and delta subunits may normally be a rate-limiting step in assembly of AChRs.
2
Bangalore, R., G. Mehrke, K. Gingrich, F. Hofmann, and R. S. Kass. "Influence of L-type Ca channel alpha 2/delta-subunit on ionic and gating current in transiently transfected HEK 293 cells." American Journal of Physiology-Heart and Circulatory Physiology 270, no. 5 (May 1, 1996): H1521—H1528. http://dx.doi.org/10.1152/ajpheart.1996.270.5.h1521.
Повний текст джерелаАнотація:
We have measured ionic and gating currents in human embryonic kidney (HEK 293) cells transiently transfected with cDNAs encoding subunits of the cardiac voltage-gated L-type Ca2+ channel. Robust recombinant ionic current and associated nonlinear charge movement could be measured over a broad voltage range without contamination by endogenous channel activity. Coexpression of the alpha 2/delta-subunit along with alpha 1- and beta 2-subunits speeded activation and deactivation kinetics and significantly increased the maximal conductance of ionic current. Charge movement was measured at voltages negative to the threshold for activation of ionic current, and gating charge could be immobilized at positive holding potentials that did not inactivate ionic current. The ratio of maximal ionic conductance to maximal charge moved remained the same in the absence or presence of the alpha 2/delta-subunit. However, the maximal amount of charge moved was increased about twofold in the presence of the alpha 2/delta-subunit. These results suggest that coexpression of the alpha 2/delta-subunit enhances the expression of functional L-type channels and, in addition, provide evidence that most of the L-type channel-associated nonlinear charge movement is caused by transitions between nonconducting states of the channel protein that precede the open and inactivated states.
3
Paudel, H. K., and G. M. Carlson. "The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin." Biochemical Journal 268, no. 2 (June 1, 1990): 393–99. http://dx.doi.org/10.1042/bj2680393.
Повний текст джерелаАнотація:
Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, β- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, β- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only β-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.
4
Blount, P., and J. P. Merlie. "Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly." Journal of Cell Biology 111, no. 6 (December 1, 1990): 2613–22. http://dx.doi.org/10.1083/jcb.111.6.2613.
Повний текст джерелаАнотація:
The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin-treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits accumulate, and ultimately make functional receptors in AChR-expressing cells.
5
Paulson, H. L., A. F. Ross, W. N. Green, and T. Claudio. "Analysis of early events in acetylcholine receptor assembly." Journal of Cell Biology 113, no. 6 (June 15, 1991): 1371–84. http://dx.doi.org/10.1083/jcb.113.6.1371.
Повний текст джерелаАнотація:
Mammalian cell lines expressing nicotinic acetylcholine receptor (AChR) subunit cDNAs from Torpedo californica were used to study early events in AChR assembly. To test the hypothesis that individual subunits form homooligomeric intermediates before assembling into alpha 2 beta gamma delta pentamers, we analyzed the sedimentation on sucrose density gradients of each subunit expressed separately in cell lines. We have shown previously that the acute temperature sensitivity of Torpedo AChR subunit assembly is due, in part, to misfolding of the polypeptide chains (Paulson, H.L., and T. Claudio. 1990. J. Cell Biol. 110:1705-1717). We use this phenomenon to further analyze putative assembly-competent intermediates. In nonionic detergent at an assembly-permissive temperature, the majority of alpha, beta, gamma, and delta subunits sediment neither as 3-4S monomers nor as 9S complexes, but rather as 6S species whether synthesized in fibroblasts, myoblasts, or differentiated myosyncytia. Several results indicate that the 6S species are complexes comprised predominantly of incorrectly folded subunit polypeptides. The complexes represent homoaggregates which form rapidly within the cell, are stable to mild SDS treatment and, in the case of alpha, contain some disulfide-linked subunits. The coprecipitation of alpha subunit with BiP or GRP78, a resident protein of the ER, further indicates that at least some of these internally sequestered subunits also associated with an endogenous protein implicated in protein folding. The majority of subunits expressed in these cell lines appear to be aggregates of subunits which are not assembly intermediates and are not assembly-competent. The portion which migrates as monomer, in contrast, appears to be the fraction which is assembly competent. This fraction increases at temperatures more permissive for assembly, further indicating the importance of the monomer as the precursor to assembly of alpha 2 beta gamma delta pentamers.
6
Tami, J. A., O. E. Urso, and K. A. Krolick. "T cell hybridomas reactive with the acetylcholine receptor and its subunits." Journal of Immunology 138, no. 3 (February 1, 1987): 732–38. http://dx.doi.org/10.4049/jimmunol.138.3.732.
Повний текст джерелаАнотація:
Abstract A panel of thirty cloned rat-mouse T cell hybridomas was prepared by fusion of acetylcholine receptor (AChR)-reactive rat T cells with the mouse thymoma BW5147. The T cell hybrids were demonstrated to be AChR reactive by their ability to secrete IL 2 in response to either AChR itself or by purified AChR subunits (alpha,beta,gamma, or delta). Various patterns of AChR subunit reactivity were observed, suggesting a predominant recognition of the alpha subunit, and also a considerable cross-reactivity from one subunit to another.
7
Vandaele, S. F., and F. Rieger. "Co-localization of 1,4-dihydropyridine receptor alpha 2/delta subunit and N-CAM during early myogenesis in vitro." Journal of Cell Science 107, no. 5 (May 1, 1994): 1217–27. http://dx.doi.org/10.1242/jcs.107.5.1217.
Повний текст джерелаАнотація:
The surface distribution of the alpha 2/delta subunit of the 1,4-dihydropyridine receptor and its topographical relationship with the neural cell adhesion molecule (N-CAM) were investigated during early myogenesis in vitro, by double immunocytochemical labeling with the monoclonal antibody 3007 and an anti-N-CAM polyclonal antiserum. The monoclonal antibody 3007 has been previously shown to immunoprecipitate dihydropyridine receptor from skeletal muscle T-tubules. In further immunoprecipitation experiments on such preparations and muscle cell cultures, it was demonstrated here that the monoclonal antibody 3007 exclusively recognizes the alpha 2/delta subunit of the 1,4-dihydropyridine receptor. In rabbit muscle cell cultures, the labeling for both alpha 2/delta and N-CAM was first detected on myoblasts, in the form of spots on the membrane and perinuclear patches. Spots of various sizes organized in aggregates were then found on the membrane of myotubes. At fusion (T0), aggregates of N-CAM spots alone were found at the junction between fusing cells. At T6 and later stages, all alpha 2/delta aggregates present on myotubes co-localized with N-CAM, while less than 3% of N-CAM aggregates did not co-localize with alpha 2/delta. A uniform N-CAM staining also made its appearance. At T12, when myotubes showed prominent contractility, alpha 2/delta-N-CAM aggregates diminished in size. Dispersed alpha 2/delta spots of a small regular size spread over the whole surface of the myotubes and alignments of these spots became visible. Corresponding N-CAM spots were now occasionally seen, and uniform N-CAM staining was prominent. These results show that alpha 2/delta and N-CAM are co-localized and that their distributions undergo concomitant changes during early myogenesis until the T-tubule network starts to be organized. This suggest that these two proteins might jointly participate in morphogenetic events preceding the formation of T-tubules.
8
Yoshii, K., L. Yu, K. M. Mayne, N. Davidson, and H. A. Lester. "Equilibrium properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes." Journal of General Physiology 90, no. 4 (October 1, 1987): 553–73. http://dx.doi.org/10.1085/jgp.90.4.553.
