• Toxicological Research
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• v.8 no.2
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• pp.343-357
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• 1992

Tumor necrosis factor-${\alpha}$(TNF), a polypeptide hormone secreted primarily by activated macrophages, was originally identified on the basis of its ability to cause hemorrhagic necrosis and tumor regression in vivo. Subsequently, TNF has been shown to be an important component of the host responses to infection and cancer and may mediate the wasting syndrome known as cachexia. These systemic actions of TNF are reflected in its diverse effects on target cells in vitro. TNF initiates its diverse cellular actions by binding to specific cell surface receptors. Although TNF receptors have been identified on most of animal cells, regulation of these receptors and the mechanisms which transduce TNF receptor binding into cellular responses are not well understood. Therefore, in the present study, the mechanisms how TNF receptors are being regulated and how TNF receptor binding is being transduced into cellular responses were investigated in rat liver plasma membranes (PM) and ME-180 human cervical carcinoma cell lines. $^{125}I$-TNF bound to high ($K_d=1.51{\pm}0.35nM$)affinity receptors in rat liver PM. Solubilization of PM with 1% Triton X-100 increased both high affinity (from $0.33{\pm}0.04\;to\;1.67{\pm}0.05$ pmoles/mg protein) and low affinity (from $1.92{\pm}0.16\;to\;7.57{\pm}0.50$ pmoles/mg protein) TNF binding without affecting the affinities for TNF, suggesting the presence of a large latent pool of TNF receptors. Affinity labeling of receptors whether from PM or solubilized PM resulted in cross-linking of $^{125}I$-TNF into $M_r$ 130 kDa, 90 kDa and 66kDa complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, $^{125}I$-TNF binding to control and TNF-pretreated membranes were assayed. Specific binding was increased by pretreatment with TNF (P<0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF. As a next step, the post-receptor events induced by TNF were examined. Although the signal transduction pathways for TNF have not been delineated clearly, the actions of many other hormones are mediated by the reversible phosphorylation of specific enzymes or target proteins. The present study demonstrated that TNF induces phosphorylation of 28 kDa protein (p28). Two dimensional soidum dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) resolved the 28kDa phosphoprotein into two isoforms having pIs of 6.2 and 6.1. The pIs and relative molecular weight of p28 were consistent with those of a previously characterized mRNA cap binding protein. mRNA cap binding proteins are a class of translation initiation factors that recognize the 7-methylguanosine cap structure found on the 5' end of eukaryotic mRNAs. In vitro, these proteins are defined by their specific elution from affinity columns composed of 7-methylguanosine 5'-triphosphate($m^7$GTP)-Sepharose. Affinity purification of mRNA cap binding proteins from control and TNF treated ME-180 cells proved that TNF rapidly stimulates phosphorylation of an mRNA cap binding protein. Phosphorylation occurred in several cell types that are important in vitro models of TNF action. The mRNA cap binding protein phosphorylated in response to TNF treatment was purifice, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor-4E(eIF-4E). These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the diverse cellular actions of TNF.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.63 no.1
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• pp.25-34
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• 2018

This study was conducted to investigate the germination and proteome profile characteristics of wheat seeds treated under various concentrations of abscisic acid (ABA). After-ripening, the seeds of three wheat cultivars (Baegjoong, Keumkang, and Uri) showing different levels of dormancy were used. Germination index and germination rate of the cultivars was higher than 0.95% and 98%, respectively, and these were not significantly different under 0, 10, 30, and $50{\mu}M$ ABA at 7 d after germination. However, the growth of the shoot and radicle was significantly inhibited at 10, 30, and $50{\mu}M$ ABA compared to that at $0{\mu}M$ ABA. Mean ABA content of the embryos of seeds germinated at 0 and $50{\mu}M$ ABA for 7 d was 0.8 and $269.0ngmg^{-1}DW$, respectively. Proteins extracted from embryos germinated for 4 d were analyzed by two-dimensional gel electrophoresis, and proteins showing a difference of 1.5-fold or greater in their spot volume relative to that of $0{\mu}M$ ABA were identified. The expression of four protein spots increased at $50{\mu}M$ ABA and two protein spots were detected only at $50{\mu}M$ ABA; these six proteins were all identified as globulin types. Conversely, the expression of three protein spots decreased at $50{\mu}M$ ABA and were identified as cytosolic glutamine sysnthetase, isocitrate dehydrogenase, and S-adenosylmethionine synthetase 2. In conclusion, ABA did not inhibit the germination rate regardless of pre-harvest sprouting characteristics of the cultivars. However, the growth of the shoot and radicle was significantly inhibited by ABA, most likely through the down regulation of glutamine, methyl group donor, and polyamines biosynthesis, among others, while accompanied by globulin accumulation in the embryos.