Soy-Derived Equol Induces Antioxidant Activity in Zebrafish in an Nrf2-Independent Manner

Antioxidant effects of soy-derived isoflavones are predicted to be mediated by the Keap1-Nrf2 pathway. Recently, we constructed an assay system to evaluate the antioxidant effects of dietary phytochemicals in zebrafish and revealed a relationship between these effects and the Keap1-Nrf2 pathway. In this study, we used this system to examine the antioxidant effects of seven isoflavones. Among those seven, equol showed strong antioxidant effects when arsenite was used as an oxidative stressor. The antioxidant effect of equol was also shown in Nrf2-mutant zebrafish nfe2l2a^fh318, suggesting that this effect was not mediated by the Keap1-Nrf2 pathway. To elucidate this unidentified mechanism, the gene expression profiles of equol-treated larvae were analyzed using RNA-seq and qRT-PCR, while no noticeable changes were detected in the expression of genes related to antioxidant effects, except weak induction of Nrf2 target genes. Because nfe2l2a^fh318 is an amino acid-substitution mutant (Arg485Lue), we considered that the antioxidant effect of equol in this mutant might be due to residual Nrf2 activity. To examine this possibility, we generated an Nrf2-knockout zebrafish nfe2l2a^it321 using CRISPR-Cas9 and analyzed the antioxidant effect of equol. As a result, equol showed strong antioxidant effects even in Nrf2-knockout larvae, suggesting that equol indeed upregulates antioxidant activity in zebrafish in an Nrf2-independent manner.     

Nrf2 Activation Sensitizes K-Ras Mutant Pancreatic Cancer Cells to Glutaminase Inhibition

Pancreatic cancer remains intractable owing to the lack of effective therapy for unresectable cases. Activating mutations of K-ras are frequently found in pancreatic cancers, but these have not yet been targeted by cancer therapies. The Keap1-Nrf2 system plays a crucial role in mediating the oxidative stress response, which also contributes to cancer progression. Nrf2 activation reprograms the metabolic profile to promote the proliferation of cancer cells. A recent report suggested that K-ras- and Nrf2-active lung cancer cells are sensitive to glutamine depletion. This finding led to the recognition of glutaminase inhibitors as novel anticancer agents. In the current study, we used murine pancreatic cancer tissues driven by mutant K-ras and p53 to establish cell lines expressing constitutively activated Nrf2. Genetic or pharmacological Nrf2 activation in cells via Keap1 deletion or Nrf2 activation sensitized cells to glutaminase inhibition. This phenomenon was confirmed to be dependent on K-ras activation in human pancreatic cancer cell lines harboring mutant K-ras, i.e., Panc-1 and MiaPaCa-2 in response to DEM pretreatment. This phenomenon was not observed in BxPC3 cells harboring wildtype K-ras. These results indicate the possibility of employing Nrf2 activation and glutaminase inhibition as novel therapeutic interventions for K-ras mutant pancreatic cancers.     

Loss of Heterozygosity for KrasG12D Promotes Malignant Phenotype of Pancreatic Ductal Adenocarcinoma by Activating HIF-2α-c-Myc-Regulated Glutamine Metabolism

Loss of heterozygosity (LOH) for KRAS, in which a wild-type KRAS allele is progressively lost, promotes invasive and migratory abilities of pancreatic ductal adenocarcinoma (PDAC) cells and tissues. Moreover, the occurrence of Kras^G12D-LOH activates nonclassical glutamine metabolism, which is related to the malignant behavior of PDAC cells. Herein, we aim to demonstrate the regulatory link between hypoxia-inducible factor-2α (HIF-2α) and glutamine metabolism that mediates malignant phenotypes in Kras^G12D-LOH PDAC cells. HIF-2α-shRNA knockdown lentivirus transfection and metabolite analysis were performed in Kras^G12D-LOH and Kras^G12D cell lines, respectively. Cell proliferation, migration, and invasion were examined using Cell Counting Kit-8, colony formation, and Transwell assays. Cell cycle phase and apoptosis were determined using flow cytometry. Western blotting and real-time quantitative PCR were also performed. Additionally, a subcutaneous xenograft mouse model was established. LOH stimulated HIF-2α activity and transactivated c-Myc, which has a central regulatory effect on glutamine metabolism independent of hypoxia. Meanwhile, HIF-2α silencing repressed Kras^G12D-LOH PDAC cell proliferation, invasion, and migration. HIF-2α knockdown inhibited glutamine uptake and GOT1 expression via a c-Myc-dependent pathway. Collectively, Kras^G12D-LOH can activate HIF-2α to regulate c-Myc-mediated glutamine metabolism and promote malignant phenotypes. Moreover, targeting HIF-2α-c-Myc regulated nonclassical glutamine metabolism, providing a new therapeutic perspective for Kras^G12D-LOH PDAC.     

