Lactosylceramide-Induced Phosphorylation Signaling to Group IVA Phospholipase A2 via Reactive Oxygen Species in Tumor Necrosis Factor-α-Treated Cells.

The activity of α-type cytosolic phospholipase A2 (cPLA2 α, group IVA PLA2 ), which releases arachidonic acid (AA), is mainly regulated by the Ca2+ -induced intracellular translocation/attachment of the enzyme to substrate membranes and its phosphorylation. We previously reported that tumor necrosis factor-α (TNFα) stimulated the formation of lactosylceramide (LacCer) in L929 fibroblast cells, and this lipid directly bound with and activated cPLA2 α [Nakamura et al. [2013] J. Biol. Chem. 288:23264-23272]. We herein investigated the role of phosphorylation signaling in the TNFα/LacCer-induced activation of cPLA2 α in cells. TNFα-treated L929 cells released AA via the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cPLA2 α, while a treatment with LacCer alone released AA in a similar manner. The TNFα-induced responses including release of AA were decreased by the inhibition of LacCer synthesis. The treatment with TNFα and LacCer increased the levels of reactive oxygen species (ROS), and the reduction/scavenging of ROS decreased the phosphorylation cascade and release of AA in TNFα/LacCer-treated L929 cells. In the cell line CHO, the treatment with LacCer stimulated the phosphorylation cascade and release of AA via the formation of ROS. Treatments with the anti-LacCer antibody and 4ß-phorbol 12-myristate 13-acetate stimulated the phosphorylation cascade, but did not release AA by itself. When combined with the Ca2+ ionophore A23187, treatments with the anti-LacCer antibody and 4ß-phorbol 12-myristate 13-acetate released AA. These results, including our previous findings, showed that LacCer alone simultaneously stimulates two processes to activate cPLA2 α: a phosphorylation signal and attachment of the enzyme to substrate membranes. J. Cell. Biochem. 118: 4370-4382, 2017. © 2017 Wiley Periodicals, Inc.

The key role of a basic domain of histone H2B N-terminal tail in the action of 5-bromodeoxyuridine to induce cellular senescence.

5-Bromodeoxyuridine (BrdU), a thymidine analogue, is an interesting reagent that modulates various biological phenomena. BrdU, upon incorporation into DNA, causes destabilized nucleosome positioning which leads to changes in heterochromatin organization and gene expression in cells. We have previously shown that BrdU effectively induces cellular senescence, a phenomenon of irreversible growth arrest in mammalian cells. Identification of the mechanism of action of BrdU would provide a novel insight into the molecular mechanisms of cellular senescence. Here, we showed that a basic domain in the histone H2B N-terminal tail, termed the HBR (histone H2B repression) domain, is involved in the action of BrdU. Notably, deletion of the HBR domain causes destabilized nucleosome positioning and derepression of gene expression, as does BrdU. We also showed that the genes up-regulated by BrdU significantly overlapped with those by deletion of the HBR domain, the result of which suggested that BrdU and deletion of the HBR domain act in a similar way. Furthermore, we showed that decreased HBR domain function induced cellular senescence or facilitated the induction of cellular senescence. These findings indicated that the HBR domain is crucially involved in the action of BrdU, and also suggested that disordered nucleosome organization may be involved in the induction of cellular senescence.

N-butyldeoxynojirimycin (miglustat) ameliorates pulmonary fibrosis through inhibition of nuclear translocation of Smad2/3.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease. The disease involves excessive accumulation of fibroblasts and myofibroblasts, and myofibroblasts differentiated by pro-fibrotic factors promote the deposition of extracellular matrix proteins such as collagen and fibronectin. Transforming growth factor-ß1 is a pro-fibrotic factor that promotes fibroblast-to-myofibroblast differentiation (FMD). Therefore, inhibition of FMD may be an effective strategy for IPF treatment. In this study, we screened the anti-FMD effects of various iminosugars and showed that some compounds, including N-butyldeoxynojirimycin (NB-DNJ, miglustat, an inhibitor of glucosylceramide synthase (GCS)), a clinically approved drug for treating Niemann-Pick disease type C and Gaucher disease type 1, inhibited TGF-ß1-induced FMD by inhibiting the nuclear translocation of Smad2/3. N-butyldeoxygalactonojirimycin having GCS inhibitory effect did not attenuate the TGF-ß1-induced FMD, suggesting that NB-DNJ exerts the anti-FMD effects by GCS inhibitory effect independent manner. N-butyldeoxynojirimycin did not inhibit TGF-ß1-induced Smad2/3 phosphorylation. In a mouse model of bleomycin (BLM)-induced pulmonary fibrosis, intratracheal or oral administration of NB-DNJ at an early fibrotic stage markedly ameliorated lung injury and deterioration of respiratory functions, such as specific airway resistance, tidal volume, and peak expiratory flow. Furthermore, the anti-fibrotic effects of NB-DNJ in the BLM-induced lung injury model were similar to those of pirfenidone and nintedanib, which are clinically approved drugs for the treatment of IPF. These results suggest that NB-DNJ may be effective for IPF treatment.

Experience of perampanel monotherapy beyond initial titration to achieve seizure freedom in patients with focal-onset seizures with newly diagnosed or currently untreated recurrent epilepsy: A post hoc analysis of the open-label Study 342 (FREEDOM).

