Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry & Molecular Biology (ASBMB)  

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate units that are linked by phosphoanhydride bonds and is involved in various pathophysiological processes. However, the role of polyP in immune cell dysfunction is not well-understood. In this study, using several biochemical and cell biology approaches, including cytokine assays, immunofluorescence microscopy, receptor-binding assays with quartz crystal microbalance, and dynamic light scanning, we investigated the effect of polyP on in vitro lipopolysaccharide (LPS)-induced macrophage inflammatory response. PolyP up-regulated LPS-induced production of the inflammatory cytokines, such as tumor necrosis factor α, interleukin-1β, and interleukin-6, in macrophages, and the effect was polyP dose- and chain length-dependent. However, orthophosphate did not exhibit this effect. PolyP enhanced the LPS-induced intracellular macrophage inflammatory signals. Affinity analysis revealed that polyP interacts with LPS, inducing formation of small micelles, and the polyP-LPS complex enhanced the binding affinity of LPS to Toll-like receptor 4 (TLR4) on macrophages. These results suggest that inorganic polyP plays a critical role in promoting inflammatory response by enhancing the interaction between LPS and TLR4 in macrophages.

DOI： 10.1074/jbc.RA119.011763

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41586-019-1707-0

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Other Link： http://www.nature.com/articles/s41586-019-1707-0

Language：English   Publishing type：Research paper (scientific journal)   Publisher：S. Karger AG  

Background/Aims: Accumulation of protein-bound uremic toxins (PBUTs) is associated with mortality due to various systemic disorders in patients with chronic kidney disease (CKD), especially in those undergoing dialysis treatment. The clinical outcomes of such patients could be improved by removing sufficient amounts of PBUTs; however, conventional dialysis lacks this ability. We examined the efficacy of activated carbon in adsorbing circulating PBUTs through direct hemoperfusion (DHP) in vitro. Methods: An in vitro blood circulating system was constructed with 8.5 mL blood circulating around a column containing activated carbon (50, 100, or 200 mg). Bovine blood containing a kind of PBUT (at the same concentration as that found in the blood of dialysis patients) and blood from hemodialysis patients (n = 8) were used. After circulation for the designated amount of time, sera were collected and the levels of PBUTs, including indoxyl sulfate (IS), p-cresyl sulfate, indole acetic acid (IAA), phenyl sulfate, and hippuric acid, were analyzed with mass spectrometry. Results: Activated carbon decreased the PBUT level in bovine blood in a dose-dependent manner (e.g., reduction rate of IS: 67.9 +/- 3.8, 83.3 +/- 1.9, and 94.5 +/- 1.1% after 60-min circulation in columns containing 50, 100, and 200 mg activated carbon respectively). IS, PCS, and IAA were dramatically adsorbed by activated carbon from the blood of patients undergoing hemodialysis (pre vs. post 240-min reaction: IS 2.835 +/- 0.876 vs. 0.455 +/- 0.108 mg/dL [p < 0.01], PCS 3.208 +/- 2.876 vs. 0.768 +/- 0.632 mg/dL [p < 0.01], IAA 0.082 +/- 0.045 vs. 0.016 +/- 0.005 mg/dL [p < 0.01]). Conclusion: Activated carbon effectively adsorbed blood PBUTs in vitro. DHP with activated carbon could be a promising strategy for removing circulating PBUTs from the blood of patients with CKD.

DOI： 10.1159/000500014

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

System xc - was recently described as the most upstream node in a novel form of regulated necrotic cell death, called ferroptosis. In this context, the small molecule erastin was reported to target and inhibit system xc -, leading to cysteine starvation, glutathione depletion and consequently ferroptotic cell death. Although the inhibitory effect of erastin towards system xc - is well-documented, nothing is known about its mechanism of action. Therefore, we sought to interrogate in more detail the underlying mechanism of erastin's pro-ferroptotic effects. When comparing with some well-known inhibitors of system xc -, erastin was the most efficient inhibitor acting at low micromolar concentrations. Notably, only a very short exposure of cells with low erastin concentrations was sufficient to cause a strong and persistent inhibition of system xc -, causing glutathione depletion. These inhibitory effects towards system xc - did not involve cysteine modifications of the transporter. More importantly, short exposure of tumor cells with erastin strongly potentiated the cytotoxic effects of cisplatin to efficiently eradicate tumor cells. Hence, our data suggests that only a very short pre-treatment of erastin suffices to synergize with cisplatin to efficiently induce cancer cell death, findings that might guide us in the design of novel cancer treatment paradigms.

