Infection perturbs Bach2- and Bach1-dependent erythroid lineage 'choice' to cause anemia.

Elucidation of how the differentiation of hematopoietic stem and progenitor cells (HSPCs) is reconfigured in response to the environment is critical for understanding the biology and disorder of hematopoiesis. Here we found that the transcription factors (TFs) Bach2 and Bach1 promoted erythropoiesis by regulating heme metabolism in committed erythroid cells to sustain erythroblast maturation and by reinforcing erythroid commitment at the erythro-myeloid bifurcation step. Bach TFs repressed expression of the gene encoding the transcription factor C/EBPß, as well as that of its target genes encoding molecules important for myelopoiesis and inflammation; they achieved the latter by binding to their regulatory regions also bound by C/EBPß. Lipopolysaccharide diminished the expression of Bach TFs in progenitor cells and promoted myeloid differentiation. Overexpression of Bach2 in HSPCs promoted erythroid development and inhibited myelopoiesis. Knockdown of BACH1 or BACH2 in human CD34+ HSPCs impaired erythroid differentiation in vitro. Thus, Bach TFs accelerate erythroid commitment by suppressing the myeloid program at steady state. Anemia of inflammation and myelodysplastic syndrome might involve reduced activity of Bach TFs.

The transcription repressors Bach2 and Bach1 promote B cell development by repressing the myeloid program.

Mature lymphoid cells express the transcription repressor Bach2, which imposes regulation on humoral and cellular immunity. Here we found critical roles for Bach2 in the development of cells of the B lineage, commencing from the common lymphoid progenitor (CLP) stage, with Bach1 as an auxiliary. Overexpression of Bach2 in pre-pro-B cells deficient in the transcription factor EBF1 and single-cell analysis of CLPs revealed that Bach2 and Bach1 repressed the expression of genes important for myeloid cells ('myeloid genes'). Bach2 and Bach1 bound to presumptive regulatory regions of the myeloid genes. Bach2(hi) CLPs showed resistance to myeloid differentiation even when cultured under myeloid conditions. Our results suggest that Bach2 functions with Bach1 and EBF1 to promote B cell development by repressing myeloid genes in CLPs.

A Case Report of Changes in Regional Cortical Blood Volume and Oxygenation During Induction of General Anaesthesia with Sevoflurane Versus Propofol in a Paediatric Moyamoya Disease, Evaluated by Near-Infrared Spectroscopy.

A safe induction method of general anaesthesia for paediatric moyamoya disease patients has not been fully established. We had the opportunity to administer general anaesthesia twice to a two-year-old girl diagnosed with moyamoya disease. We used different induction methods for general anaesthesia at each session, i.e. slow induction with sevoflurane and rapid induction with propofol, and were able to evaluate changes in her left regional cortical blood volume (rCBV) and oxygenation (rCBO) during both anaesthesia inductions using near-infrared spectroscopy (NIRS). The mean change value of total-Hb (rCBV) (mean ± SD; µmol/L) in the rapid induction was lower than that in the slow induction (-0.54 ± 1.43 vs. 1.82 ± 1.74). However, the TOI (rCBO) levels during both anaesthesia inductions were constantly higher than these respective baseline values (64% in the slow induction, 71% in the rapid induction), and these mean change values in each of the anaesthesia induction were about the same. The present results suggested that both the slow induction method with sevoflurane and the rapid induction method with propofol might be safe and effective for anaesthesia induction in paediatric patients with moyamoya disease.

Changes in Blood Volume and Oxygenation in Lower Limb Tissue in Patients Maintaining the Lithotomy Position Under General Anaesthesia.

To investigate the effect of the lithotomy position on lower limb circulation under general anaesthesia, near-infrared spectroscopy (NIRS) was used to measure changes in blood volume and oxygenation in thigh tissue in patients (n = 35) under general anaesthesia in the lithotomy position. The NIRS measurement items, including total haemoglobin concentration (total-Hb), tissue haemoglobin index (nTHI), and tissue oxygenation index (TOI) in the thigh, were recorded for 60 min, while the patients were in the lithotomy position. The correlation between changes in each measurement item and patient characteristics, elevation of the lower extremities, and cardiorespiratory indices were evaluated. Data obtained from 24 patients were analysed. The median values (quartile deviation) of changes in total-Hb, nTHI, and TOI during the 60-min period from baseline were + 3.09 (1.99) µmol/L, +0.08 (0.03) a.u., and + 2.25 (1.75) %, respectively, all of which were significantly increased (p < 0.05). Regression analysis showed that no factor was significantly associated with the increase in any measurement item. The present results suggest that circulation in thigh tissue tends to shift towards hyperaemia during 60 min of general anaesthesia in the lithotomy position, regardless of patient background factors or changes in cardiorespiratory conditions.

