Kaempferol Suppresses the Activation of Mast Cells by Modulating the Expression of FcεRI and SHIP1.

In the present study, we evaluated the effects of kaempferol on bone marrow-derived mast cells (BMMCs). Kaempferol treatment significantly and dose-dependently inhibited IgE-induced degranulation, and cytokine production of BMMCs under the condition that cell viability was maintained. Kaempferol downregulated the surface expression levels of FcεRI on BMMCs, but the mRNA levels of FcεRIα, ß, and γ-chains were not changed by kaempferol treatment. Furthermore, the kaempferol-mediated downregulation of surface FcεRI on BMMCs was still observed when protein synthesis or protein transporter was inhibited. We also found that kaempferol inhibited both LPS- and IL-33-induced IL-6 production from BMMCs, without affecting the expression levels of their receptors, TLR4 and ST2. Although kaempferol treatment increased the protein amount of NF-E2-related factor 2 (NRF2)-a master transcription factor of antioxidant stress-in BMMCs, the inhibition of NRF2 did not alter the suppressive effect of kaempferol on degranulation. Finally, we found that kaempferol treatment increased the levels of mRNA and protein of a phosphatase SHIP1 in BMMCs. The kaempferol-induced upregulation of SHIP1 was also observed in peritoneal MCs. The knockdown of SHIP1 by siRNA significantly enhanced IgE-induced degranulation of BMMCs. A Western blotting analysis showed that IgE-induced phosphorylation of PLCγ was suppressed in kaempferol-treated BMMCs. These results indicate that kaempferol inhibited the IgE-induced activation of BMMCs by downregulating FcεRI and upregulating SHIP1, and the SHIP1 increase is involved in the suppression of various signaling-mediated stimulations of BMMCs, such as those associated with TLR4 and ST2.

First description of a clinical glutamine-dependent Escherichia coli with a missense mutation in the glnA.

INTRODUCTION: Small-colony variants (SCVs) of bacteria are subpopulations with a small colony size, low growth rate, and atypical colony morphology. The purpose of this study was to comprehensively elucidate the characteristics and underlying mechanism of the development of a glutamine-dependent SCV of E. coli, GU-92SCV, isolated from the blood of a patient with pyelonephritis. METHODS: The GU-92SCV strain was tested for auxotrophy testing for glutamine. DNA mutations in genes related to glutamine synthesis were analysed by sequencing. The isolate's proliferation and antimicrobial susceptibility in Mueller-Hinton II medium supplemented with glutamine were examined. RESULTS: The colony of the GU-92SCV strain did not grow on Mueller-Hinton II agar, but growth around the filter paper containing l-glutamine was enhanced on Mueller-Hinton II agar. The GU-92SCV strain had a single nucleotide substitution in glnA, c.193G>A, corresponding to p.Asp65Asn. Changing c.193G>A to the wild-type sequence in glnA restored these phenotypes. Because GU-92SCV did not grow in Mueller-Hinton II broth, antimicrobial susceptibility test results were not obtained; however, in the presence of 10 mg mL-1l-glutamine, the results were consistent with those of the revertant strain GU-92REV. CONCLUSION: To the best of our knowledge, this is the first clinical isolation of a glutamine-dependent E. coli SCV from a patient blood culture. Our data showed that glnA was important for the growth of E. coli in Mueller-Hinton II medium, which also required the presence of glutamine when performing antimicrobial susceptibility testing for glutamine-dependent SCV strains.

LC-MS/MS Assay for the Measurement of Cannabidiol Profiling in CBD Oil from Japanese Market and Application for Convertible Tetrahydrocannabinol in Acetic Acid Condition.

Cannabidiol (CBD), a major non-psychoactive cannabinoid, has a lot of attention due to its potential relaxing properties and led the trend in commercial CBD aroma/oral hemp seed oil from the Japanese market. In this study, a routine assay for evaluating CBD oil samples was performed using LC coupled with tandem mass spectrometry (LC-MS/MS) and was used to apply the convertible tetrahydrocannabinol (THC) in acetic acid conditions. Based on the electrospray positive ion mode, the detection of cannabidiolic acid (CBDA; m/z 359 > 219), cannabigerolic acid (CBGA; m/z 361 > 343), cannabigerol (CBG; m/z 317 > 193), CBD (m/z 315 > 193), THC (m/z 315 > 193) and cannabinol (CBN; m/z 311 > 223) was performed by satisfying separation with high density of C18 column. Oil samples (50 mg) were diluted with isopropanol (5 mL), to which stable isotope internal standards were added by dilution with methanol/water (50/50), and accuracy rates ranged from 97.8 to 102.2%. This method was used to evaluate the CBD oil products (5 kinds) from the Japanese market. Our survey found obvious counterfeit (non-detectable CBD) CBD oil from Japanese market. Following that, we investigated the conversion of THC in CBD oil samples in simple conditions such as 10% acetic acid and 70 °C for 6 h and discovered that converts THC proportions are approximately 5% ((THC content/CBD content) × 100) and <1.0%. Thus, our developed LC-MS/MS assay could be applied to monitor the CBD concentration and convertible THC from CBD oil.