DOI： 10.1038/s41598-018-19213-4

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Other Link： http://www.nature.com/articles/s41598-018-19213-4.pdf

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

The amino acid transport system xc − is important for maintaining intracellular glutathione levels and extracellular redox balance. The main component of system xc −, xCT, is strongly induced by various stimuli, including oxidative stress and bacterial lipopolysaccharides (LPS) in macrophages. In the present study, we investigated the production of nitric oxide by LPS-stimulated mouse peritoneal macrophages isolated from both xCT-deficient and wild-type mice. After culturing macrophages in the presence of LPS for 24–48 h, nitrite levels in the medium of xCT-deficient macrophages were significantly decreased compared to that of wild-type cells. However, the transport activity of arginine, a precursor of nitric oxide, and the expression of nitric oxide synthase 2 in xCT-deficient macrophages were similar to those of wild-type cells. When wild-type macrophages were cultured in the medium that contained no cystine, nitric oxide production was decreased to the level similar to that of the xCT-deficient macrophages. When xCT-deficient macrophages were cultured with 2-mercaptoethanol, intracellular cysteine levels were increased and nitrite accumulation in the medium was significantly increased. On the other hand, when these cells were cultured with buthionine sulfoximine, an inhibitor of glutathione synthesis, nitrite accumulation in the medium was essentially unchanged, although intracellular glutathione levels were very low. Reactive oxygen species levels in xCT-deficient macrophages were higher than those of wild-type cells, and treatment with LPS caused an increase in oxidative stress in both cells. These results suggest that intracellular cysteine supplied by xCT contributes to nitric oxide production and the reduction of oxidative stress in macrophages.

DOI： 10.1016/j.niox.2018.05.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Inc.  

An accumulation of protein-bound uremic toxins (PBUTs) is one of major reasons for development of uremia-related complications. We examined the PBUT removal ability of a hexadecyl-immobilized cellulose bead (HICB)-containing column for patients undergoing hemodialysis. Adsorption of indoxyl sulfate (IS), a representative PBUT, to HICBs was examined in vitro. The HICB column was used in patients undergoing hemodialysis for direct hemoperfusion with a regular hemodialyzer. The serum IS, indole acetic acid (IAA), phenyl sulfate (PhS), and p-cresyl sulfate (PCS) levels were measured before and after passing the column. HICBs adsorbed protein-free (free) IS in a dose- and time-dependent manner in vitro (55.4 ± 1.4% adsorption of 1 millimolar, 251 µg/mL, IS for 1 h). In clinical studies, passing the HICB-containing column decreased the serum level of free IS, IAA, PhS, and PCS levels significantly (by 34.4 ± 30.0%, 34.8 ± 25.4%, 28.4 ± 18.0%, and 34.9 ± 22.1%, respectively), but not protein-bound toxins in maintenance hemodialysis patients. HICBs absorbed some amount of free PBUTs, but the clinical trial to use HICB column did not show effect to reduce serum PBUTs level in hemodialysis patients. Adsorption treatment by means of direct hemoperfusion with regular hemodialysis may become an attractive blood purification treatment to increase PBUT removal when more effective materials to adsorb PBUTs selectively will be developed.

DOI： 10.1111/aor.12961

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Language：Japanese   Publisher：(公社)日本透析医会  

背景・目的:蛋白結合尿毒症物質(protein-bound uremic toxins;PBUTs)の血中濃度高値は種々の透析関連合併症の原因となる。我々はヘキサデキル基固定セルロースビーズ(hexadecyl-immobilized cellulose bead;HICB)のPBUTsの吸着効果について臨床的に検討した。方法:HICBを含有したカラム(リクセルS-35)を維持血液透析患者に使用した。カラム通過前後と2週間使用前後の血清インドキシル硫酸(indoxyl sulfate;IS)、インドール酢酸(indole acetic acid;IAA)、フェニル硫酸(phenyl sulfate;PhS)、そしてpクレシル硫酸(p-cresyl sulfate;PCS)濃度を質量分析により測定した。結果:リクセルS-35通過後に蛋白結合していない血中フリーIS、IAA、PhSとPCS濃度は34.4±30.0%、34.8±25.4%、28.4±18.0%と34.9±22.1%減少した。しかし、蛋白結合したPBUTsはカラム通過後に有意に減少しなかった。リクセルを2週間使用したが、血中PBUT値は有意に低下しなかった。結論:HICBsはPBUTsを部分的に吸着した。この吸着効果は臨床的に十分でなく、今後PBUTs吸着効果のより優れた血液浄化器の開発が望まれる。(著者抄録)

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry and Molecular Biology Inc.  

The cystine/glutamate transporter, designated as system x&lt
inf&gt
c&lt
/inf&gt
&lt
sup&gt
-&lt
/sup&gt
, is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system x&lt
inf&gt
c&lt
/inf&gt
&lt
sup&gt
-&lt
/sup&gt
, xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system x&lt
inf&gt
c&lt
/inf&gt
&lt
sup&gt
-&lt
/sup&gt
and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine β-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation.Wethus conclude that cystathionine is a novel physiological substrate of system x&lt
inf&gt
c&lt
/inf&gt
&lt
sup&gt
-&lt
/sup&gt
and that the accumulation of cystathionine in immune tissues is exclusively mediated by system x&lt
inf&gt
c&lt
/inf&gt
&lt
sup&gt
-&lt
/sup&gt
.

DOI： 10.1074/jbc.M114.625053

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