Повний текст джерелаАнотація:
This study used messenger RNA encoding each subunit (alpha, beta, gamma and delta) of the nicotinic acetylcholine (ACh) receptor from mouse BC3H-1 cells and from Torpedo electric organ. The mRNA was synthesized in vitro by transcription with SP6 polymerase from cDNA clones. All 16 possible combinations that include one mRNA for each of alpha, beta, gamma, and delta were injected into oocytes. After allowing 2-3 d for translation and assembly, we assayed each oocyte for (a) receptor assembly, measured by the binding of [125I]alpha-bungarotoxin to the oocyte surface, and (b) ACh-induced conductance, measured under voltage clamp at various membrane potentials. All combinations yielded detectable assembly (30-fold range among different combinations) and ACh-induced conductances (greater than 1,000-fold range at 1 microM). On double-logarithmic coordinates, the dose-response relations all had a slope near 2 for low concentrations of ACh. Data were corrected for variations in efficiency of translation among identically injected oocytes by expressing ACh-induced conductance per femtomole of alpha-bungarotoxin-binding sites. Five combinations were tested for d-tubocurarine inhibition by the dose-ratio method; the apparent dissociation constant ranged from 0.08 to 0.27 microM. Matched responses and geometric means are used for describing the effects of changing a particular subunit (mouse vs. Torpedo) while maintaining the identity of the other subunits. A dramatic subunit-specific effect is that of the beta subunit on voltage sensitivity of the response: gACh(-90 mV)/gACh(+30 mV) is always at least 1, but this ratio increases by an average of 3.5-fold if beta M replaces beta T. Also, combinations including gamma T or delta M usually produce greater receptor assembly than combinations including the homologous subunit from the other species. Finally, EACh is defined as the concentration of ACh inducing 1 microS/fmol at -60 mV; EACh is consistently lower for alpha M. We conclude that receptor assembly, voltage sensitivity, and EACh are governed by different properties.
9
Paturle-Lafanechere, L., M. Manier, N. Trigault, F. Pirollet, H. Mazarguil, and D. Job. "Accumulation of delta 2-tubulin, a major tubulin variant that cannot be tyrosinated, in neuronal tissues and in stable microtubule assemblies." Journal of Cell Science 107, no. 6 (June 1, 1994): 1529–43. http://dx.doi.org/10.1242/jcs.107.6.1529.
Повний текст джерелаАнотація:
Tubulin is the major protein component of brain tissue. It normally undergoes a cycle of tyrosination-detyrosination on the carboxy terminus of its alpha-subunit and this results in subpopulations of tyrosinated tubulin and detyrosinated tubulin. Brain tubulin preparations also contain a third major tubulin subpopulation, composed of a non-tyrosinatable variant of tubulin that lacks a carboxy-terminal glutamyl-tyrosine group on its alpha-subunit (delta 2-tubulin). Here, the abundance of delta 2-tubulin in brain tissues, its distribution in developing rat cerebellum and in a variety of cell types have been examined and compared with that of total alpha-tubulin and of tyrosinated and detyrosinated tubulin. Delta 2-tubulin accounts for approximately 35% of brain tubulin. In rat cerebellum, delta 2-tubulin appears early during neuronal differentiation and is detected only in neuronal cells. This apparent neuronal specificity of delta 2-tubulin is confirmed by examination of its distribution in cerebellar cells in primary cultures. In such cultures, neuronal cells are brightly stained with anti-delta 2-tubulin antibody while glial cells are not. Delta 2-tubulin is apparently present in neuronal growth cones. As delta 2-tubulin, detyrosinated tubulin is enriched in neuronal cells, but in contrast with delta 2-tubulin, detyrosinated tubulin is not detectable in Purkinje cells and is apparently excluded from neuronal growth cones. In a variety of cell types such as cultured fibroblasts of primary culture of bovine adrenal cortical cells, delta 2-tubulin is confined to very stable structures such as centrosomes and primary cilia. Treatment of such cells with high doses of taxol leads to the appearance of delta 2-tubulin in microtubule bundles. Delta 2-tubulin also occurs in the paracrystalline bundles of protofilamentous tubulin formed after vinblastine treatment. Delta 2-tubulin is present in sea urchin sperm flagella and it appears in sea urchin embryo cilia during development. Thus, delta 2-tubulin is apparently a marker of very long-lived microtubules. It might represent the final stage of alpha-tubulin maturation in long-lived polymers.
10
Horovitz, O., D. Knaack, T. R. Podleski, and M. M. Salpeter. "Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization." Journal of Cell Biology 108, no. 5 (May 1, 1989): 1823–32. http://dx.doi.org/10.1083/jcb.108.5.1823.
Повний текст джерелаАнотація:
Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.
11
Adams, L., B. M. Carlson, L. Henderson, and D. Goldman. "Adaptation of nicotinic acetylcholine receptor, myogenin, and MRF4 gene expression to long-term muscle denervation." Journal of Cell Biology 131, no. 5 (December 1, 1995): 1341–49. http://dx.doi.org/10.1083/jcb.131.5.1341.
Повний текст джерелаАнотація:
Muscle activity alters the expression of functionally distinct nicotinic acetylcholine receptors (nAChR) via regulation of subunit gene expression. Denervation increases the expression of all subunit genes and promotes the expression of embryonic-type (alpha 2 beta delta gamma) nAChRs, while electrical stimulation of denervated muscle prevents this induction. We have discovered that the denervation-induced increases in alpha, beta, gamma, and delta subunit gene expression do not persist in muscles that have been denervated for periods extending beyond a couple of months. However, expression of RNA encoding the epsilon-subunit remains elevated suggesting a return to expression of predominantly adult-type (alpha 2 beta delta epsilon) nAChR in long-term denervated muscles; a finding confirmed by single channel patch-clamp analysis. Since the nAChR subunit genes are regulated by the MyoD family of muscle regulatory factors, and the genes encoding these factors are also induced following short-term muscle denervation, we determined their level of expression in long-term denervated muscle. Although MyoD and myf-5 RNA levels remained elevated, myogenin and MRF4 RNAs were induced only transiently by muscle denervation. Surprisingly, Id-1, a negative regulator of transcription, was gradually induced in denervated muscle with RNA levels peaking about two months after denervation. It is likely that this maintained level of increased Id expression, in conjunction with the returning levels of myogenin and MRF4 expression, account for the reduced level of embryonic receptors in long-term denervated muscle. These changing patterns of gene expression may have important consequences for the ability of muscle to recover function after denervation.
12
Jeromin, A., R. L. Huganir, and D. J. Linden. "Suppression of the glutamate receptor delta 2 subunit produces a specific impairment in cerebellar long-term depression." Journal of Neurophysiology 76, no. 5 (November 1, 1996): 3578–83. http://dx.doi.org/10.1152/jn.1996.76.5.3578.
Повний текст джерелаАнотація:
1. The role of the glutamate receptor subunit delta 2 in the induction of cerebellar long-term depression (LTD) was investigated by application of antisense oligonucleotides. The delta 2 subunit is selectively localized to Purkinje cells (PCs), with the highest levels being in the PC dendritic spines, where parallel fibers are received and where cerebellar LTD is expressed. 2. Immunocytochemical analysis of calbindin-positive PCs revealed that both the dendritic and somatic expression of delta 2 was reduced in antisense-but not in sense-treated cultures. An antisense oligonucleotide directed against the related subunit delta 1 did not affect the expression of delta 2 in PCs. 3. Cerebellar LTD may be reliably induced in a preparation of cultured embryonic cerebellar neurons from the mouse when parallel and climbing fiber stimulation are replaced by brief glutamate pulses and strong, direct depolarization of the PC, respectively. Application of an antisense oligonucleotide directed against delta 2 completely blocked the induction of LTD produced by glutamate/ depolarization conjunctive stimulation. A delta 2 sense oligonucleotide or an antisense oligonucleotide directed against the related delta 1 subunit had no effect. 4. The effect of the delta 2 antisense oligonucleotide was not related to attenuation of calcium influx via voltage-gated channels or calcium mobilization via metabotropic glutamate receptors, as assessed with fura-2 microfluorimetry. Current flow through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor-associated ion channels also appeared unaltered. All three of these processes have previously been shown to be required for cerebellar LTD induction. The observation that delta 2 is involved in a metabotropic-glutamate-receptor-independent signaling pathway that is required for LTD induction supports the view that delta 2 participates in the formation of a novel postsynaptic receptor complex.
