The NF-κB signaling system plays an important regulatory role in the control of many biological processes. The activities of NF-κB signaling networks and the expression of their target genes are frequently elevated in pathophysiological situations including inflammation, infection, and cancer. In these conditions, the outcome of NF-κB activity can vary according to (i) differential activation states, (ii) the pattern of genomic recruitment of the NF-κB subunits, and (iii) cellular heterogeneity. Additionally, the cytosolic NF-κB activation steps leading to the liberation of DNA-binding dimers need to be distinguished from the less understood nuclear pathways that are ultimately responsible for NF-κB target gene specificity. This raises the need to more precisely determine the NF-κB activation status not only for the purpose of basic research, but also in (future) clinical applications. Here we review a compendium of different methods that have been developed to assess the NF-κB activation status in vitro and in vivo. We also discuss recent advances that allow the assessment of several NF-κB features simultaneously at the single cell level.
Background:The kernel number of maize (Zea mays L.) at maturity is mainly determined at the time around pollination. Kernel abortion frequently occurs during this period, leading to grain yield depressions. Plasma membrane (PM) H + -ATPase was identified as a key enzyme responsible for supply of assimilates to the developing maize kernels shortly after pollination. Aims:This study aimed at stimulating PM H + -ATPase activity in the kernels by in vivo application of the auxin indole-3-acetic acid (IAA) to maize plants at flowering, leading to an improved hexose uptake and finally to a better kernel set.Methods: Maize plants were cultivated under well-watered conditions using the container technique. IAA was applied to unstressed maize plants twice, 2 days before controlled pollination and at pollination (application rate per plant: 1.9 mL of 1.5 mM IAA).The developing kernels were harvested 2 days after pollination, and PM vesicles were isolated and purified using two-phase partitioning. Results:The in vitro hydrolytic activity of the PM H + -ATPase was significantly stimulated by 22% due to in vivo IAA application (control: 0.99 ± 0.05, IAA treatment: 1.21 ± 0.03* μmol inorganic phosphate mg -1 protein min -1 ). V max was significantly increased by IAA treatment, whereas K m was reduced. The maximal pH gradient (ΔA 492 ) at the PM was increased by 10% (control: 0.071 ± 0.002, IAA treatment: 0.078 ± 0.002*). IAA caused a significant increase of PM H + -ATPase abundance in the vesicles. Concentrations of sucrose and hexoses as well as acid invertase activity in the kernels were unaffected by IAA treatment. However, at maturity kernel numbers per cob were significantly decreased causing grain yield reductions of 19%. Conclusions:Increased PM H + -ATPase activity could not be translated into grain yield improvements. Probably the auxin application occurred too early during kernel development. As cytokinins play a key role during pollination, auxin application at this stage may have disturbed the phytohormone balance, causing disruption of cell division and a rather early onset of cell extension due to increased IAA concentrations. In further studies, itThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
The NF-κB system is a key transcriptional pathway that regulates innate and adaptive immunity because it triggers the activation and differentiation processes of lymphocytes and myeloid cells during immune responses. In most instances, binding to cytoplasmic inhibitory IκB proteins sequesters NF-κB into an inactive state, while a plethora of external triggers activate three complex signaling cascades that mediate the release and nuclear translocation of the NF-κB DNA-binding subunits. In addition to these cytosolic steps (level 1 of NF-κB regulation), NF-κB activity is also controlled in the nucleus by signaling events, cofactors and the chromatin environment to precisely determine chromatin recruitment and the specificity and timing of target gene transcription (level 2 of NF-κB regulation). Here, we discuss an additional layer of the NF-κB system that manifests in various steps of post-transcriptional gene expression and protein secretion. This less-studied regulatory level allows reduction of (transcriptional) noise and signal integration and endows time-shifted control of the secretion of inflammatory mediators. Detailed knowledge of these steps is important, as dysregulated post-transcriptional NF-κB signaling circuits are likely to foster chronic inflammation and contribute to the formation and maintenance of a tumor-promoting microenvironment.
The family of hypoxia-inducible transcription factors (HIF) is activated to adapt cells to low oxygen conditions, but is also known to regulate some biological processes under normoxic conditions. Here we show that HIF-1α protein levels transiently increase during the G1 phase of the cell cycle (designated as G1-HIF) in an AMP-activated protein kinase (AMPK)-dependent manner. The transient elimination of G1-HIF by a degron system revealed its contribution to cell survival under unfavorable metabolic conditions. Indeed, G1-HIF plays a key role in the cell cycle-dependent expression of genes encoding metabolic regulators and the maintenance of mTOR activity under conditions of nutrient deprivation. Accordingly, transient elimination of G1-HIF led to a significant reduction in the concentration of key proteinogenic amino acids and carbohydrates. These data indicate that G1-HIF acts as a cell cycle-dependent surveillance factor that prevents the onset of starvation-induced apoptosis.
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