Reversal of Rocuronium-Induced Muscle Relaxation with Sugammadex Enhances Oxygen Metabolism in Skeletal Muscle.

We measured changes in blood flow and oxygenation in the brachioradialis muscle using near-infrared spectroscopy (NIRS) during reversal of rocuronium-induced muscle relaxation with administration of sugammadex in patients (n = 25) under general anaesthesia, to investigate whether reversal of muscle relaxant-induced muscle relaxation increases oxygen metabolism in skeletal muscle under general anaesthesia. NIRS measurements, including oxy-haemoglobin (Hb), deoxyHb, total Hb concentration, tissue oxygen index, and various cardiopulmonary parameters, were recorded at four timepoints: T0 (baseline), 3 min before sugammadex administration; T1, immediately before sugammadex administration; T2, at complete recovery of muscle contractility; and T3, 3 min after T2. All measured values at each timepoint were compared using multiple comparison tests. The median values (quartile deviation; QD) (µmol/L) of oxyHb and deoxyHb at T0, T1, T2, and T3 were 0, -0.01 (0.14), -1.15 (0.54), and -1.52 (0.54), and 0, 0.11 (0.06), 0.86 (0.5), and 1.36 (0.61), respectively. The levels of oxyHb were significantly lower and those of deoxyHb were significantly higher at T2 and T3 when compared to those at T1, respectively (P < 0.01). There were no significant changes in other measurements. These results suggest that reversal of rocuronium-induced muscle relaxation by sugammadex slightly increases oxygen metabolism in the brachioradialis muscle. This study might support the clinical finding that administration of neuromuscular blockers decreases whole body oxygen consumption in patients receiving mechanical ventilation under general anaesthesia.

Transcriptional Up-Regulation of FBXW7 by KCa1.1 K+ Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model.

The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.

Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2-Nrf2-CEBPB Transcriptional Axis in THP-1-Derived M2 Macrophages.

M2-polarized, tumor-associated macrophages (TAMs) produce pro-tumorigenic and angiogenic mediators, such as interleukin-8 (IL-8) and IL-10. Leucine-rich repeat-containing protein 8 members (LRRC8s) form volume-regulated anion channels and play an important role in macrophage functions by regulating cytokine and chemokine production. We herein examined the role of LRRC8A in IL-8 and IL-10 expression in THP-1-differentiated M2-like macrophages (M2-MACs), which are a useful tool for investigating TAMs. In M2-MACs, the pharmacological inhibition of LRRC8A led to hyperpolarizing responses after a transient depolarization phase, followed by a slight elevation in the intracellular concentration of Ca2+. Both the small interfering RNA-mediated and pharmacological inhibition of LRRC8A repressed the transcriptional expression of IL-8 and IL-10, resulting in a significant reduction in their secretion. The inhibition of LRRC8A decreased the nuclear translocation of phosphorylated nuclear factor-erythroid 2-related factor 2 (Nrf2), while the activation of Nrf2 reversed the LRRC8A inhibition-induced transcriptional repression of IL-8 and IL-10 in M2-MACs. We identified the CCAAT/enhancer-binding protein isoform B, CEBPB, as a downstream target of Nrf2 signaling in M2-MACs. Moreover, among several upstream candidates, the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) suppressed the Nrf2-CEBPB transcriptional axis in M2-MACs. Collectively, the present results indicate that the inhibition of LRRC8A repressed IL-8 and IL-10 transcription in M2-MACs through the NOX2-Nrf2-CEBPB axis and suggest that LRRC8A inhibitors suppress the IL-10-mediated evasion of tumor immune surveillance and IL-8-mediated metastasis and neovascularization in TAMs.

TANK Binding Kinase 1 Promotes BACH1 Degradation through Both Phosphorylation-Dependent and -Independent Mechanisms without Relying on Heme and FBXO22.