Advanced Glycation End Product-Induced Astrocytic Differentiation of Cultured Neurospheres through Inhibition of Notch-Hes1 Pathway-Mediated Neurogenesis

This study aims to investigate the roles of the Notch-Hes1 pathway in the advanced glycation end product (AGE)-mediated differentiation of neural stem cells (NSCs). We prepared pLentiLox3.7 lentiviral vectors that express short hairpin RNA (shRNA) against Notch1 and transfected it into NSCs. Cell differentiation was analyzed under confocal laser-scanning microscopy. The percentage of neurons and astrocytes was quantified by normalizing the total number of TUJ1⁺ (Neuron-specific class III β-tubulin) and GFAP⁺ (Glial fibrillary acidic protein) cells to the total number of Hoechst 33342-labeled cell nuclei. The protein and gene expression of Notch-Hes1 pathway components was examined via western blot analysis and real-time PCR. After 1 week of incubation, we found that AGE-bovine serum albumin (BSA) (400 μg/mL) induced the astrocytic differentiation of cultured neurospheres and inhibited neuronal formation. The expression of Notch-Hes1 pathway components was upregulated in the cells in the AGE-BSA culture medium. Immunoblot analysis indicated that shRNA silencing of Notch1 expression in NSCs significantly increases neurogenesis and suppresses astrocytic differentiation in NSCs incubated with AGE-BSA. AGEs promote the astrocytic differentiation of cultured neurospheres by inhibiting neurogenesis through the Notch-Hes1 pathway, providing a potential therapeutic target for hyperglycemia-related cognitive deficits.     

Influence of nitrogen nutrition and metabolism on ammonia volatilization in plants

Barley (Hordeum vulgare L. cv. Golf) was grown in solution culture with controlled nitrogen availability in order to study the influence of nitrogen nutrition on ammonia emission from the leaves. Ammonia emission measured in cuvettes connected to an automatic NH₃ monitor was close to zero for nitrate grown plants but increased to 0.9–1.3 nmol NH₃ m^-2 leaf area s^-1 after 3–5 days of ammonium nutrition. Increasing concentrations from 0.5 to 10 mM NH₄ ⁺ in the root medium increased NH₃ emission from the shoots, root glutamine synthetase activity and NH₄ ⁺ concentrations in apoplast, xylem sap and bulk tissue, while apoplastic pH values decreased.Inhibition of glutamine synthetase in nitrate grown barley plants by addition of 1 mM methionine sulfoximine (MSO) to the root medium caused ammonia emission to increase 5 to 10-fold after 2–3 hours. At the same time shoot tissue ammonium concentrations started to increase. Addition of an inhibitor of photorespiration, 1 mM pyrid-2-yl hydroxymethane sulfonate (HPMS) reduced this increase in ammonia emission showing a relation between NH₃ emission and photorespiration.Oil seed rape (Brassica napus L. cv. Global) plants grown at 3 different nitogen levels (2N, 4N and 7N) in a sand/soil mixture showed increasing NH₃ compensation points with increasing N level. This increase was highly correlated with increasing NH₄ ⁺ concentrations in the leaf apoplast and total leaf tissue. The NH₃ compensation points could be succesfully predicted on basis of the pH and NH₄ ⁺ concentration in the leaf apoplast.     

Spadin Modulates Astrocytic Passive Conductance via Inhibition of TWIK-1/TREK-1 Heterodimeric Channels

Astrocytes, the most abundant cell type in the brain, are non-excitable cells and play critical roles in brain function. Mature astrocytes typically exhibit a linear current–voltage relationship termed passive conductance, which is believed to enable astrocytes to maintain potassium homeostasis in the brain. We previously demonstrated that TWIK-1/TREK-1 heterodimeric channels mainly contribute to astrocytic passive conductance. However, the molecular identity of astrocytic passive conductance is still controversial and needs to be elucidated. Here, we report that spadin, an inhibitor of TREK-1, can dramatically reduce astrocytic passive conductance in brain slices. A series of gene silencing experiments demonstrated that spadin-sensitive currents are mediated by TWIK-1/TREK-1 heterodimeric channels in cultured astrocytes and hippocampal astrocytes from brain slices. Our study clearly showed that TWIK-1/TREK-1-heterodimeric channels can act as the main molecular machinery of astrocytic passive conductance, and suggested that spadin can be used as a specific inhibitor to control astrocytic passive conductance.     