13
Zambello, R., L. Trentin, G. Pizzolo, P. Bulian, M. Masciarelli, C. Feruglio, C. Agostini, R. Raimondi, T. Chisesi, and G. Semenzato. "Cell membrane expression and functional role of the p75 subunit of interleukin-2 receptor in lymphoproliferative disease of granular lymphocytes." Blood 76, no. 10 (November 15, 1990): 2080–85. http://dx.doi.org/10.1182/blood.v76.10.2080.2080.
Повний текст джерелаАнотація:
Abstract The cell membrane expression and functional role of the interleukin-2 receptor (IL-2R) was analyzed in nine patients with lymphoproliferative disease of granular lymphocytes (LDGL) using monoclonal antibodies (MoAbs) specific for the p75 (TU27) and the p55 (anti-Tac) subunits of IL-2R. Four patients were characterized by the proliferation of CD3+CD8+ granular lymphocytes (GL) expressing the alpha/beta T-cell receptor (T alpha beta) and one case by the proliferation of CD3+CD4- CD8- GL expressing the gamma/delta T-cell receptor (T gamma delta); in four additional cases proliferating cells were CD3 negative GL. Consistent with data observed on normal GL, phenotypic analysis demonstrated that patients' GL lack the expression of the p55 IL-2R, whereas the p75 subunit is constitutionally expressed by expanding GL of both T-cell (either T alpha beta and T gamma delta) and natural killer (NK) origin in variable proportions (11% to 94% of cells). The analysis of the cytotoxic and proliferative activity demonstrated that the anti-p55 MoAb failed to inhibit IL-2-mediated activation, whereas a marked inhibition of both cytotoxicity and proliferation were obtained using the anti-p75 chain specific MoAb. These data indicate that the p75 chain of IL-2R is responsible for IL-2 signal transduction in both CD3+ and CD3- LDGL patients' GL.
14
Zambello, R., L. Trentin, G. Pizzolo, P. Bulian, M. Masciarelli, C. Feruglio, C. Agostini, R. Raimondi, T. Chisesi, and G. Semenzato. "Cell membrane expression and functional role of the p75 subunit of interleukin-2 receptor in lymphoproliferative disease of granular lymphocytes." Blood 76, no. 10 (November 15, 1990): 2080–85. http://dx.doi.org/10.1182/blood.v76.10.2080.bloodjournal76102080.
Повний текст джерелаАнотація:
The cell membrane expression and functional role of the interleukin-2 receptor (IL-2R) was analyzed in nine patients with lymphoproliferative disease of granular lymphocytes (LDGL) using monoclonal antibodies (MoAbs) specific for the p75 (TU27) and the p55 (anti-Tac) subunits of IL-2R. Four patients were characterized by the proliferation of CD3+CD8+ granular lymphocytes (GL) expressing the alpha/beta T-cell receptor (T alpha beta) and one case by the proliferation of CD3+CD4- CD8- GL expressing the gamma/delta T-cell receptor (T gamma delta); in four additional cases proliferating cells were CD3 negative GL. Consistent with data observed on normal GL, phenotypic analysis demonstrated that patients' GL lack the expression of the p55 IL-2R, whereas the p75 subunit is constitutionally expressed by expanding GL of both T-cell (either T alpha beta and T gamma delta) and natural killer (NK) origin in variable proportions (11% to 94% of cells). The analysis of the cytotoxic and proliferative activity demonstrated that the anti-p55 MoAb failed to inhibit IL-2-mediated activation, whereas a marked inhibition of both cytotoxicity and proliferation were obtained using the anti-p75 chain specific MoAb. These data indicate that the p75 chain of IL-2R is responsible for IL-2 signal transduction in both CD3+ and CD3- LDGL patients' GL.
15
Cohen, B. N., C. Labarca, L. Czyzyk, N. Davidson, and H. A. Lester. "Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region." Journal of General Physiology 99, no. 4 (April 1, 1992): 545–72. http://dx.doi.org/10.1085/jgp.99.4.545.
Повний текст джерелаАнотація:
Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.
16
Paulson, H. L., and T. Claudio. "Temperature-sensitive expression of all-Torpedo and Torpedo-rat hybrid AChR in mammalian muscle cells." Journal of Cell Biology 110, no. 5 (May 1, 1990): 1705–17. http://dx.doi.org/10.1083/jcb.110.5.1705.
Повний текст джерелаАнотація:
When the four subunits of the Torpedo californica nicotinic acetylcholine receptor (AChR) are expressed in mammalian fibroblasts, they properly assembly into alpha 2 beta gamma delta pentamers only at temperatures lower than 37 degrees C (Claudio, T., W. N. Green, D. S. Hartman, D. Hayden, H. L. Paulson, F. J. Sigworth, S. M. Sine, and A. Swedlund. 1987. Science (Wash. DC). 238:1688-1694). Experiments here with rat L6 myoblast cell lines indicate that this temperature sensitivity is not specific to fibroblasts, but is intrinsic to Torpedo subunits. A clonal isolate of L6 cells cotransfected with the four Torpedo subunit cDNAs synthesizes the exogenous AChR subunits at 37 degrees and 26 degrees C, but expresses Torpedo AChR complexes only at the lower temperature. When Torpedo alpha alone is expressed in L6 myotubes, hybrid AChRs are formed, again only at temperatures below 37 degrees C. These hybrid AChRs can contain either two Torpedo alpha subunits or one each of rat and Torpedo alpha, proving that the two alpha subunits in an AChR pentamer need not derive from the same polysome. Further analysis of hybrid and all-Torpedo AChR established that there is no internally sequestered pool of AChR at the nonpermissive temperature, and that the AChR, once formed, is thermostable. Two lines of experimentation with alpha subunits expressed in fibroblasts indicate that alpha polypeptides exhibit different conformations at 26 degrees and 37 degrees C, favoring the hypothesis that the temperature-sensitive step occurs before assembly and reflects, at least in part, misfolding of subunits: at 37 degrees C, there is a reduction in the fraction of alpha subunits that (a) bind the AChR antagonist alpha-bungarotoxin with high affinity; and (b) bind a monoclonal antibody that recognizes correctly folded and/or assembled alpha subunit.
17
He, Y. W., and T. R. Malek. "Interleukin-7 receptor alpha is essential for the development of gamma delta + T cells, but not natural killer cells." Journal of Experimental Medicine 184, no. 1 (July 1, 1996): 289–93. http://dx.doi.org/10.1084/jem.184.1.289.
Повний текст джерелаАнотація:
Mice that lack a functional gamma c subunit of the receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 display profound defects in lymphoid development. The IL-7/IL-7R system represents a critical interaction for conventional T and B cell development. In this report, the role of IL-7R alpha in the development of lymphoid lineages other than conventional T and B cells was examined. We demonstrate that gamma delta + T cells were absent in IL-7R alpha-deficient mice, whereas the development and function of natural killer cells were normal. Thus, IL-7R alpha function is required for the development of gamma delta + T cells but not natural killer cells.
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Koyasu, S. "CD3+CD16+NK1.1+B220+ large granular lymphocytes arise from both alpha-beta TCR+CD4-CD8- and gamma-delta TCR+CD4-CD8- cells." Journal of Experimental Medicine 179, no. 6 (June 1, 1994): 1957–72. http://dx.doi.org/10.1084/jem.179.6.1957.