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. While BACH1 is rapidly degraded when it is bound to heme, it remains unclear how BACH1 degradation is regulated under other conditions. We found that FBXO22, a ubiquitin ligase previously reported to promote BACH1 degradation, polyubiquitinated BACH1 only in the presence of heme in a highly purified reconstitution assay. In parallel to this regulatory mechanism, TANK binding kinase 1 (TBK1), a protein kinase that activates innate immune response and regulates iron metabolism via ferritinophagy, was found to promote BACH1 degradation when overexpressed in 293T cells. While TBK1 phosphorylated BACH1 at multiple serine and threonine residues, BACH1 degradation was observed with not only the wild-type TBK1 but also catalytically impaired TBK1. The BACH1 degradation in response to catalytically impaired TBK1 was not dependent on FBXO22 but involved both autophagy-lysosome and ubiquitin-proteasome pathways judging from its suppression by using inhibitors of lysosome and proteasome. Chemical inhibition of TBK1 in hepatoma Hepa1 cells showed that TBK1 was not required for the heme-induced BACH1 degradation. Its inhibition in Namalwa B lymphoma cells increased endogenous BACH1 protein. These results suggest that TBK1 promotes BACH1 degradation in parallel to the FBXO22- and heme-dependent pathway, placing BACH1 as a downstream effector of TBK1 in iron metabolism or innate immune response.

Down-Regulation of CYP3A4 by the KCa1.1 Inhibition Is Responsible for Overcoming Resistance to Doxorubicin in Cancer Spheroid Models.

The large-conductance Ca2+-activated K+ channel, KCa1.1, plays a pivotal role in cancer progression, metastasis, and the acquisition of chemoresistance. Previous studies indicated that the pharmacological inhibition of KCa1.1 overcame resistance to doxorubicin (DOX) by down-regulating multidrug resistance-associated proteins in the three-dimensional spheroid models of human prostate cancer LNCaP, osteosarcoma MG-63, and chondrosarcoma SW-1353 cells. Investigations have recently focused on the critical roles of intratumoral, drug-metabolizing cytochrome P450 enzymes (CYPs) in chemoresistance. In the present study, we examined the involvement of CYPs in the acquisition of DOX resistance and its overcoming by inhibiting KCa1.1 in cancer spheroid models. Among the CYP isoforms involved in DOX metabolism, CYP3A4 was up-regulated by spheroid formation and significantly suppressed by the inhibition of KCa1.1 through the transcriptional repression of CCAAT/enhancer-binding protein, CEBPB, which is a downstream transcription factor of the Nrf2 signaling pathway. DOX resistance was overcome by the siRNA-mediated inhibition of CYP3A4 and treatment with the potent CYP3A4 inhibitor, ketoconazole, in cancer spheroid models. The phosphorylation levels of Akt were significantly reduced by inhibiting KCa1.1 in cancer spheroid models, and KCa1.1-induced down-regulation of CYP3A4 was reversed by the treatment with Akt and Nrf2 activators. Collectively, the present results indicate that the up-regulation of CYP3A4 is responsible for the acquisition of DOX resistance in cancer spheroid models, and the inhibition of KCa1.1 overcame DOX resistance by repressing CYP3A4 transcription mainly through the Akt-Nrf2-CEBPB axis.

Urinary FABP1 is a biomarker for impaired proximal tubular protein reabsorption and is synergistically enhanced by concurrent liver injury.

Urinary fatty acid binding protein 1 (FABP1, also known as liver-type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1-deficient mice with liver-specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver-derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT-R) either in a hepatocyte- or in a PTEC-specific manner, or both. Upon administration of diphtheria toxin (DT), massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver-derived FABP1 through a megalin-mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

KCa3.1 inhibition-induced activation of the JNK/c-Jun signaling pathway enhances IL-10 expression in peripherally-induced regulatory T cells.

The KCa3.1 inhibition up-regulates IL-10 expression in regulatory T (Treg) cells in the recovery phase of inflammatory bowel disease (IBD) model mice; however, the underlying signaling pathway remains unclear. We investigated the involvement of AP-1 (Fos/Jun) and NF-κB in the expression of IL-10 and its transcription factors (TFs) in in vitro-induced mouse splenic Treg cells. The pharmacological inhibition of JNK reversed KCa3.1 inhibition-induced increases in the expression of IL-10 and its TFs. The inhibition of KCa3.1 increased phosphorylated JNK and c-Jun levels. Therefore, the JNK/c-Jun signaling pathway may contribute to the KCa3.1 inhibition-induced up-regulation of IL-10 in peripherally-induced Treg cells.