Study on the Synthesis of Methylated Reference and Their Application in the Quantity of Curcuminoids Using Single Reference Liquid Chromatography Based on Relative Molar Sensitivity.

We report on the recommendation of the simple and versatility of methylated reference (MR) to improve applications in the single reference (SR)-LC based on relative molar sensitivity (RMS). Three curcuminoids (Curs) such as curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric products were determined using authentic standards and methylated curcumin. In addition, high-speed countercurrent chromatography (HSCCC) purification is necessary to separate Curs for indicating the RMS. For HSCCC separation, a biphasic solvent system was used to obtain these fractions, which were then subjected to 1H quantitative NMR to determine their contents in each test solution. Using these solutions, the RMS of Curs are calculated from slopes ratios of calibration curves (three ranges from 0-100 µmol/L, r2 > 0.998). The averaged RMS of Curs were 8.92 (relative standard deviation (RSD), 1.17%), 8.97 (2.18%), and 9.61 (0.77%), respectively. Cur concentrations in turmeric products can be determined using RMS, peak area, and MR content added in these samples. This proposed method, which is based on chemical methylation and the SR-LC assay has been successfully applied for the simple and reliable estimation of Curs in turmeric products.

Changes in the Expression of Smooth Muscle Cell-Related Genes in Human Dermal Sheath Cup Cells Associated with the Treatment Outcome of Autologous Cell-Based Therapy for Male and Female Pattern Hair Loss.

In a clinical study of autologous cell-based therapy using dermal sheath cup (DSC) cells, the treatment of hair loss showed improvements. However, the outcomes were variable. Here, correlations between marker gene expression in DSC cells and treatment outcomes were assessed to predict therapeutic efficacy. Overall, 32 DSC cell lines were used to evaluate correlations between marker gene expression and treatment outcomes. Correlations between vascular pericyte and preadipocyte marker expression and treatment outcomes were inconsistent. As smooth muscle cell markers, MYOCD correlated negatively with treatment outcomes and SRF consistently demonstrated an inverse correlation. Additionally, CALD1 correlated negatively and ACTA2 correlated inversely with treatment outcomes. DSC cell lines were divided into good and moderate/poor responders to further investigate the correlations. SRF and CALD1 were lower in a good responder compared with a moderate responder. Next, DSC cells were differentiated toward dermal papilla cells. Dermal papilla markers SOX2 and LEF1 before differentiation had moderate positive and inverse correlations with the treatment outcome, respectively. SOX2 after differentiation more consistently demonstrated a positive correlation. Significant downregulation of smooth muscle-related genes was also observed after differentiation. These findings revealed putative markers for preclinical evaluation of DSC cells to improve hair loss.

Monitoring Analysis of Perfluoroalkyl Substances and F-53B in Bottled Water, Tea and Juice Samples by LC-MS/MS.

Monitoring analysis of 14 per- and polyfluoroalkyl substances (PFAS), 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate (F-53B) and dodecafluoro-3H-4,8-dioxanonanoate (ADONA) in bottled drinking water, tea and juice samples was performed using LC coupled with tandem mass spectrometry (LC-MS/MS) and solid-phase extraction (SPE). In the electrospray negative ion mode, the limit of detection and limit of quantification (LOQ) values were 0.1 to 0.8 ng/mL and 0.2 to 1.6 ng/mL, respectively. The calibration curves were linear from LOQ to 50 ng/mL (r2 > 0.999). The SPE procedure (Presep PFC-II) was utilized for sample preparation and recovery rates for three standards (35, 70 and 140 ng/L) were 80.4-118.8% with relative standard deviation (RSD) ≤ 0.6%. Using the developed method, various samples (n = 54) from Japanese markets were investigated for PFAS and F-53B contamination, and values below the LOQ were observed. It is concluded that for monitoring products in the Japanese market, our method represents a significant improvement over complex techniques for the quantification of PFAS and related compounds from various foods.