Повний текст джерелаАнотація:
Cultivation of CD4-CD8- double negative (DN) mouse thymocytes and splenocytes with recombinant interleukin 2 (IL2) in the absence of other stimulation results in the generation of DN-CD3/TCR+CD16+NK1.1+B220+ large granular lymphocytes (LGL). Purified DN alpha-beta TCR+ thymocytes and splenocytes are CD16+IL2R alpha-IL2R beta+NK1.1+B220-CD5high. These cells are unique in that they express both CD16 and T cell receptor (TCR) which are usually mutually exclusive. In addition, they express the natural killer (NK) marker, NK1.1. Cultivation of these cells with IL2 for several days results in the generation of DN alpha-beta TCR+CD16+NK1.1+B220+CD5- LGL, suggesting that DN alpha-beta TCR+ cells in thymus and spleen are the precursors of the DN LGL reported previously. DN gamma-delta TCR+CD16-NK1.1-B220-CD5high thymocytes and splenocytes also give rise to DN gamma-delta TCR+CD16+NK1.1+B220+CD5- LGL which, as shown previously with DN alpha-beta TCR+ LGL cells, are cytotoxic against NK-sensitive YAC-1 cells. Cytotoxic activity is also induced through either CD16 or the gamma-delta TCR. DN alpha-beta TCR+ and DN gamma-delta TCR+ LGL cells are thus similar in phenotype to TCR- NK cells. DN alpha-beta TCR+ thymocytes express low levels of the gamma subunit of the high affinity immunoglobulin E receptor (Fc epsilon RI gamma) molecule, an essential component of CD16 expression. Fc epsilon RI gamma expression is greatly enhanced after cultivation with IL2, resulting in a higher surface expression of CD16. In contrast to DN alpha-beta TCR+ thymocytes, DN gamma-delta TCR+ thymocytes do not express detectable CD16 or Fc epsilon RI gamma mRNA but expression of both is induced by cultivation with IL2, leading to the expression of CD16 on the surface. Whereas CD16 molecules on both DN alpha-beta TCR+ and DN gamma-delta TCR+ LGL are associated with only Fc epsilon RI gamma homodimers, the TCR on these cells are associated with an Fc epsilon RI gamma homodimer and/or CD3 zeta-Fc epsilon RI gamma heterodimers. These results demonstrate that the Fc epsilon RI gamma subunit is a component of the TCR in a fraction of T lineage cells.
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Honorio, S., K. Gordon, D. MacCartney, A. Agathanggelou, and F. Latif. "Identification of a single nucleotide polymorphism in the human alpha 2 delta 2 calcium channel subunit gene." Molecular and Cellular Probes 15, no. 6 (December 2001): 391–93. http://dx.doi.org/10.1006/mcpr.2001.0382.
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O'Toole, TE, Y. Katagiri, RJ Faull, K. Peter, R. Tamura, V. Quaranta, JC Loftus, SJ Shattil, and MH Ginsberg. "Integrin cytoplasmic domains mediate inside-out signal transduction." Journal of Cell Biology 124, no. 6 (March 15, 1994): 1047–59. http://dx.doi.org/10.1083/jcb.124.6.1047.
Повний текст джерелаАнотація:
We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.
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Buitkamp, J., D. Ewald, J. Masabanda, M. D. Bishop, and R. Fries. "FISH and RH mapping of the bovine alpha (2)/delta calcium channel subunit gene (CACNA2D1)." Animal Genetics 34, no. 4 (July 22, 2003): 309–10. http://dx.doi.org/10.1046/j.1365-2052.2003.01030.x.
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Bonifacino, J. S., C. K. Suzuki, J. Lippincott-Schwartz, A. M. Weissman, and R. D. Klausner. "Pre-Golgi degradation of newly synthesized T-cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly." Journal of Cell Biology 109, no. 1 (July 1, 1989): 73–83. http://dx.doi.org/10.1083/jcb.109.1.73.
Повний текст джерелаАнотація:
The T cell antigen receptor (TCR) is a multisubunit complex composed of at least seven transmembrane chains. The predominant species in most T cells has the composition alpha beta gamma delta epsilon zeta 2. The roles of subunit assembly in transport out of the ER and in the recently described process of pre-Golgi degradation of newly synthesized TCR chains were analyzed in a T-cell line deficient in the synthesis of delta chains (delta 2) and in COS-1 fibroblasts transfected with genes encoding individual TCR chains. Studies with the delta-deficient T-cell line showed that, in the absence of delta, the other TCR chains were synthesized at normal rates, but, instead of being transported to the cell surface, they were retained in the ER. Analysis of the fate of TCR chains retained in the ER showed that they were degraded at vastly different rates by a nonlysosomal pathway. Whereas the alpha chains were degraded rapidly, gamma, zeta, and epsilon were relatively long-lived. To analyze whether this selective degradation was because of different intrinsic susceptibility of the individual chains to degradation or to the formation of resistant oligomers, TCR chains were expressed alone or in combinations in COS-1 fibroblasts. These studies showed that (a) individual TCR chains were degraded at different rates when expressed alone in COS-1 cells, and (b) sensitive chains could be stabilized by coexpression with a resistant chain. Taken together, these observations indicate that both intrinsic sensitivity and subunit assembly play a role in determining the rates at which newly synthesized TCR chains are degraded in the ER.
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Jay, S. D., A. H. Sharp, S. D. Kahl, T. S. Vedvick, M. M. Harpold, and K. P. Campbell. "Structural characterization of the dihydropyridine-sensitive calcium channel alpha 2-subunit and the associated delta peptides." Journal of Biological Chemistry 266, no. 5 (February 1991): 3287–93. http://dx.doi.org/10.1016/s0021-9258(18)49986-3.
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Zhou, C., and Z. D. Luo. "Electrophysiological characterization of spinal neuron sensitization by elevated calcium channel alpha-2-delta-1 subunit protein." European Journal of Pain 18, no. 5 (October 23, 2013): 649–58. http://dx.doi.org/10.1002/j.1532-2149.2013.00416.x.
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Kurachi, Y. "G protein regulation of cardiac muscarinic potassium channel." American Journal of Physiology-Cell Physiology 269, no. 4 (October 1, 1995): C821—C830. http://dx.doi.org/10.1152/ajpcell.1995.269.4.c821.
Повний текст джерелаАнотація:
Several ion channels can be regulated by G proteins in a “membrane-delimited” manner. The cardiac muscarinic K+ (KACh) channel, which is responsible for the acetylcholine (ACh) or adenosine-induced deceleration of heart beat and atrioventricular conduction, is the prototype of this type of receptor-dependent regulation of ion channels. Because similar transduction mechanisms are utilized by various membrane receptors, such as somatostatin, 5-hydroxytryptamine-1, alpha 2-adrenergic, mu-and delta-opioid, D2-dopamine, and gamma-aminobutyric acid B receptors, in neuronal, hormone-secreting, renal, or smooth muscle cells, the G protein (GK)-KACh channel system illustrates the principles underlying one of the most important cell signaling mechanisms (B. Hille. Neuron 9: 187-195, 1992). It seems that both alpha- and beta gamma-subunits of GK may be involved in the regulation of the KACh channel of mammalian atrial muscle. A general consensus of opinion has emerged, after some years of controversy, to support the notion that physiological activation of the channel by GK is the responsibility of the beta gamma-subunits. Recent evidence suggests that the KACh channel interacts with the alpha-subunit in the terminating process of activation.
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Cohen, B. N., C. Labarca, N. Davidson, and H. A. Lester. "Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations." Journal of General Physiology 100, no. 3 (September 1, 1992): 373–400. http://dx.doi.org/10.1085/jgp.100.3.373.
Повний текст джерелаАнотація:
We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222.
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Neisig, A., A. Vangsted, J. Zeuthen, and C. Geisler. "Assembly of the T-cell antigen receptor. Participation of the CD3 omega chain." Journal of Immunology 151, no. 2 (July 15, 1993): 870–79. http://dx.doi.org/10.4049/jimmunol.151.2.870.
Повний текст джерелаАнотація:
Abstract The human TCR is composed of the Ti alpha beta heterodimer in association with the CD3 chains CD3 gamma delta epsilon zeta 2. Another chain, referred to as CD3 omega, has recently been described in T cells. CD3 omega is an intracellular protein transiently associated with the CD3 complex during the assembly of the TCR in the endoplasmic reticulum (ER) and it is not expressed on the cell surface. The function of CD3 omega is unknown but it has been suggested that it plays an important role in the assembly of the TCR. We have studied the possible function of CD3 omega in the human leukemic T-cell line Jurkat and different variants of this cell line. Cells were metabolically labeled, subjected to lysis, immunoprecipitated, and analyzed by SDS-PAGE. The results indicate that: 1) CD3 omega associates primarily with the CD3 delta epsilon complex; 2) CD3 omega is not associated with single Ti alpha or Ti beta chains, but is present in complexes composed of both the CD3 and the Ti chains; 3) CD3 omega is part of the complete, intracellular receptor complex Ti alpha beta/CD3 gamma epsilon delta omega zeta 2; and 4) CD3 omega dissociates from the Ti/CD3 complex in the ER before maturation of the Ti alpha beta heterodimer. On the basis of these results, we propose a model for the assembly and subunit stoichiometry of the TCR complex which includes the participation of the CD3 omega chain.