Downregulation of IL-8 and IL-10 by the Activation of Ca2+-Activated K+ Channel KCa3.1 in THP-1-Derived M2 Macrophages.

THP-1-differentiated macrophages are useful for investigating the physiological significance of tumor-associated macrophages (TAMs). In the tumor microenvironment (TME), TAMs with the M2-like phenotype play a critical role in promoting cancer progression and metastasis by inhibiting the immune surveillance system. We examined the involvement of Ca2+-activated K+ channel KCa3.1 in TAMs in expressing pro-tumorigenic cytokines and angiogenic growth factors. In THP-1-derived M2 macrophages, the expression levels of IL-8 and IL-10 were significantly decreased by treatment with the selective KCa3.1 activator, SKA-121, without changes in those of VEGF and TGF-ß1. Furthermore, under in vitro experimental conditions that mimic extracellular K+ levels in the TME, IL-8 and IL-10 levels were both significantly elevated, and these increases were reversed by combined treatment with SKA-121. Among several signaling pathways potentially involved in the transcriptional regulation of IL-8 and IL-10, respective treatments with ERK and JNK inhibitors significantly repressed their transcriptions, and treatment with SKA-121 significantly reduced the phosphorylated ERK, JNK, c-Jun, and CREB levels. These results strongly suggest that the KCa3.1 activator may suppress IL-10-induced tumor immune surveillance escape and IL-8-induced tumorigenicity and metastasis by inhibiting their production from TAMs through ERK-CREB and JNK-c-Jun cascades.

Polymorphisms in glia maturation factor ß gene are markers of cellulose ether effectiveness in prion-infected mice.

Anti-prion effects of cellulose ether (CE) are reported in rodents, but the molecular mechanism is fully unknown. Here, we investigated the genetic background of CE effectiveness by proteomic and genetic analysis in mice. Proteomic analysis in the two mouse lines showing a dramatic difference in CE effectiveness revealed a distinct polymorphism in the glia maturation factor ß gene. This polymorphism was significantly associated with the CE effectiveness in various prion-infected mouse lines. Sequencing of this gene and its vicinity genes also revealed several other polymorphisms that were significantly related to the CE effectiveness. These polymorphisms are useful as genetic markers for finding more suitable mouse lines and exploring the genetic factors of CE effectiveness.

Increased Interleukin-10 Expression by the Inhibition of Ca2+-Activated K+ Channel KCa3.1 in CD4+CD25+ Regulatory T Cells in the Recovery Phase in an Inflammatory Bowel Disease Mouse Model.

Inflammatory bowel diseases (IBD) are chronic inflammatory diseases of the gastrointestinal tract arising from abnormal responses of the innate and adaptative immune systems. Interleukin (IL)-10-producing CD4+CD25+ regulatory T (Treg) cells play a protective role in the recovery phase of IBD. In the present study, the effects of the administration of the selective Ca2+-activated K+ channel KCa3.1 inhibitor TRAM-34 on disease activities were examined in chemically induced IBD model mice. IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation, and crypt damage in the colon, was significantly lower in mice administered 1 mg/kg TRAM-34 than in vehicle-administered mice. Quantitative real-time polymerase chain reaction examinations showed that IL-10 expression levels in the recovery phase were markedly increased by the inhibition of KCa3.1 in mesenteric lymph node (mLN) Treg cells of IBD model mice compared with vehicle-administered mice. Among several positive and negative transcriptional regulators (TRs) for IL-10, three positive TRs-E4BP4, KLF4, and Blimp1-were upregulated by the inhibition of KCa3.1 in the mLN Treg cells of IBD model mice. In mouse peripheral CD4+CD25+ Treg cells induced by lectin stimulation, IL-10 expression and secretion were enhanced by the treatment with TRAM-34, together with the upregulation of E4BP4, KLF4, and Blimp1. Collectively, the present results demonstrated that the pharmacological inhibition of KCa3.1 decreased IBD symptoms in the IBD model by increasing IL-10 production in peripheral Treg cells and that IL-10high Treg cells produced by the treatment with KCa3.1 inhibitor may contribute to efficient Treg therapy for chronic inflammatory disorders, including IBD. SIGNIFICANCE STATEMENT: Pharmacological inhibition of Ca2+-activated K+ channel KCa3.1 increased IL-10 expression in peripheral Treg cells, together with the upregulation of the transcriptional regulators of IL-10: Krüppel-like factor 4, E4 promoter-binding protein 4, and/or B lymphocyte-induced maturation protein 1. The manipulation of IL-10high-producing Treg cells by the pharmacological inhibition of KCa3.1 may be beneficial in the treatment of chronic inflammatory diseases such as inflammatory bowel disease.

KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model.

Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.

Inhibition of Interleukin 10 Transcription through the SMAD2/3 Signaling Pathway by Ca2+-Activated K+ Channel KCa3.1 Activation in Human T-Cell Lymphoma HuT-78 Cells.

The hyperpolarization induced by intermediate-conductance Ca2+-activated K+ channel (KCa3.1) activation increases the driving force for Ca2+ influx, which generally promotes cell proliferation, migration, and cytokine production in immunocompetent cells. Interleukin-10 (IL-10) from tumor-infiltrating lymphocytes and macrophages, lymphoma, and carcinoma cells facilitates escape from cancer immune surveillance; however, the role of KCa3.1 in IL-10 production remains unclear. The objective of the present study was to elucidate the involvement of KCa3.1 in IL-10 expression and production using the human T-cell lymphoma HuT-78 cells. In HuT-78 cells, IL-10 gene expression and production were reduced by treatment with the KCa3.1 activator, as 6-hour Western blotting showed that the protein expression ratio of phosphorylated Smad2 (P-Smad2)/Smad2, but not P-Smad3/Smad3, was decreased by the treatment with KCa3.1 activator in HuT-78 cells. Concomitant with this, the nuclear translocation of P-Smad2 was inhibited by KCa3.1 activator. Furthermore, the KCa3.1 activator-induced transcriptional repression of IL-10 disappeared with pretreatment with the calmodulin kinase II (CaMKII) inhibitor KN-62 for 1 hour, and KCa3.1 activator-induced decreases in the nuclear translocation of P-Smad2 were also prevented by pretreatment with KN-62. Taken together, the KCa3.1 activator-induced transcriptional repression of IL-10 is due to the inhibition of the nuclear translocation of P-Smad2 in HuT-78 cells, resulting in the prevention of P-Smad2/3 complex formation in nuclei, and the activation of CaMKII induced by KCa3.1 activators suppresses the constitutive activation of P-Smad2/3 in HuT-78 cells. Therefore, KCa3.1 activators have potential as a therapeutic option to suppress the tumor-promoting activities of IL-10.

Biophysical characterization of heme binding to the intrinsically disordered region of Bach1.

Transcriptional repressor Bach1 plays an important role in antioxidant response. Bach1 function is regulated by heme binding to the four cysteine-proline (CP) motifs in Bach1, which leads to inhibition of its activity. Three of these CP motifs are located N-terminal to the bZip (basic leucine zipper) domain that is responsible for DNA binding. Based on sequence analysis, the region surrounding these CP motifs was expected to be intrinsically disordered. Bach1 is one of few known intrinsically disordered proteins that accept multiple heme molecules for functional regulation, but the molecular mechanisms of heme binding and functional regulation remain unclear. Uncovering these mechanisms is important for understanding Bach1-mediated antioxidant response. Biophysical characterization revealed that 5-coordinated heme binding was unique to the CP motifs within the heme-binding region of Bach1, whereas 6-coordinated binding occurred nonspecifically. Comparison of the wild-type protein and a CP motif mutant indicated that the level of 6-coordinated heme binding was reduced in the absence of 5-coordinated heme binding. Analytical ultracentrifugation showed that the CP motif mutant protein had a more elongated conformation than the wild-type protein, suggesting that cysteines within the CP motifs contribute to intramolecular interactions in Bach1. Thus, heme binding at the CP motifs induces a global conformational change in the Bach1 heme-binding region, and this conformational change, in turn, regulates the biological activity of Bach1.

Exercise endurance capacity is markedly reduced due to impaired energy homeostasis during prolonged fasting in FABP4/5 deficient mice.

