Fluorescent Auxin Analogs Report Two Auxin Binding Sites with Different Subcellular Distribution and Affinities: A Cue for Non-Transcriptional Auxin Signaling

The complexity of auxin signaling is partially due to multiple auxin receptors that trigger differential signaling. To obtain insight into the subcellular localization of auxin-binding sites, we used fluorescent auxin analogs that can undergo transport but do not deploy auxin signaling. Using fluorescent probes for different subcellular compartments, we can show that the fluorescent analog of 1-naphthaleneacetic acid (NAA) associates with the endoplasmic reticulum (ER) and tonoplast, while the fluorescent analog of indole acetic acid (IAA) binds to the ER. The binding of the fluorescent NAA analog to the ER can be outcompeted by unlabeled NAA, which allows us to estimate the affinity of NAA for this binding site to be around 1 μM. The non-transportable auxin 2,4-dichlorophenoxyacetic acid (2,4-D) interferes with the binding site for the fluorescent NAA analog at the tonoplast but not with the binding site for the fluorescent IAA analog at the ER. We integrate these data into a working model, where the tonoplast hosts a binding site with a high affinity for 2,4-D, while the ER hosts a binding site with high affinity for NAA. Thus, the differential subcellular localization of binding sites reflects the differential signaling in response to these artificial auxins

Thermospermine suppresses auxin-inducible xylem differentiation in Arabidopsis thaliana

Thermospermine, a structural isomer of spermine, is synthesized by a thermospermine synthase designated ACAULIS5 (ACL5). Thermospermine-deficient acl5 mutant of Arabidopsis thaliana shows severe dwarfism and excessive xylem differentiation. By screening for compounds that affect xylem differentiation in the acl5 mutant, we identified auxin analogs that remarkably enhanced xylem vessel differentiation in the acl5 mutant but not in the wild type. The xylem-inducing effect of auxin analogs was clearly suppressed by thermospermine, indicating that auxin-inducible xylem differentiation is normally limited by thermospermine. Here, we further characterized xylem-inducing effect of auxin analogs in various organs. Auxin analogs promoted protoxylem differentiation in roots and cotyledons in the acl5 mutant. Our results indicate that the opposite action between thermospermine and auxin in xylem differentiation is common in different organs and also suggest that thermospermine might be required for the suppression of protoxylem differentiation

A chemical biology approach reveals an opposite action between thermospermine and auxin in xylem development in Arabidopsis thaliana

Thermospermine, a structural isomer of spermine, is produced through the action of ACAULIS5 (ACL5) and suppresses xylem differentiation in Arabidopsis thaliana. To elucidate the molecular basis of the function of thermospermine, we screened chemical libraries for compounds that can modulate xylem differentiation in the acl5 mutant, which is deficient in thermospermine and shows a severe dwarf phenotype associated with excessive proliferation of xylem vessels. We found that the isooctyl ester of a synthetic auxin, 2,4-D, remarkably enhanced xylem vessel differentiation in acl5 seedlings. 2,4-D, 2,4-D analogs and IAA analogs, including 4-chloro IAA (4-Cl-IAA) and IAA ethyl ester, also enhanced xylem vessel formation, while IAA alone had little or no obvious effect on xylem differentiation. These effects of auxin analogs were observed only in the acl5 mutant but not in the wild type, and were suppressed by the anti-auxin, p-chlorophenoxyisobutyric acid (PCIB) and alpha-(phenyl ethyl-2-one)-IAA (PEO-IAA), and also by thermospermine. Furthermore, the suppressor of acaulis51-d (sac51-d) mutation, which causes SAC51 overexpression in the absence of thermospermine and suppresses the dwarf phenotype of acl5, also suppressed the effect of auxin analogs in acl5. These results suggest that the auxin signaling that promotes xylem differentiation is normally limited by SAC51-mediated thermospermine signaling but can be continually stimulated by exogenous auxin analogs in the absence of thermospermine. The opposite action between thermospermine and auxin may fine-tune the timing and spatial pattern of xylem differentiation

Novel protein extraction approach using micro-sized chamber for evaluation of proteins eluted from formalin-fixed paraffin-embedded tissue sections

We describe a novel antigen-retrieval method using a micro-sized chamber for mass spectrometry (MS) analysis to identify proteins that are preferentially eluted from formalin-fixed paraffin-embedded (FFPE) samples. This approach revealed that heat-induced antigen retrieval (HIAR) from an FFPE sample fixed on a glass slide not only improves protein identification, but also facilitates preferential elution of protein subsets corresponding to the properties of antigen-retrieval buffers. Our approach may contribute to an understanding of the mechanism of HIAR

Yokonolide B, a Novel Inhibitor of Auxin Action, Blocks Degradation of AUX/IAA Factors