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Deckers-Hebestreit, G., and K. Altendorf. "The Fo complex of the proton-translocating F-type ATPase of Escherichia coli." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 451–59. http://dx.doi.org/10.1242/jeb.172.1.451.
Повний текст джерелаАнотація:
The ATP synthase (F1Fo) of Escherichia coli consists of two structurally and functionally distinct entities. The F1 part is composed of five subunits alpha, beta, gamma, delta and epsilon (3:3:1:1:1) and carries the catalytic centres of the enzyme. The membrane-bound Fo complex functions as a proton channel and consists of the three subunits a, b and c (1:2:10 +/- 1). Subunit c (8288 M(r)) exhibits a hairpin-like structure within the membrane. A conserved acidic residue (Asp-61) in the C-terminal hydrophobic segment is absolutely required for proton translocation through Fo, whereas the hydrophilic loop region is necessary for F1 binding. Expression of the chloroplast proteolipid together with subunits a and b of E. coli did not produce an active Fo hybrid complex. Therefore, the construction of hybrid c subunits consisting of parts of the proteolipid from both organisms is in progress to determine those parts of subunit c that are essential for a functional interplay with subunits a and b. Subunit a (30,276 M(r)), which is also involved in proton translocation, is an extremely hydrophobic protein with 5-8 membrane-spanning helices. Studies with alkaline phosphatase fusion proteins resulted in controversial conclusions about the localization of the N and C termini of the protein. A foreign epitope (13 amino acids) has been inserted into the N- or C-terminal region of subunit a without affecting the function of Fo. Binding studies with a monoclonal antibody against this epitope are now under investigation to determine the orientation of subunit a.(ABSTRACT TRUNCATED AT 250 WORDS)
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Piiper, A., D. Stryjek-Kaminska, R. Klengel, and S. Zeuzem. "CCK, carbachol, and bombesin activate distinct PLC-beta isoenzymes via Gq/11 in rat pancreatic acinar membranes." American Journal of Physiology-Gastrointestinal and Liver Physiology 272, no. 1 (January 1, 1997): G135—G140. http://dx.doi.org/10.1152/ajpgi.1997.272.1.g135.
Повний текст джерелаАнотація:
Four different isoforms of phospholipase C-beta (PLC-beta 1-4) have been discovered, raising the important question of whether a distinct receptor activates a single PLC-beta isoenzyme or a subset of PLC-beta isoenzymes. The present study was designed to investigate activation of PLC-beta isoenzymes by three different PLC-activating agonists that bind to different receptor entities, i.e., cholecystokinin octapeptide (CCK-8), bombesin, and carbachol in rat pancreatic acinar membranes. PLC activity was measured using exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate. Western blot analysis of pancreatic acinar membranes revealed the presence of PLC-beta 1, -beta 3, -gamma 1, and -delta 1, but not of PLC-beta 2, -beta 4, -gamma 2, and -delta 2. Preincubation of the membranes with anti-PLC-beta 1 or -beta 3 antibody reduced agonist-induced activation of PLC. The order of sensitivity toward inhibition by anti-PLC-beta 1 antibody was CCK-8 > bombesin > carbachol. An opposite order of sensitivity was found for inhibition of PLC activity by anti-PLC-beta 3 antibody (carbachol > bombesin > CCK-8). Anti-PLC-beta 2, -beta 4, -gamma 1, -gamma 2, -delta 1, and -delta 2 antibodies had no effect. Preincubation of the membranes with an antibody raised against the COOH terminus of the alpha-subunit of Gq/11 proteins inhibited PLC activity in response to all three different receptor agonists to a similar extent, whereas anti-Gi alpha 1-2 and anti-Gi alpha 3 antibodies had no effect. In conclusion, the data of the present study indicate that CCK-8 and carbachol activate PLC-beta 1 and PLC-beta 3, respectively, whereas bombesin activates both PLC-beta 1 and PLC-beta 3. Activation of PLC-beta by these receptor agonists is mediated by Gq/11.
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Peter, K., and T. E. O'Toole. "Modulation of cell adhesion by changes in alpha L beta 2 (LFA-1, CD11a/CD18) cytoplasmic domain/cytoskeleton interaction." Journal of Experimental Medicine 181, no. 1 (January 1, 1995): 315–26. http://dx.doi.org/10.1084/jem.181.1.315.
Повний текст джерелаАнотація:
The integrin alpha L beta 2 (leukocyte function-associated molecule 1, CD11a/CD18) mediates activation-dependent adhesion of leukocytes. The cytoplasmic domains of alpha L beta 2 have been demonstrated to modulate adhesiveness of alpha L beta 2. Affinity changes of alpha L beta 2 for its ligand or postreceptor events can be responsible for this modulation of adhesiveness. To investigate the possible role of the alpha L beta 2 cytoplasmic domains in postreceptor events we constructed cDNA encoding chimeric proteins with intracellular alpha L beta 2 domains, which are responsible for alpha L beta 2 specific intracellular interactions, and extracellular alpha IIIb beta 3 (GP IIb/IIIa) domains, which allow the assessment of the receptor affinity state. The cDNA was stably transfected in Chinese hamster ovary cells and chimeric heterodimer formation proven by immunoprecipitations and flow cytometry. The chimeric receptors mediate adhesion to immobilized fibrinogen, and this adhesion is increased by phorbol myristate acetate and abolished by cytochalasin D. However, neither treatment affects the affinity state of the chimeric receptor, suggesting involvement of the cytoskeleton in the regulation of alpha L beta 2 mediated cell adhesion. To exclude the possibility of postoccupancy affinity changes of the chimeric receptors, we locked the receptors into a high affinity state by creating a deletion variant. The region deleted (VGFFK) is highly conserved in integrin alpha subunit cytoplasmic domains. Cotransfection of this deletion variant with a beta subunit truncation (beta 3 delta 724) and a triple mutation at 758-760 (TTT to AAA) of beta 2 abolishes adhesion without changing the affinity state. A single mutation (TTT to TAT) reduces adhesion by half without affinity change. Scanning electron microscopy reveals impaired spreading of these truncated/mutated chimeras. Immunofluorescence microscopy demonstrates a correlation between impaired adhesion and a decrease in the ability to form focal adhesions and to organize the cytoskeleton into stress fibers. These results describe the integrin/cytoskeleton interaction, the organization of the cytoskeleton, and cell spreading as postreceptor events modulating alpha L beta 2 cytoplasmic domain mediated cell adhesion. Furthermore, we demonstrate that the cytoplasmic domain of the beta 2 subunit, and within it the TTT region, are required for these postreceptor events. Additionally, we present a new approach, using deletion variants to lock integrins in a high affinity state without interfering with the investigated integrin/cytoskeleton interaction. This approach may be generally useful to investigate the role of postreceptor events in integrin-mediated cell adhesion and migration.
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Feng, L., H. Yoon, and T. F. Donahue. "Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae." Molecular and Cellular Biology 14, no. 8 (August 1994): 5139–53. http://dx.doi.org/10.1128/mcb.14.8.5139-5153.1994.