The E3 Ubiquitin-Protein Ligase RNF4 Promotes TNF-α-Induced Cell Death Triggered by RIPK1.

Receptor-interacting protein kinase 1 (RIPK1) is a key component of the tumor necrosis factor (TNF) receptor signaling complex that regulates both pro- and anti-apoptotic signaling. The reciprocal functions of RIPK1 in TNF signaling are determined by the state of the posttranslational modifications (PTMs) of RIPK1. However, the underlying mechanisms associated with the PTMs of RIPK1 are unclear. In this study, we found that RING finger protein 4 (RNF4), a RING finger E3 ubiquitin ligase, is required for the RIPK1 autophosphorylation and subsequent cell death. It has been reported that RNF4 negatively regulates TNF-α-induced activation of the nuclear factor-κB (NF-κB) through downregulation of transforming growth factor ß-activated kinase 1 (TAK1) activity, indicating the possibility that RNF4-mediated TAK1 suppression results in enhanced sensitivity to cell death. However, interestingly, RNF4 was needed to induce RIPK1-mediated cell death even in the absence of TAK1, suggesting that RNF4 can promote RIPK1-mediated cell death without suppressing the TAK1 activity. Thus, these observations reveal the existence of a novel mechanism whereby RNF4 promotes the autophosphorylation of RIPK1, which provides a novel insight into the molecular basis for the PTMs of RIPK1.

Quantification of tea-derived catechins without the requirement for respective calibration curves by single reference liquid chromatography based on relative molar sensitivity.

BACKGROUND: Many studies report the monitoring of catechins in tea samples by chromatographic techniques. Unfortunately, only a small number of screening assays for catechins exist as a result of the complexity of authentic standards for the respective calibration curves. In the present study, a single reference (SR) exhaustive assay for the simultaneous quantification of tea-derived catechins by liquid chromatography (LC) with photodiode array and fluorescence detectors based on relative molar sensitivity (RMS) was developed as a screening assay of common tea samples without respective calibration curves using authentic standards. RESULTS: Three original SR standards were proposed based on flavonoid structures, evaluated by quantitative 1 H-NMR based on an indirect standard (1,4-bis(trimethylsilyl) benzene-d4 ) and successfully separated in a LC chromatogram. In tea samples with these added SR calculated based on RMS, the concentrations of eight tea-derived catechins could be measured with a relative SD of < 8.5% by a single LC run. CONCLUSION: This LC screening assay based on RMS allows reliable quantification without the requirement for respective calibration curves using authentic standards. © 2020 Society of Chemical Industry.

trans-Fatty acids promote proinflammatory signaling and cell death by stimulating the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway.

Food-borne trans-fatty acids (TFAs) are mainly produced as byproducts during food manufacture. Recent epidemiological studies have revealed that TFA consumption is a major risk factor for various disorders, including atherosclerosis. However, the underlying mechanisms in this disease etiology are largely unknown. Here we have shown that TFAs potentiate activation of apoptosis signal-regulating kinase 1 (ASK1) induced by extracellular ATP, a damage-associated molecular pattern leaked from injured cells. Major food-associated TFAs such as elaidic acid (EA), linoelaidic acid, and trans-vaccenic acid, but not their corresponding cis isomers, dramatically enhanced extracellular ATP-induced apoptosis, accompanied by elevated activation of the ASK1-p38 pathway in a macrophage-like cell line, RAW264.7. Moreover, knocking out the ASK1-encoding gene abolished EA-mediated enhancement of apoptosis. We have reported previously that extracellular ATP induces apoptosis through the ASK1-p38 pathway activated by reactive oxygen species generated downstream of the P2X purinoceptor 7 (P2X7). However, here we show that EA did not increase ATP-induced reactive oxygen species generation but, rather, augmented the effects of calcium/calmodulin-dependent kinase II-dependent ASK1 activation. These results demonstrate that TFAs promote extracellular ATP-induced apoptosis by targeting ASK1 and indicate novel TFA-associated pathways leading to inflammatory signal transduction and cell death that underlie the pathogenesis and progression of TFA-induced atherosclerosis. Our study thus provides insight into the pathogenic mechanisms of and proposes potential therapeutic targets for these TFA-related disorders.