BACKGROUND: Skeletal muscle prefers carbohydrate use to fatty acid (FA) use as exercise intensity increases. In contrast, skeletal muscle minimizes glucose use and relies more on FA during fasting. In mice deficient for FABP4 and FABP5 (double knockout (DKO) mice), FA utilization by red skeletal muscle and the heart is markedly reduced by the impairment of trans-endothelial FA transport, with an increase in glucose use to compensate for reduced FA uptake even during fasting. We attempted to determine whether prolonged fasting affects exercise performance in DKO mice, where constant glucose utilization occurs. RESULTS: A single bout of treadmill exercise was performed in the fed and fasted states. The initial speed was 10 m/min, and gradually increased by 5 m/min every 5 min up to 30 m/min until the mice stopped running. Running distance was significantly reduced by DKO genotype and prior fasting, leading to the shortest distance in fasted DKO mice. Levels of glycogen in skeletal muscle and the liver were nearly depleted in both WT and DKO mice during prolonged fasting prior to exercise. Levels of TG in skeletal muscle were not reduced by exercise in fasted DKO mice, suggesting that intramuscular TG was not utilized during exercise. Hypoglycaemia was accelerated in fasted DKO mice, and this acceleration could be due to constant glucose utilization by red skeletal muscle and the heart where FA uptake is diminished due to defective trans-endothelial FA transport. Taken together, energy supply from serum and storage in skeletal muscle were very low in fasted DKO mice, which could lead to a significant reduction in exercise performance. CONCLUSIONS: FABP4/5 have crucial roles in nutrient homeostasis during prolonged fasting for maintaining exercise endurance capacity.

Reversibility of Rocuronium-Induced Deep Neuromuscular Block with Sugammadex in Infants and Children-A Randomized Study.

Sugammadex 4 mg·kg-1 is recommended for reversal from rocuronium-induced deep neuromuscular block. However, there is limited data regarding the dose-response of sugammadex required for reversal from deep neuromuscular block in pediatric patients. The aim of this study was to determine the reversibility of rocuronium-induced deep neuromuscular block with sugammadex in infants and children. Seventy-five children (48 infants and 27 children, mean standard deviation (S.D.), age: 11.6 (6.7) months) were enrolled in this study. After induction of anesthesia and administration of 0.6 mg·kg-1 rocuronium, neuromuscular block was acceleromyographically evaluated by observing contractions of the adductor pollicis muscle to ulnar nerve train-of-four (TOF) stimulation. Subsequently, the intensity of rocuronium-induced block was determined every 6 min using post-tetanic count (PTC) stimulation during sevoflurane and remifentanil anesthesia. When the first response to the PTC stimulus was detected, either 1, 2 or 4 mg·kg-1 sugammadex was administered and the time required for facilitated recovery to a TOF ratio of 0.9 following each dose was compared. The time [mean (S.D.)] from the administration of 1 mg·kg-1 sugammadex until recovery to a TOF ratio of 0.9 was significantly longer [129.1 (83.5) s, p < 0.001] than that with 2 and 4 mg·kg-1 sugammadex [70.3 (26.7) s and 68.2 (34.5) s, respectively]. Incomplete reversal was seen in 3 patients in the 1 mg·kg-1 group. The results suggested that a 4 mg·kg-1 sugammadex dose is recommended for reversal from rocuronium-induced deep neuromuscular block even in infants and children.

Phosphorylation of BACH1 switches its function from transcription factor to mitotic chromosome regulator and promotes its interaction with HMMR.

The transcription repressor BACH1 performs mutually independent dual roles in transcription regulation and chromosome alignment during mitosis by supporting polar ejection force of mitotic spindle. We now found that the mitotic spindles became oblique relative to the adhesion surface following endogenous BACH1 depletion in HeLa cells. This spindle orientation rearrangement was rescued by re-expression of BACH1 depending on its interactions with HMMR and CRM1, both of which are required for the positioning of mitotic spindle, but independently of its DNA-binding activity. A mass spectrometry analysis of BACH1 complexes in interphase and M phase revealed that BACH1 lost during mitosis interactions with proteins involved in chromatin and gene expression but retained interactions with HMMR and its known partners including CHICA. By analyzing BACH1 modification using stable isotope labeling with amino acids in cell culture, mitosis-specific phosphorylations of BACH1 were observed, and mutations of these residues abolished the activity of BACH1 to restore mitotic spindle orientation in knockdown cells and to interact with HMMR. Detailed histological analysis of Bach1-deficient mice revealed lengthening of the epithelial fold structures of the intestine. These observations suggest that BACH1 performs stabilization of mitotic spindle orientation together with HMMR and CRM1 in mitosis, and that the cell cycle-specific phosphorylation switches the transcriptional and mitotic functions of BACH1.