Повний текст джерелаАнотація:
Previous studies have demonstrated that the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe that yeast eIF-2 alpha is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism. 32Pi labeling and isoelectric focusing analysis of a SUI2+ delta gcn2 strain identifies eIF-2 alpha as radiolabeled and a single isoelectric protein species. Treatment of SUI2+ delta gcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2 alpha that correspond to the stepwise removal of three phosphates from the protein. Mutational analysis of SUI2 coupled with biochemical analysis of eIF-2 alpha maps the sites to the carboxyl region of SUI2 that correspond to Ser residues at amino acid positions 292, 294, and 301 that compose consensus casein kinase II sequences. 32Pi labeling or isoelectric focusing analysis of eIF-2 alpha from conditional casein kinase II mutants indicated that phosphorylation of eIF-2 alpha is abolished or dephosphorylated forms of eIF-2 alpha are detected when these strains are grown at the restrictive growth conditions. Furthermore, yeast casein kinase II phosphorylates recombinant wild-type eIF-2 alpha protein in vitro but does not phosphorylate recombinant eIF-2 alpha that contains Ser-to-Ala mutations at all three consensus casein kinase II sequences. These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2 alpha at one or all of these Ser amino acids in vivo. Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S. cerevisiae. Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2 alpha protein are required for optimal eIF-2 function in S. cerevisiae.
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Feng, L., H. Yoon, and T. F. Donahue. "Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae." Molecular and Cellular Biology 14, no. 8 (August 1994): 5139–53. http://dx.doi.org/10.1128/mcb.14.8.5139.
Повний текст джерелаАнотація:
Previous studies have demonstrated that the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe that yeast eIF-2 alpha is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism. 32Pi labeling and isoelectric focusing analysis of a SUI2+ delta gcn2 strain identifies eIF-2 alpha as radiolabeled and a single isoelectric protein species. Treatment of SUI2+ delta gcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2 alpha that correspond to the stepwise removal of three phosphates from the protein. Mutational analysis of SUI2 coupled with biochemical analysis of eIF-2 alpha maps the sites to the carboxyl region of SUI2 that correspond to Ser residues at amino acid positions 292, 294, and 301 that compose consensus casein kinase II sequences. 32Pi labeling or isoelectric focusing analysis of eIF-2 alpha from conditional casein kinase II mutants indicated that phosphorylation of eIF-2 alpha is abolished or dephosphorylated forms of eIF-2 alpha are detected when these strains are grown at the restrictive growth conditions. Furthermore, yeast casein kinase II phosphorylates recombinant wild-type eIF-2 alpha protein in vitro but does not phosphorylate recombinant eIF-2 alpha that contains Ser-to-Ala mutations at all three consensus casein kinase II sequences. These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2 alpha at one or all of these Ser amino acids in vivo. Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S. cerevisiae. Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2 alpha protein are required for optimal eIF-2 function in S. cerevisiae.
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Cheng, Jen-Kun, Yu-Jun Lai, Chien-Chuan Chen, Ching-Rong Cheng, and Lih-Chu Chiou. "Magnesium Chloride and Ruthenium Red Attenuate the Antiallodynic Effect of Intrathecal Gabapentin in a Rat Model of Postoperative Pain." Anesthesiology 98, no. 6 (June 1, 2003): 1472–79. http://dx.doi.org/10.1097/00000542-200306000-00026.
Повний текст джерелаАнотація:
Background Gabapentin, a gamma-aminobutyric acid analog anticonvulsant, has been shown to possess antinociceptive effects in animal models and clinical trials. An endogenous binding site of [3H]gabapentin has been revealed to be the alpha(2)delta subunit of voltage-dependent Ca2+ channels. Magnesium chloride, ruthenium red, and spermine have been shown to modulate [3H]gabapentin binding to this binding site in vitro. In this study, the authors examined whether intrathecal magnesium chloride, ruthenium red, or spermine could affect the antiallodynic effect of intrathecal gabapentin in a rat model of postoperative pain. Methods Under isoflurane anesthesia, male Sprague-Dawley rats received an incision over the plantar surface of the right hind paw to produce punctate mechanical allodynia. Withdrawal thresholds to von Frey filament stimulation near the incision site were measured before incision, 2 h after incision, and every 30 min after intrathecal coadministration of gabapentin with normal saline or different doses of magnesium chloride, ruthenium red, or spermine for 2 h. Results Intrathecal gabapentin (30, 100, 200 microg) dose-dependently reduced incision-induced allodynia. Hexahydrated magnesium chloride (5, 10, 20 microg) and ruthenium red (0.2, 2, 20 ng) noncompetitively inhibited the antiallodynic effect of gabapentin. Spermine at doses not inducing motor weakness (30, 60 microg) did not affect the antiallodynic effect of gabapentin. The antiallodynic effect of intrathecal morphine (1.5 microg) was not affected by hexahydrated magnesium chloride (20 microg), ruthenium red (20 ng), or spermine (60 microg). Conclusions These results provide behavioral evidence to support that the alpha(2)delta subunit of Ca2+ channels may be involved in the antiallodynic action of intrathecal gabapentin in the postoperative pain model.
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Marton, M. J., D. Crouch, and A. G. Hinnebusch. "GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2." Molecular and Cellular Biology 13, no. 6 (June 1993): 3541–56. http://dx.doi.org/10.1128/mcb.13.6.3541-3556.1993.
Повний текст джерелаАнотація:
Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1. Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.
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Marton, M. J., D. Crouch, and A. G. Hinnebusch. "GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2." Molecular and Cellular Biology 13, no. 6 (June 1993): 3541–56. http://dx.doi.org/10.1128/mcb.13.6.3541.
Повний текст джерелаАнотація:
Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1. Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.
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Silberstein, S., P. G. Collins, D. J. Kelleher, and R. Gilmore. "The essential OST2 gene encodes the 16-kD subunit of the yeast oligosaccharyltransferase, a highly conserved protein expressed in diverse eukaryotic organisms." Journal of Cell Biology 131, no. 2 (October 15, 1995): 371–83. http://dx.doi.org/10.1083/jcb.131.2.371.
Повний текст джерелаАнотація:
Oligosaccharyltransferase catalyzes the transfer of a preassembled high mannose oligosaccharide from a dolichol-oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six non-identical subunits (alpha-zeta). The alpha, beta, gamma, and delta subunits of the oligosaccharyltransferase are encoded by the OST1, WBP1, OST3, and SWP1 genes, respectively. Here we describe the functional characterization of the OST2 gene that encodes the epsilon-subunit of the oligosaccharyltransferase. Genomic disruption of the OST2 locus was lethal in haploid yeast showing that expression of the Ost2 protein is essential for viability. Overexpression of the Ost2 protein suppresses the temperature-sensitive phenotype of the wbp1-2 allele and increases in vivo and in vitro oligosaccharyltransferase activity in a wbp1-2 strain. An analysis of a series of conditional ost2 mutants demonstrated that defects in the Ost2 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins. Microsomal membranes isolated from ost2 mutant yeast show marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Surprisingly, the Ost2 protein was found to be 40% identical to the DAD1 protein (defender against apoptotic cell death), a highly conserved protein initially identified in vertebrate organisms. The protein sequence of ost2 mutant alleles revealed mutations at highly conserved residues in the Ost2p/DAD1 protein sequence.
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Muramatsu, M., K. Kaibuchi, and K. Arai. "A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester." Molecular and Cellular Biology 9, no. 2 (February 1989): 831–36. http://dx.doi.org/10.1128/mcb.9.2.831-836.1989.
Повний текст джерелаАнотація:
We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.
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Muramatsu, M., K. Kaibuchi, and K. Arai. "A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester." Molecular and Cellular Biology 9, no. 2 (February 1989): 831–36. http://dx.doi.org/10.1128/mcb.9.2.831.
Повний текст джерелаАнотація:
We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.
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Chang, E., X. Chen, M. Kim, N. Gong, S. Bhatia, and Z. D. Luo. "Differential effects of voltage-gated calcium channel blockers on calcium channel alpha-2-delta-1 subunit protein-mediated nociception." European Journal of Pain 19, no. 5 (August 27, 2014): 639–48. http://dx.doi.org/10.1002/ejp.585.
Повний текст джерела
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BEESON, D., S. JEREMIAH, L. F. WEST, S. POVEY, and J. NEWSOM-DAVIS. "Assignment of the human nicotinic acetylcholine receptor genes: the alpha and delta subunit genes to chromosome 2 and the beta subunit gene to chromosome 17." Annals of Human Genetics 54, no. 3 (July 1990): 199–208. http://dx.doi.org/10.1111/j.1469-1809.1990.tb00378.x.