Role of intensive nutrition support and prophylactic percutaneous endoscopic gastrostomy during concomitant chemoradiotherapy for oropharyngeal cancer.

BACKGROUND: Concomitant chemoradiotherapy (CCRT) produces severe mucositis and swallowing dysfunction, often resulting in malnutrition. Intensive nutrition support (INS) in addition to percutaneous endoscopic gastrostomy (PEG) is reported to decrease adverse effects during CCRT. PATIENTS AND METHODS: Fifty-eight patients with oropharyngeal cancer treated by CDDP-based CCRT were retrospectively analyzed. Twenty-nine patients treated with INS in addition to PEG were classified as INS group, and other 29 patients treated with PEG but without INS were classified as control group. RESULTS: INS in addition to PEG significantly increased calorie intake in the second half of CCRT and reduced adverse events including mucositis (p = 0.0019), leukopenia (p = 0.04), and renal function (p = 0.006). Moreover, 21 out of 29 patients had successfully administration of 200 mg/m2 or more of CDDP, while only 10 out of 29 patients had enough amount of CDDP in control group. CONCLUSIONS: These results suggest that INS in addition to prophylactic PEG not only decreases adverse effects but also may potentially improve oncological outcome of the patients with oropharyngeal cancer treated by CCRT.

Impact of Perkinsus olseni infection on a wild population of Manila clam Ruditapes philippinarum in Ariake Bay, Japan.

Many studies have addressed the production decline of Manila clam, Ruditapes philippinarum, in Japan, but infection of clams with Perkinsus olseni has received scarce attention. To evaluate the impact of P. olseni, infection levels and host density of a wild, unexploited clam population were monitored monthly or bimonthly on a tidal flat from June 2009 to January 2013. Real-time PCR analysis discriminating P. olseni and Perkinsus honshuensis detected only P. olseni in tested clams. The prevalence of P. olseni was 100% or nearly 100% in 7 cohorts throughout the study period, except in newly recruited clams. Infection intensity remained low and seldom reached 106 cells/g wet tissue in newly recruited clams until the month of September. Infection intensity reached 106 cells/g in autumn and remained high at 104-106 cells/g until each cohort of clams disappeared. Clam density began to decrease in the autumn when the infection intensities reached ca. 106 cells/g. Density was relatively stable in winter, increased in spring and decreased again in clams aged 1 year or older during summer and autumn in the following years. Survival of clams experimentally infected with P. olseni at ca. 106 cells/g and placed in a cage in the tidal flat for 1 or 2 months was significantly lower than survival of uninfected control clams. Our results suggested that heavy infection with P. olseni was a major cause of the clam density decrease, although other environmental and biological factors also may have contributed to the decline in density. In addition, uninfected clams were deployed in cages for 1-2 months from June 2010 to January 2013 and prevalence and infection intensity were monitored. Parasite transmission and infection progression increased in summer and autumn.

Biochanin A enhances RORγ activity through STAT3-mediated recruitment of NCOA1.

Interleukin (IL)-17-producing T cells play important roles in autoimmunity, chronic inflammation and host protection against extracellular bacteria and fungi. The retinoic acid receptor-related orphan receptors (ROR) α and γ are key regulators of the IL-17-producing phenotype. We previously showed that the isoflavone biochanin A enhanced ROR-mediated transcriptional activity. Here, we investigated the possible mechanisms underlying this ROR activation. Biochanin A-treated murine thymoma EL4 and primary splenocytes demonstrated enhanced induction of IL-17. Biochanin A also induced tyrosine-phosphorylation of signal transducer and activator of transcription 3 (STAT3) in these cells. Stable knockdown of either RORγ or STAT3 in EL4 cells canceled biochanin A-induced upregulation of IL-17 expression. Importantly, biochanin A enhanced complex formation between RORγ and STAT3 or nuclear-receptor coactivator 1 (NCOA1). Furthermore, the biochanin A-induced RORγ-NCOA1 complex was disrupted by a dominant negative mutant of STAT3 or by the STAT3 specific inhibitor Stattic. These results suggest that biochanin A activates RORγ-dependent IL-17 transcription through the enhancement of STAT3 phosphorylation and STAT3-mediated recruitment of NCOA1 to RORγ.

PXR stimulates growth factor-mediated hepatocyte proliferation by cross-talk with the FOXO transcription factor.