OBJECTIVE: This post hoc analysis evaluated whether continued treatment with perampanel monotherapy beyond initial titration may be appropriate for patients with focal-onset seizures (FOS) with currently untreated epilepsy to achieve seizure freedom with an effective dose. METHODS: Study 342 (NCT03201900; FREEDOM) is a single-arm, open-label, Phase III study of perampanel monotherapy. Patients aged ≥12 years with untreated FOS received perampanel 4 mg/d in a 32-week Treatment Phase (6-week Titration and 26-week Maintenance Periods); in case of seizure(s) during Maintenance Period, patients could enter a 30-week Treatment Phase (4-week Titration and 26-week Maintenance Periods) to be up-titrated to perampanel 8 mg/d. The primary endpoint was seizure-freedom rate during Maintenance Period in the modified Intent-to-Treat (mITT) Analysis Set (patients who had ≥1 post-dose efficacy measurement during Maintenance Period); safety was monitored. This analysis of 4-mg/d efficacy data assessed the proportion of patients achieving seizure freedom during the Maintenance Period (responders) relative to patients with an early/later response (depending on seizure status during the Titration Period). RESULTS: In the mITT population (n = 73), 46 patients were 4-mg/d responders; of whom, 37 (80.4%) were early responders and nine (19.6%) were later responders. The mean (standard deviation) percent reductions in FOS frequency from baseline at the end of the 4-mg/d Titration Period were 100.0% (0.0%; early responders) and 46.3% (97.3%; later responders). Among the 27 4-mg/d nonresponders, nine (33.3%) patients who had an early response experienced seizure(s) during the subsequent 4-mg/d Maintenance Period. Safety outcomes were similar, regardless of responder status, without new safety concerns. SIGNIFICANCE: Some patients with untreated FOS may benefit from continued treatment beyond initial titration of perampanel monotherapy to achieve seizure freedom, suggesting that it may not be appropriate to make treatment decisions to discontinue or switch from perampanel monotherapy solely based on seizure response before an effective dose has been reached.

[Efficacy of the Extract from Fermentation of Soybean and Rice Bran on Hyperglycemia].

In recent years, lifestyle-related diseases such as hypertension and diabetes have been on the rise. These conditions can cause serious conditions such as myocardial and cerebral infarctions. Therefore, proper control of blood pressure and blood glucose levels is important issues in preventive medicine. Traditional fermented foods have been shown to have various functions, and their effects on lifestyle-related diseases have attracted particular attention. In this study, we investigated the effects of fermented soybeans and rice bran (OE-1) and supplements containing OE-1 on blood glucose levels and weight changes. We identified an inhibitory effect on elevated blood glucose levels upon administration of OE-1, and this effect was thought to be due to digestive enzyme inhibition. These effects of foods containing OE-1 are expected to have a positive effect on the prevention and improvement of lifestyle-related diseases as health foods.

Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria.

Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and -16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.

Association between the point-rating system used for oral health and the prevalence of Gram-negative bacilli in hematological inpatients: A retrospective cohort study.

ABSTRACT: Gram-negative bacteremia is a major cause of death among hematology inpatients who require heavy-dose chemotherapy and hematopoietic stem cell transplantation. Gram-negative bacillus (GNB) is more likely to be detected when the oral health is poor. However, there is a dearth of studies on the relationship between oral assessment and prevalence of GNB in hematology inpatients.This retrospective study aimed to evaluate the relationship between the original point-rating system for oral health examinations (point-oral exam) and the prevalence of GNB in hematology inpatients at the hematology ward of the Yamanashi University Hospital. GNB was detected by cultivating samples from the sputum and blood of each patient.A total of 129 subjects underwent a medical checkup and point-oral exam. The sputum and blood culture results of 55 patients were included in this study. The total points of patients positive for GNB (n = 25, 45.5%) were significantly higher than those who were negative for GNB (total score: median, 25th, 75th, percentile; 6 [4, 7] vs 2 [1, 4]; P = .00016). Based on the receiver operating characteristic analysis, a cutoff score of 5 proved to be most useful to detect GNB.An oral evaluation with a cutoff value of 5 or higher in the point-oral exam might indicate the need for a more thorough oral management to prevent the development of systemic infections from GNB.

FGFRL1 deficiency reduces motility and tumorigenic potential of cells derived from oesophageal squamous cell carcinomas.

Oesophageal squamous cell carcinoma (ESCC) is an aggressive cancer that resulted in ~400,000 mortalities worldwide in 2012. It was reported previously that fibroblast growth factor receptor-like 1 (FGFRL1) is highly expressed in ESCC patients with lymph node metastasis and poor prognosis accordingly. FGFRL1 is an FGFR that lacks tyrosine kinase activity, whereas the activity is critical for other FGFRs to activate intracellular signalling. The mechanism by which FGFRL1 promotes the aggressiveness of ESCCs is unknown. In the present study, two independent FGFRL1-deficient cell lines were generated from human ESCC KYSE520 cells, in order to investigate the relationship of FGFRL1 with the aggressiveness of ESCCs. FGFRL1-deficiency did not affect proliferation of KYSE520 cells in vitro. However, a xenograft mouse model demonstrated that FGFRL1-deficiency decelerated tumour growth in vivo. The haematoxylin-eosin staining identified that FGFRL1-deficient cells formed well-differentiated squamous cell carcinomas, whereas wild-type cells formed moderately differentiated squamous cell carcinomas. Microarray analysis of mRNA expression revealed that FGFRL1-depletion resulted in decreased expression of proteins associated with motility and invasion of tumour cells, matrix metalloproteinase-1 and fibroblast growth factor binding protein 1. The wound-healing assay indicated that depleting FGFRL1 reduced cell motility. Furthermore, the invasiveness of FGFRL1-deficient cells was lesser than that of wild-type KYSE520 cells. In the FGFRL1-deficient KYSE520 cells, actin filaments around the nucleus were observed sparsely, whereas the filaments along the plasma membranes were observed as frequently as those in the parent KYSE520 cells. These results demonstrate that FGFRL1 may be involved in regulation of protein expression, actin filament assembly and tumorigenic potential of ESCC cells.

Tim23-Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import.

Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23-Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23-Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.