Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response

And Now for Something Completely Different: Diversity in Ligand-Dependent Activation of Ah Receptor Responses.

Ligand-dependent activation of the Ah receptor (AhR) can result in an extremely diverse spectrum of biological and toxic effects that occur in a ligand-, species- and tissue-specific manner. While the classical mechanism of AhR-dependent signal transduction is directly related to its ability to modulate gene expression, the dramatic diversity in responses observed following AhR activation or inhibition is inconsistent with a single molecular mechanism of AhR action. Recent studies have revealed that key molecular events underlying the AhR signaling pathway are significantly more varied and complex than previously established, and the specificity and diversity in AhR response can be selectively modulated by a variety of factors. Here we describe new insights into the mechanistic diversity in AhR signal transduction that can contribute to ligand-, species- and tissue-specific differences in AhR reponse

Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response

Fast synaptic transmission in the goldfish CNS mediated by multiple nicotinic receptors

In this study, fast synaptic transmission at vertebrate CNS connections mediated by several different nicotinic ACh receptors (nAChRs) was investigated with paired recordings from pre- and postsynaptic neurons. Analysis of the response kinetics at the axo-axonic connections between the Mauthner (M-) axon and cranial relay neurons (CRN) indicates up to three main components are present and can be characterized by fast, ∼1.5 ms, intermediate, ∼6 ms and slow, ∼15 ms, decay time constants. Further analysis indicates most responses have multiexponential decays and each response falls into one of six classes dependent on the weight and combination of kinetic components. Pharmacological results suggest that up to three nAChRs, α7*, α3β2* and α3β4*, mediate the postsynaptic responses and correspond to the fast, intermediate and slow decay components, respectively. The fast decay component is blocked by ∼35 nm methyllycaconitine (MLA), 100 nmα-bungarotoxin (α-Btx) or 150 nmα-conotoxin (α-Ctx) ArIB. The intermediate decay component is blocked by 2 μm dihydro-beta-erythroidine (DHβE) or 200 nmα-Ctx GIC. The slow decay component is blocked by 10 μmα-Ctx AuIB, but not by 7.25 μm DHβE. Intriguingly, the mEPSPs (minis) at connections with evoked EPSPs best fitted by multiple exponentials, were not composite; rather, there were multiple populations of minis, each with single exponential decay times corresponding to those of the different evoked EPSP components. This indicates that the different receptors are topographically segregated at the connection between the M-axon and CRN axon. These results suggest that, as with glutamate, fast nicotinic synaptic transmission in the CNS can be mediated by multiple receptors in the same postsynaptic neuron. The coexistence of EPSPs of different durations may have implications for network function and plasticity

Coronal Heating as Determined by the Solar Flare Frequency Distribution Obtained by Aggregating Case Studies

Flare frequency distributions represent a key approach to addressing one of the largest problems in solar and stellar physics: determining the mechanism that counter-intuitively heats coronae to temperatures that are orders of magnitude hotter than the corresponding photospheres. It is widely accepted that the magnetic field is responsible for the heating, but there are two competing mechanisms that could explain it: nanoflares or Alfv\'en waves. To date, neither can be directly observed. Nanoflares are, by definition, extremely small, but their aggregate energy release could represent a substantial heating mechanism, presuming they are sufficiently abundant. One way to test this presumption is via the flare frequency distribution, which describes how often flares of various energies occur. If the slope of the power law fitting the flare frequency distribution is above a critical threshold, $\alpha=2$ as established in prior literature, then there should be a sufficient abundance of nanoflares to explain coronal heating. We performed $>$600 case studies of solar flares, made possible by an unprecedented number of data analysts via three semesters of an undergraduate physics laboratory course. This allowed us to include two crucial, but nontrivial, analysis methods: pre-flare baseline subtraction and computation of the flare energy, which requires determining flare start and stop times. We aggregated the results of these analyses into a statistical study to determine that $\alpha = 1.63 \pm 0.03$. This is below the critical threshold, suggesting that Alfv\'en waves are an important driver of coronal heating.Comment: 1,002 authors, 14 pages, 4 figures, 3 tables, published by The Astrophysical Journal on 2023-05-09, volume 948, page 7