Growth factor-mediated hepatocyte proliferation is crucial in liver regeneration and the recovery of liver function after injury. The nuclear receptor, pregnane X receptor (PXR), is a key transcription factor for the xenobiotic-induced expression of genes associated with various liver functions. Recently, we reported that PXR activation stimulates xenobiotic-induced hepatocyte proliferation. In the present study, we investigated whether PXR activation also stimulates growth factor-mediated hepatocyte proliferation. In G0 phase-synchronized, immortalized mouse hepatocytes, serum or epidermal growth factor treatment increased cell growth and this growth was augmented by the expression of mouse PXR and co-treatment with pregnenolone 16α-carbonitrile (PCN), a PXR ligand. In a liver regeneration model using carbon tetrachloride, PCN treatment enhanced the injury-induced increase in the number of Ki-67-positive nuclei as well as Ccna2 and Ccnb1 mRNA levels in wild-type (WT) but not Pxr-null mice. Chronological analysis of this model demonstrated that PCN treatment shifted the maximum cell proliferation to an earlier time point and increased the number of M-phase cells at those time points. In WT but not Pxr-null mice, PCN treatment reduced hepatic mRNA levels of genes involved in the suppression of G0/G1- and G1/S-phase transition, e.g. Rbl2, Cdkn1a and Cdkn1b. Analysis of the Rbl2 promoter revealed that PXR activation inhibited its Forkhead box O3 (FOXO3)-mediated transcription. Finally, the PXR-mediated enhancement of hepatocyte proliferation was inhibited by the expression of dominant active FOXO3 in vitro. The results of the present study suggest that PXR activation stimulates growth factor-mediated hepatocyte proliferation in mice, at least in part, through inhibiting FOXO3 from accelerating cell-cycle progression.

Post-Translational Modifications of the TAK1-TAB Complex.

Transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family that is activated by growth factors and cytokines such as TGF-ß, IL-1ß, and TNF-α, and mediates a wide range of biological processes through activation of the nuclear factor-κB (NF-κB) and the mitogen-activated protein (MAP) kinase signaling pathways. It is well established that activation status of TAK1 is tightly regulated by forming a complex with its binding partners, TAK1-binding proteins (TAB1, TAB2, and TAB3). Interestingly, recent evidence indicates the importance of post-translational modifications (PTMs) of TAK1 and TABs in the regulation of TAK1 activation. To date, a number of PTMs of TAK1 and TABs have been revealed, and these PTMs appear to fine-tune and coordinate TAK1 activities depending on the cellular context. This review therefore focuses on recent advances in the understanding of the PTMs of the TAK1-TAB complex.

Determination and purification of sesamin and sesamolin in sesame seed oil unsaponified matter using reversed-phase liquid chromatography coupled with photodiode array and tandem mass spectrometry and high-speed countercurrent chromatography.

In Asian countries, sesame seed oil unsaponified matter is used as a natural food additive due to its associated antioxidant effects. We determined and purified the primary lignans sesamin and sesamolin in sesame seed oil unsaponified matter using reversed-phase liquid chromatography coupled with photodiode array and tandem mass spectrometry and high-speed countercurrent chromatography. Calibration curves showed good correlation coefficients (r2 > 0.999, range 0.08 and/or 0.15 to 5 µg/mL) with a limit of detection (at 290 nm) of 0.02 µg/mL for sesamin and 0.04 µg/mL for sesamolin. Sesame seed oil unsaponified matter contained 2.82% sesamin and 2.54% sesamolin, respectively. Direct qualitative analysis of sesamin and sesamolin was achieved using quadrupole mass spectrometry with positive-mode electrospray ionization. Pure (>99%) sesamin and sesamolin standards were obtained using high-speed countercurrent chromatographic purification (hexane/ethyl acetate/methanol/water; 7:3:7:3). An effective method for determining and purifying sesamin and sesamolin from sesame seed oil unsaponified matter was developed by combining these separation techniques for standardized food additives.

[A clinical analysis of elderly dementia patients with physical comorbidities].