The Catalytic Mechanism of Pdx2 Glutaminase Driven by a Cys-His-Glu Triad: A Computational Study

The catalytic mechanism of Pdx2 was studied with atomic detail employing the computational ONIOM hybrid QM/MM methodology. Pdx2 employs a Cys-His-Glu catalytic triad to deaminate glutamine to glutamate and ammonia – the source of the nitrogen of pyridoxal 5’-phosphate (PLP). This enzyme is, therefore, a rate-limiting step in the PLP biosynthetic pathway of Malaria and Tuberculosis pathogens that rely on this mechanism to obtain PLP. For this reason, Pdx2 is considered a novel and promising drug target to treat these diseases. The results obtained show that the catalytic mechanism of Pdx2 occurs in six steps that can be divided into four stages: (i) activation of Cys₈₇, (ii) deamination of glutamine with the formation of the glutamyl-thioester intermediate, (iii) hydrolysis of the formed intermediate, and (iv) enzymatic turnover. The kinetic data available in the literature (19.1–19.5 kcal mol⁻¹) agree very well with the calculated free energy barrier of the hydrolytic step (18.2 kcal.mol⁻¹¹), which is the rate-limiting step of the catalytic process when substrate is readily available in the active site. This catalytic mechanism differs from other known amidases in three main points: i) it requires the activation of the nucleophile Cys₈₇ to a thiolate; ii) the hydrolysis occurs in a single step and therefore does not require the formation of a second tetrahedral reaction intermediate, as it is proposed, and iii) Glu₁₉₈ does not have a direct role in the catalytic process. Together, these results can be used for the synthesis of new transition state analogue inhibitors capable of inhibiting Pdx2 and impair diseases like Malaria and Tuberculosis.     

Identification and Structural Analysis of New Nrf2 Activators by Mechanism‐Based Chemical Transformation of 15‐Deoxy‐Δ12, 14‐PGJ2

Mechanism‐based chemical transformation of 15‐deoxy‐Δ^{12, 14}‐PGJ₂ (15d‐PGJ₂) resulted in a series of new NF‐E2‐related factor‐2 (Nrf2) activators and detailed elucidation of the function of each electrophilic binding site. In addition, HO‐1 expression resulting from Nrf2 activation through enhanced dissociation of the Keap1–Nrf2 complex by the new activators was proved.     

Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism

The plasma membrane transporter ASCT2 is a well-known Na⁺-dependent obligatory antiporter of neutral amino acids. The crucial role of the residue C467 in the recognition and binding of the ASCT2 substrate glutamine, has been highlighted by structure/function relationship studies. The reconstitution in proteoliposomes of the human ASCT2 produced in P. pastoris is here employed to unveil another role of the C467 residue in the transport reaction. Indeed, the site-directed mutant C467A displayed a novel property of the transporter, i.e., the ability of mediating a low but measurable unidirectional transport of [³H]-glutamine. This reaction conforms to the main features of the ASCT2-mediated transport, namely the Na⁺-dependence, the pH dependence, the stimulation by cholesterol included in the proteoliposome membrane, and the specific inhibition by other common substrates of the reconstituted human ASCT2. Interestingly, the WT protein cannot catalyze the unidirectional transport of [³H]-glutamine, demonstrating an unspecific phenomenon. This difference is in favor of a structural conformational change between a WT and C467A mutant that triggers the appearance of the unidirectional flux; this feature has been investigated by comparing the available 3D structures in two different conformations, and two homology models built on the basis of hEAAT1 and GLT_Ph.     

NLRP2 Is Overexpressed in Spinal Astrocytes at the Peak of Mechanical Pain Sensitivity during Complete Freund Adjuvant-Induced Persistent Pain

Our earlier findings revealed that interleukin-1 receptor type-1 (IL-1R1) was overexpressed in spinal neurons, and IL-1R1-deficient mice showed significant attenuation of thermal and mechanical allodynia during the course of the Complete Freund adjuvant (CFA)-induced persistent pain model. In the present study, we found that a ligand of IL-1R1, termed interleukin-1β (IL-1β), is also significantly overexpressed at the peak of mechanical pain sensitivity in the CFA-evoked pain model. Analysis of cellular distribution and modeling using IMARIS software showed that in the lumbar spinal dorsal horn, IL-1β is significantly elevated by astrocytic expression. Maturation of IL-1β to its active form is facilitated by the formation of the multiprotein complex called inflammasome; thus, we tested the expression of NOD-like receptor proteins (NLRPs) in astrocytes. At the peak of mechanical allodynia, we found expression of the NLRP2 inflammasome sensor and its significantly elevated co-localization with the GFAP astrocytic marker, while NLRP3 was moderately present and NLRP1 showed total segregation from the astrocytic profiles. Our results indicate that peripheral CFA injection induces NLRP2 inflammasome and IL-1β expression in spinal astrocytes. The release of mature IL-1β can contribute to the maintenance of persistent pain by acting on its neuronally expressed receptor, which can lead to altered neuronal excitability.     