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Piromjitpong, Juthamart, Jantana Wongsantichon, and Albert J. Ketterman. "Differences in the subunit interface residues of alternatively spliced glutathione transferases affects catalytic and structural functions." Biochemical Journal 401, no. 3 (January 12, 2007): 635–44. http://dx.doi.org/10.1042/bj20060603.
Повний текст джерелаАнотація:
GSTs (glutathione transferases) are multifunctional widespread enzymes. Currently there are 13 identified classes within this family. Previously most structural characterization has been reported for mammalian Alpha, Mu and Pi class GSTs. In the present study we characterize two enzymes from the insect-specific Delta class, adGSTD3-3 and adGSTD4-4. These two proteins are alternatively spliced products from the same gene and have very similar tertiary structures. Several major contributions to the dimer interface area can be separated into three regions: conserved electrostatic interactions in region 1, hydrophobic interactions in region 2 and an ionic network in region 3. The four amino acid side chains studied in region 1 interact with each other as a planar rectangle. These interactions are highly conserved among the GST classes, Delta, Sigma and Theta. The hydrophobic residues in region 2 are not only subunit interface residues but also active site residues. Overall these three regions provide important contributions to stabilization and folding of the protein. In addition, decreases in yield as well as catalytic activity changes, suggest that the mutations in these regions can disrupt the active site conformation which decreases binding affinity, alters kinetic constants and alters substrate specificity. Several of these residues have only a slight effect on the initial folding of each subunit but have more influence on the dimerization process as well as impacting upon appropriate active site conformation. The results also suggest that even splicing products from the same gene may have specific features in the subunit interface area that would preclude heterodimerization.
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Röck, Jürgen, Jürgen Schmitz, and Gregor Winkels. "CD303 (BDCA-2) mediated inhibition of interferon type I production in plasmacytoid dendritic cells is linked to SYK, BLNK and PKC delta signaling (89.25)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S153. http://dx.doi.org/10.4049/jimmunol.178.supp.89.25.
Повний текст джерелаАнотація:
Abstract CD303 is a calcium dependent type II lectin also known as Blood-Dendritic-Cell-Antigen-2 (BDCA-2) specifically expressed by human plasmacytoid dendritic cells (PDC). We have previously shown that monoclonal antibody (mAb) ligation of CD303 induces mAb endocytosis, calcium mobilization, protein tyrosine phosphorylation and inhibition of type I interferon (IFN I) production in stimulated PDC. Here we show that CD303 signaling and internalization in many aspects resembles B cell receptor (BCR) signaling and internalization. Instead of CD79a and CD79b, CD303 appears to use the Fc-receptor-common-gamma-chain as transmembrane adaptor protein. Like BCR signaling, CD303 triggering leads to SYK and BLNK phosphorylation. Signal transduction most likely involves activation of the phospholipase C-gamma 2, the phosphoinositide-3 kinase and the protein kinase C delta. Therefore, inhibition of IFN I production in stimulated PDC can be mimicked by PMA. Western blotting and peptide mass fingerprinting show that tyrosine phosphorylation occurs at cytoskeletal proteins (actin, alpha/beta-tubulin, profilin, alpha-actinin), proteasome activator subunit and the clathrin heavy chain, indicating clathrin-mediated endocytosis and vesicle trafficking. Finally, CD303 triggering inhibits TNF-alpha and CpG ODN-induced NF-kappa B activation in PDC.
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Craddock, B. L., N. T. Price та C. G. Proud. "Cloning and expression of cDNAs for the β subunit of eukaryotic initiation factor-2B, the guanine nucleotide exchange factor for eukaryotic initiation factor-2". Biochemical Journal 309, № 3 (1 серпня 1995): 1009–14. http://dx.doi.org/10.1042/bj3091009.
Повний текст джерелаАнотація:
A key control point in the initiation of protein synthesis in mammalian cells is the recycling of eukaryotic initiation factor (eIF)-2 by the guanine nucleotide exchange factor eIF-2B. In mammalian cells, eIF-2B is a complex of five different subunits termed epsilon, delta, gamma, beta and alpha. To clone cDNAs for the beta subunit of rabbit eIF-2B, amino acid sequence data was first obtained and used to design redundant oligonucleotide primers for use in PCR. PCR products were used to screen a rabbit liver cDNA library in lambda gt11 to obtain full-length cDNAs for eIF-2B beta. The cDNAs were sequenced completely on both strands and revealed an open reading frame encoding a predicted 351-amino acid polypeptide of 39.0 kDa. The molecular mass and pI (5.99) of the predicted protein agree well with the properties of eIF-2B beta purified from rabbit reticulocytes. In vitro transcription/-translation of the cDNAs gave rise to a product that migrated at a position indistinguishable from that of this subunit of the purified protein. The amino acid sequence shows a high degree of similarity to that of GCD7, a Saccharomyces cerevisiae protein thought to be equivalent to mammalian eIF-2B beta. Northern-blot analysis revealed a single major mRNA species for eIF-2B beta in each of the four rabbit tissues tested.
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Tsay, H. J., and J. Schmidt. "Skeletal muscle denervation activates acetylcholine receptor genes." Journal of Cell Biology 108, no. 4 (April 1, 1989): 1523–26. http://dx.doi.org/10.1083/jcb.108.4.1523.
Повний текст джерелаАнотація:
Transcriptional activity of acetylcholine receptor subunit genes was investigated in innervated and denervated chick skeletal muscle. The sciatic nerve of 3-d-old White Leghorn chicks was sectioned unilaterally; after various intervals, nuclei were isolated from operated and sham-operated animals, and run-on assays performed. Nuclei were incubated with 32P-UTP, and total RNA was extracted and hybridized onto filters containing an excess of subunit-specific DNA. Specific transcripts were detected by autoradiography and quantitated densitometrically. A sharp increase in transcriptional activity was observed to begin approximately 1/2 d after the operation and peak 1 d later when transcriptional rates reached approximately seven-, six-, and fivefold control levels for the alpha-, delta-, and gamma-subunit genes, respectively. The specificity of the effect was ascertained by normalization to total RNA synthesis and by the demonstration that several nonreceptor genes respond differently to denervation. These results suggest that a denervation signal reaches the genome to induce receptor expression. In addition, since the increase in mRNA levels significantly exceeds what can be accounted for by increased gene activity, posttranscriptional effects are suggested.
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Chen, Liqing, Xiaoxiao Qi, Dan Liang, Guiqi Li, Xiaofang Peng, Xiaohui Li, Bixia Ke, et al. "Human Fc-Conjugated Receptor Binding Domain-Based Recombinant Subunit Vaccines with Short Linker Induce Potent Neutralizing Antibodies against Multiple SARS-CoV-2 Variants." Vaccines 10, no. 9 (September 8, 2022): 1502. http://dx.doi.org/10.3390/vaccines10091502.
Повний текст джерелаАнотація:
The coronavirus disease-19 (COVID-19) pandemic has been ongoing since December 2019, with more than 6.3 million deaths reported globally as of August 2022. Despite the success of several SARS-CoV-2 vaccines, the rise in variants, some of which are resistant to the effects of vaccination, highlights the need for a so-called pan-coronavirus (universal) vaccine. Here, we performed an immunogenicity comparison of prototype vaccines containing spike protein receptor-binding domain (RBD) residues 319–541, or spike protein regions S1, S2 and S fused to a histidine-tagged or human IgG1 Fc (hFC) fragment with either a longer (six residues) or shorter (three residues) linker. While all recombinant protein vaccines developed were effective in eliciting humoral immunity, the RBD-hFc vaccine was able to generate a potent neutralizing antibody response as well as a cellular immune response. We then compared the effects of recombinant protein length and linker size on immunogenicity in vivo. We found that a longer recombinant RBD protein (residues 319–583; RBD-Plus-hFc) containing a small alanine linker (AAA) was able to trigger long-lasting, high-titer neutralizing antibodies in mice. Finally, we evaluated cross-neutralization of wild-type and mutant RBD-Plus-hFc vaccines against wild-type, Alpha, Beta, Delta and Omicron SARS-CoV-2 variants. Significantly, at the same antigen dose, wild-type RBD-Plus-hFc immune sera induced broadly neutralizing antibodies against wild-type, Alpha, Beta, Delta and Omicron variants. Taken together, our findings provide valuable information for the continued development of recombinant protein-based SARS-CoV-2 vaccines and a basic foundation for booster vaccinations to avoid reinfection with SARS-CoV-2 variants.