Yokonolide B (YkB; also known as A82548A), a spiroketal-macrolide, was isolated from Streptomyces diastatochromogenes B59 in a screen for inhibitors of beta-glucoronidase expression under the control of an auxin-responsive promoter in Arabidopsis. YkB inhibits the expression of auxin-inducible genes as shown using native and synthetic auxin promoters as well as using expression profiling of 8300 Arabidopsis gene probes but does not affect expression of an abscisic acid- and a gibberellin A3-inducible gene. The mechanism of action of YkB is to block AUX/IAA protein degradation; however, YkB is not a general proteasome inhibitor. YkB blocks auxin-dependent cell division and auxin-regulated epinastic growth mediated by auxin-binding protein 1. Gain of function mutants such as shy2-2, slr1, and axr2-1 encoding AUX/IAA transcriptional repressors and loss of function mutants encoding components of the ubiquitin-proteolytic pathway such as axr1-3 and tir1-1, which display increased AUX/IAAs protein stability, are less sensitive to YkB, although axr1 and tir1 mutants were sensitive to MG132, a general proteasome inhibitor, consistent with a site of action downstream of AXR1 and TIR. YkB-treated seedlings displayed similar phenotypes as dominant AUX/IAA mutants. Taken together, these results indicate that YkB acts to block AUX/IAA protein degradation upstream of AXR and TIR, links a shared element upstream of AUX/IAA protein stability to auxin-induced cell division/elongation and to auxin-binding protein 1, and provides a new tool to dissect auxin signal transduction

Ultra fast quantum key distribution over a 97 km installed telecom fiber with wavelength-division multiplexing clock synchronization

We demonstrated ultra fast BB84 quantum key distribution (QKD) transmission at 625 MHz clock rate through a 97 km field-installed fiber using practical clock synchronization based on wavelength-division multiplexing (WDM). We succeeded in over-one-hour stable key generation at a high sifted key rate of 2.4 kbps and a low quantum bit error rate (QBER) of 2.9%. The asymptotic secure key rate was estimated to be 0.78-0.82 kbps from the transmission data with the decoy method of average photon numbers 0, 0.15, and 0.4 photons/pulse.Comment: 7 pages, 3 figures, v2 : We added a comment on the significance of our work, some minor corrections, and reference

A novel indole compound MA-35 attenuates renal fibrosis by inhibiting both TNF-α and TGF-β1 pathways

Renal fibrosis is closely related to chronic inflammation and is under the control of epigenetic regulations. Because the signaling of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) play key roles in progression of renal fibrosis, dual blockade of TGF-β1 and TNF-α is desired as its therapeutic approach. Here we screened small molecules showing anti-TNF-α activity in the compound library of indole derivatives. 11 out of 41 indole derivatives inhibited the TNF-α effect. Among them, Mitochonic Acid 35 (MA-35), 5-(3, 5-dimethoxybenzyloxy)-3-indoleacetic acid, showed the potent effect. The anti-TNF-α activity was mediated by inhibiting IκB kinase phosphorylation, which attenuated the LPS/GaIN-induced hepatic inflammation in the mice. Additionally, MA-35 concurrently showed an anti-TGF-β1 effect by inhibiting Smad3 phosphorylation, resulting in the downregulation of TGF-β1-induced fibrotic gene expression. In unilateral ureter obstructed mouse kidney, which is a renal fibrosis model, MA-35 attenuated renal inflammation and fibrosis with the downregulation of inflammatory cytokines and fibrotic gene expressions. Furthermore, MA-35 inhibited TGF-β1-induced H3K4me1 histone modification of the fibrotic gene promoter, leading to a decrease in the fibrotic gene expression. MA-35 affects multiple signaling pathways involved in the fibrosis and may recover epigenetic modification; therefore, it could possibly be a novel therapeutic drug for fibrosis

Metformin Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background: Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model. Methodology/Principal Findings: Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH. Conclusions/Significance: These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance. © 2012 Kita et al

Mitochonic Acid 5 (MA-5) Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases

Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model “Mitomouse” (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial

Mg-chelatase H subunit affects ABA signaling in stomatal guard cells, but is not an ABA receptor in Arabidopsis thaliana

Mg-chelatase H subunit (CHLH) is a multifunctional protein involved in chlorophyll synthesis, plastid-to-nucleus retrograde signaling, and ABA perception. However, whether CHLH acts as an actual ABA receptor remains controversial. Here we present evidence that CHLH affects ABA signaling in stomatal guard cells but is not itself an ABA receptor. We screened ethyl methanesulfonate-treated Arabidopsis thaliana plants with a focus on stomatal aperture-dependent water loss in detached leaves and isolated a rapid transpiration in detached leaves 1 (rtl1) mutant that we identified as a novel missense mutant of CHLH. The rtl1 and CHLH RNAi plants showed phenotypes in which stomatal movements were insensitive to ABA, while the rtl1 phenotype showed normal sensitivity to ABA with respect to seed germination and root growth. ABA-binding analyses using 3H-labeled ABA revealed that recombinant CHLH did not bind ABA, but recombinant pyrabactin resistance 1, a reliable ABA receptor used as a control, showed specific binding. Moreover, we found that the rtl1 mutant showed ABA-induced stomatal closure when a high concentration of extracellular Ca2+ was present and that a knockout mutant of Mg-chelatase I subunit (chli1) showed the same ABA-insensitive phenotype as rtl1. These results suggest that the Mg-chelatase complex as a whole affects the ABA-signaling pathway for stomatal movements