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα–PGC-1α–ERRα Circuit after Traumatic Brain Injury

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)–estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O₂ consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα–PGC-1α–ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα–PGC-1α–ERRα circuit and consequent oxidative phosphorylation, O₂ consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.     

Emerging Roles of Astrocyte Kir4.1 Channels in the Pathogenesis and Treatment of Brain Diseases

Inwardly rectifying Kir4.1 channels in astrocytes mediate spatial potassium (K⁺) buffering, a clearance mechanism for excessive extracellular K⁺, in tripartite synapses. In addition to K⁺ homeostasis, astrocytic Kir4.1 channels also play an essential role in regulating extracellular glutamate levels via coupling with glutamate transporters. Moreover, Kir4.1 channels act as novel modulators of the expression of brain-derived neurotrophic factor (BDNF) in astrocytes. Specifically, inhibition of astrocytic Kir4.1 channels elevates extracellular K⁺ and glutamate levels at synapses and facilitates BDNF expression in astrocytes. These changes elevate neural excitability, which may facilitate synaptic plasticity and connectivity. In this article, we summarize the functions and pharmacological features of Kir4.1 channels in astrocytes and highlight the importance of these channels in the treatment of brain diseases. Although further validation in animal models and human patients is required, astrocytic Kir4.1 channel could potentially serve as a novel therapeutic target for the treatment of depressive disorders and epilepsy.     

Abnormal Ca2+ Signals in Reactive Astrocytes as a Common Cause of Brain Diseases

In pathological brain conditions, glial cells become reactive and show a variety of responses. We examined Ca²⁺ signals in pathological brains and found that reactive astrocytes share abnormal Ca²⁺ signals, even in different types of diseases. In a neuropathic pain model, astrocytes in the primary sensory cortex became reactive and showed frequent Ca²⁺ signals, resulting in the production of synaptogenic molecules, which led to misconnections of tactile and pain networks in the sensory cortex, thus causing neuropathic pain. In an epileptogenic model, hippocampal astrocytes also became reactive and showed frequent Ca²⁺ signals. In an Alexander disease (AxD) model, hGFAP-R239H knock-in mice showed accumulation of Rosenthal fibers, a typical pathological marker of AxD, and excessively large Ca²⁺ signals. Because the abnormal astrocytic Ca²⁺ signals observed in the above three disease models are dependent on type II inositol 1,4,5-trisphosphate receptors (IP₃RII), we reanalyzed these pathological events using IP₃RII-deficient mice and found that all abnormal Ca²⁺ signals and pathologies were markedly reduced. These findings indicate that abnormal Ca²⁺ signaling is not only a consequence but may also be greatly involved in the cause of these diseases. Abnormal Ca²⁺ signals in reactive astrocytes may represent an underlying pathology common to multiple diseases.     

Ammonia sorption by peat and N fractionation in some peat-ammonia systems

Ammonia sorption is an important peat property for making composts and peat-mineral fertilizers. In this study, we investigated the influence of moisture content of fibric, hemic and sapric peat materials on ammonia sorption capacity, and we determined the N forms and the amount of solubilized humic substances following peat ammoniation. Ammonia sorption capacity increased curvilinearly with degree of decomposition from the fibric to the sapric peats, and increased with peat moisture content up to 600 g H₂O [kg wet peat]^–1 in the range of 400 to 700 g H₂O [kg wet peat]^–1. The amount of chemically sorbed ammonia was highest in the sapric peat containing [600 g H₂O] [kg wet peat]^–1. On a dry weight basis, maximum sorption capacity was 24 g NH₃-N kg^–1 for the fibric peat, 29 g NH₃-N kg^–1 for the hemic peat and 49 g NH₃-N kg^–1 for the sapric peat. Ammonia loss of physically retained ammonia was greatest at the highest moisture content and for the least decomposed peat. At ammoniation rates exceeding 30 g NH₃ [kg dry peat]^–1, exchangeable ammonium and acid-hydrolyzable N were the dominant N fractions in ammoniated peats. Peat ammoniation increased significantly (p < 0.001) the amounts of water-soluble carbon in all peat materials, of fulvic acid in the fibric peat and of both humic and fulvic acids in the hemic and the sapric peats. At maximum ammonia sorption capacity, the total amount of water-soluble and alkali-extractable organic matter reached 338 g kg^–1 in fibric peat, 683 g kg^–1 in the hemic peat and 848 g kg^–1 in the sapric peat. The hemic and sapric peats appeared more appropriate than the fibric peat for making peat-base fertilizers.