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Kusuyama, Kazuki, Toshiya Tachibana, Hiroki Yamanaka, Masamichi Okubo, Shinichi Yoshiya, and Koichi Noguchi. "Upregulation of calcium channel alpha-2-delta-1 subunit in dorsal horn contributes to spinal cord injury-induced tactile allodynia." Spine Journal 18, no. 6 (June 2018): 1062–69. http://dx.doi.org/10.1016/j.spinee.2018.01.010.
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McMillan, Christopher L. D., Armira Azuar, Jovin J. Y. Choo, Naphak Modhiran, Alberto A. Amarilla, Ariel Isaacs, Kate E. Honeyman, et al. "Dermal Delivery of a SARS-CoV-2 Subunit Vaccine Induces Immunogenicity against Variants of Concern." Vaccines 10, no. 4 (April 8, 2022): 578. http://dx.doi.org/10.3390/vaccines10040578.
Повний текст джерелаАнотація:
The ongoing coronavirus disease 2019 (COVID-19) pandemic continues to disrupt essential health services in 90 percent of countries today. The spike (S) protein found on the surface of the causative agent, the SARS-CoV-2 virus, has been the prime target for current vaccine research since antibodies directed against the S protein were found to neutralize the virus. However, as new variants emerge, mutations within the spike protein have given rise to potential immune evasion of the response generated by the current generation of SARS-CoV-2 vaccines. In this study, a modified, HexaPro S protein subunit vaccine, delivered using a needle-free high-density microarray patch (HD-MAP), was investigated for its immunogenicity and virus-neutralizing abilities. Mice given two doses of the vaccine candidate generated potent antibody responses capable of neutralizing the parental SARS-CoV-2 virus as well as the variants of concern, Alpha and Delta. These results demonstrate that this alternative vaccination strategy has the potential to mitigate the effect of emerging viral variants.
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Angeloni, D., M.-H. Wei, F.-M. Duh, BE Johnson, and MI Lerman. "A G-to-A single nucleotide polymorphism in the human Alpha 2 Delta 2 calcium channel subunit gene that maps at chromosome 3p21.3." Molecular and Cellular Probes 14, no. 1 (February 2000): 53–54. http://dx.doi.org/10.1006/mcpr.1999.0277.
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Santoli, D., R. O'Connor, A. Cesano, P. Phillips, T. L. Colt, B. Lange, S. C. Clark, and G. Rovera. "Synergistic and antagonistic effects of IL-1 alpha and IL-4, respectively, on the IL-2-dependent growth of a T cell receptor-gamma delta+ human T leukemia cell line." Journal of Immunology 144, no. 12 (June 15, 1990): 4703–11. http://dx.doi.org/10.4049/jimmunol.144.12.4703.
Повний текст джерелаАнотація:
Abstract The TALL-103/2 cell line was derived from an immature acute T lymphocytic leukemia with T-myeloid differentiating capacity. The leukemic cells were first expanded in recombinant human IL-3 in which they acquired a myeloid phenotype, and subsequently were adapted to grow in human rIL-2 in which they became lymphoid committed. The TALL-103/2 cell line expresses only T cell-specific differentiation Ag (CD2, CD3, CD7, and CD8) but has retained the CD33 myeloid Ag originally present on the IL-3 expanded population. By using mAb directed at the TCR-alpha beta or specific for framework determinants on human TCR-gamma and -delta chains, the TALL-103/2 cells were shown to be WT31-, TCR delta 1+, TCS-1+, and Ti gamma A-, thus representing a T cell subset expressing the nondisulfide-linked form of the TCR-gamma delta. The TALL-103/2 cells have been maintained for more than 1 y in the presence of human rIL-2 on which they are strictly dependent. Chemical cross-linking and immunofluorescence studies indicate the presence of both high and intermediate affinity IL-2R on the TALL-103/2 cells. Whereas mAb antiTac and H-31 with reactivity to the IL-2R alpha-chain (p55) compete only partially for the IL-2-induced proliferation of these cells, mAb TU27, specific to the IL-2R beta-subunit (p75), inhibits such growth completely even at high concentrations of IL-2. The interactions of the two T cell-stimulating factors IL-1 and IL-4 on the IL-2-dependent growth of TALL-103/2 cells were investigated. IL-1 alpha synergizes with IL-2 in supporting the short and long term growth of this cell line, whereas IL-4 abrogates its growth. These effects are, at least in part, due to the modulation of IL-2R expression induced by the two lymphokines. Functionally, the TALL-103/2 cells display MHC-nonrestricted cytotoxic activity that is significantly enhanced by addition of either IL-4, IL-6, or IFN-gamma. Because of its properties and its stable requirement for IL-2 for continuous growth, this T lymphocytic leukemia-derived cell line represents an interesting model to analyze ontogeny and function of leukemic T cells.
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Mahasirimongkol, Surakameth, Athiwat Khunphon, Oraya Kwangsukstid, Sompong Sapsutthipas, Mingkwan Wichaidit, Archawin Rojanawiwat, Nuanjun Wichuckchinda, et al. "The Pilot Study of Immunogenicity and Adverse Events of a COVID-19 Vaccine Regimen: Priming with Inactivated Whole SARS-CoV-2 Vaccine (CoronaVac) and Boosting with the Adenoviral Vector (ChAdOx1 nCoV-19) Vaccine." Vaccines 10, no. 4 (March 30, 2022): 536. http://dx.doi.org/10.3390/vaccines10040536.
Повний текст джерелаАнотація:
In response to the SARS-CoV-2 Delta variant, which partially escaped the vaccine-induced immunity provided by two doses of vaccination with CoronaVac (Sinovac), the National Vaccine Committee recommended the heterologous CoronaVac-ChAdOx1 (Oxford–AstraZeneca), a prime–boost vaccine regimen. This pilot study aimed to describe the immunogenicity and adverse events of the heterologous CoronaVac-ChAdOx1 regimen, in comparison with homologous CoronaVac, and homologous ChAdOx1. Between May and August 2021, we recruited a total of 354 participants from four vaccination groups: the CoronaVac-ChAdOx1 vaccinee (n = 155), the homologous CoronaVac vaccinee (n = 32), the homologous ChAdOx1 vaccinee (n = 47), and control group of COVID-19 patients (n = 120). Immunogenicity was evaluated by measuring the level of IgG antibodies against the receptor-binding domain (anti-SRBD) of the SARS-CoV-2 spike protein S1 subunit and the level of neutralizing antibodies (NAbs) against variants of concern (VOCs) using the plaque reduction neutralization test (PRNT) and pseudovirus neutralization test (pVNT). The safety profile was recorded by interviewing at the 1-month visit after vaccination. The anti-SRBD level after the second booster dose of the CoronaVac-ChAdOx1 group at 2 weeks was higher than 4 weeks. At 4 weeks after the second booster dose, the anti-SRBD level in the CoronaVac-ChAdOx1 group was significantly higher than either homologous CoronaVac, the homologous ChAdOx1 group, and Control group (p < 0.001). In the CoronaVac-ChAdOx1 group, the PRNT50 level against the wild-type (434.5 BAU/mL) was the highest; followed by Alpha variant (80.4), Delta variant (67.4), and Beta variant (19.8). The PVNT50 level was also found to be at its highest against the wild-type (432.1); followed by Delta variants (178.3), Alpha variants (163.9), and Beta variant (42.2), respectively. The AEs in the CoronaVac-ChAdOx1 group were well tolerated and generally unremarkable. The CoronaVac-ChAdOx1 heterologous regimen induced higher immunogenicity and a tolerable safety profile. In a situation when only CoronaVac-ChAdOx1 vaccines are available, they should be considered for use in responding to the Delta variant.