AIM: To investigate the current status of elderly dementia patients with physical illnesses and identify optimal care strategies for this growing population. METHODS: This retrospective study included elderly dementia patients who (i) received in-patient treatment for a physical comorbidity at the dementia ward of the Juntendo Tokyo Koto Geriatric Medical Center, and (ii) who were discharged from April 2009 to March 2011. RESULTS: The study population was 390 patients (144 males, 246 females), with a mean [±SD] age of 80.5 [±8.1] years. Two hundred thirteen of the patients had Alzheimer's disease; the remaining 177 had other types of dementia. The comorbidities necessitating admission were: malignant neoplasms (n=65), respiratory conditions (n=57), genitourinary conditions (n=50), trauma or fracture (n=41), and other (n=177). Among the 239 subjects who were hospitalized from their homes and who were discharged alive, 157 (65.7%) returned to their homes. The hospital stays of patients who were discharged were significantly shorter (P<0.000) and their N-ADL scores were significantly better at admission (P<0.013) and at discharge (P<0.000). The proportion of subjects who were capable of oral ingestion was significantly higher among the patients who were discharged to their homes (P<0.025). The subjects who lived in their homes alone at the time of hospitalization were significantly less likely to be discharged to their homes (P<0.018). CONCLUSIONS: Elderly dementia patients should ideally return home after hospitalization for comorbid illnesses. This was facilitated by minimizing their hospital stay. During in-patient treatment, efforts should be made to maintain their N-ADL levels and support their oral intake.

[Blood culture results in elderly febrile patients].

AIM: We retrospectively evaluated blood culture results in elderly patients (≥65 years) with a fever due to infection. METHODS: We examined the bacteria isolated from blood cultures and compared them to bacteria detected in infected lesions that caused bacteremia. We compared the types of bacteria isolated in the two groups (the community-acquired group and the hospital-acquired group). RESULTS: Blood cultures were obtained from 638 patients. Bacteria were detected in 182 patients (28.5%), including 66 (36.3%) patients in the community-acquired group and 116 (63.7%) patients in the hospital-acquired group. There were 259 positive samples (25.1%). In arterial blood specimens, 153 (30.9%) samples were positive, while in venous blood specimens, there were 106 (19.8%) positive samples (P<0.001). In the community-acquired group, the most common bacteria identified were E. coli compared to S. epidermidis in the hospital-acquired group. More than 50% of the bacteria identified in the blood cultures were of the same species identified in the respective urine samples and central venous catheter tips. CONCLUSIONS: The bacteria detection rate in this study was 28.5% for blood cultures, which is higher than the 17.5% reported by the Japan Nosocomial Infections Surveillance Program conducted by the Japanese Ministry of Health, Labour and Welfare. These results suggest that in elderly patients from whom an insufficient volume of blood can be drawn from a vein, an arterial sample may increase the detection rate. A high percentage of bacterial species isolated from the blood cultures was also detected in urinary tract infections and central venous catheter-related infections, indicating that a blood culture is useful for detecting various infectious diseases, even in elderly febrile patients.

Roquin-2 promotes oxidative stress-induced cell death by ubiquitination-dependent degradation of TAK1.

Reactive oxygen species (ROS) are highly reactive and their accumulation causes oxidative damage to cells. Cells maintain survival upon mild oxidative stress with anti-oxidative systems, such as the kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system. On the other hand, upon severe oxidative stress, cells undergo regulated cell death, including apoptosis, for eliminating damaged cells. To execute efficient cell death, cells need to turn off the anti-oxidant systems, while triggering cell death. However, it remains unknown how cells orchestrate these two conflicting systems under excessive oxidative stress. Herein, we show that when cells are exposed to excessive oxidative damage, an E3 ubiquitin ligase Roquin-2 (also known as RC3H2) plays a key role in switching cell fate from survival to death by terminating activation of transforming growth factor-ß-activated kinase 1 (TAK1), a positive regulator for Nrf2 activation. Roquin-2 interacted with TAK1 via four cysteine residues in TAK1 (C96, C302, C486, and C500) that are susceptible to oxidative stress and participate in oligomer formation via disulfide bonds, promoting K48-linked polyubiquitination and degradation of TAK1. Nrf2 was inactivated upon lethal oxidative stress in wild-type mouse embryonic fibroblast (MEF) cells, whereas it sustained activation and conferred resistance to Roquin-2 deficient cells, which was reversed by pharmacological or genetic inhibition of TAK1. These data demonstrate that in response to excessive ROS exposure, Roquin-2 promotes ubiquitination and degradation of TAK1 to suppress Nrf2 activation, and thereby contributes to an efficient cell death, providing insight into the pathogenesis of oxidative stress-related diseases, including cancer.

Unexpected T cell regulatory activity of anti-histone H1 autoantibody: its mode of action in regulatory T cell-dependent and -independent manners.

Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4(+-)Foxp3(+) Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.