Molecular hydrogen attenuates gefitinib-induced exacerbation of naphthalene-evoked acute lung injury through a reduction in oxidative stress and inflammation.

Although inhibition of epidermal growth factor receptor (EGFR)-mediated cell signaling by the EGFR tyrosine kinase inhibitor gefitinib is highly effective against advanced non-small cell lung cancer, this drug might promote severe acute interstitial pneumonia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive anti-oxidant. Here, we show that treatment with H2 effectively protects the lungs of mice from severe damage caused by oral administration of gefitinib after intraperitoneal injection of naphthalene, the toxicity of which is related to oxidative stress. Drinking H2-rich water ad libitum mitigated naphthalene/gefitinib-induced weight loss and significantly improved survival, which was associated with a decrease in lung inflammation and inflammatory cytokines in the bronchoalveolar lavage fluid. Naphthalene decreased glutathione in the lung, increased malondialdehyde in the plasma, and increased 4-hydroxy-2-nonenal production in airway cells, all of which were mitigated by H2-rich water, indicating that the H2-rich water reverses cellular damage to the bronchial wall caused by oxidative stress. Finally, treatment with H2 did not interfere with the anti-tumor effects of gefitinib on a lung cancer cell line in vitro or on tumor-bearing mice in vivo. These results indicate that H2-rich water has the potential to improve quality of life during gefitinib therapy by mitigating lung injury without impairing anti-tumor activity.

The proline-arginine repeat protein linked to C9-ALS/FTD causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

A GGGGCC repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat (DPR) proteins. Although it has been reported that DPR proteins cause neurotoxicity, the underlying mechanism has not been fully elucidated. In this study, we have first confirmed that proline-arginine repeat protein (poly-PR) reduces levels of ribosomal RNA and causes neurotoxicity and found that the poly-PR-induced neurotoxicity is repressed by the acceleration of ribosomal RNA synthesis. These results suggest that the poly-PR-induced inhibition of ribosome biogenesis contributes to the poly-PR-induced neurotoxicity. We have further identified DEAD-box RNA helicases as poly-PR-binding proteins, the functions of which are inhibited by poly-PR. The enforced reduction in the expression of DEAD-box RNA helicases causes impairment of ribosome biogenesis and neuronal cell death. These results together suggest that poly-PR causes neurotoxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

Src Family Tyrosine Kinase Signaling Regulates FilGAP through Association with RBM10.

FilGAP is a Rac-specific GTPase-activating protein (GAP) that suppresses lamellae formation. In this study, we have identified RBM10 (RNA Binding Motif domain protein 10) as a FilGAP-interacting protein. Although RBM10 is mostly localized in the nuclei in human melanoma A7 cells, forced expression of Src family tyrosine kinase Fyn induced translocation of RBM10 from nucleus into cell peripheries where RBM10 and FilGAP are co-localized. The translocation of RBM10 from nucleus appears to require catalytic activity of Fyn since kinase-negative Fyn mutant failed to induce translocation of RBM10 in A7 cells. When human breast carcinoma MDA-MB-231 cells are spreading on collagen-coated coverslips, endogenous FilGAP and RBM10 were localized at the cell periphery with tyrosine-phosphorylated proteins. RBM10 appears to be responsible for targeting FilGAP at the cell periphery because depletion of RBM10 by siRNA abrogated peripheral localization of FilGAP during cell spreading. Association of RBM10 with FilGAP may stimulate RacGAP activity of FilGAP. First, forced expression of RBM10 suppressed FilGAP-mediated cell spreading on collagen. Conversely, depletion of endogenous RBM10 by siRNA abolished FilGAP-mediated suppression of cell spreading on collagen. Second, FilGAP suppressed formation of membrane ruffles induced by Fyn and instead produced spiky cell protrusions at the cell periphery. This protrusive structure was also induced by depletion of Rac, suggesting that the formation of protrusions may be due to suppression of Rac by FilGAP. We found that depletion of RBM10 markedly reduced the formation of protrusions in cells transfected with Fyn and FilGAP. Finally, depletion of RBM10 blocked FilGAP-mediated suppression of ruffle formation induced by EGF. Taken together, these results suggest that Src family tyrosine kinase signaling may regulate FilGAP through association with RBM10.

An efficient screening system for influenza virus cap-dependent endonuclease inhibitors.

The synthesis of influenza virus mRNA is primed by capped (m(7)GpppNm-) short RNAs that are cleaved from RNA polymerase II transcripts by a virally encoded endonuclease. This cap-dependent endonuclease activity called "cap-snatching" may provide a unique target for novel anti-viral agents. To screen candidate inhibitors, it is essential to establish a method for producing efficiently a capped RNA substrate and a convenient assay for the cap-snatching activity. A 3'-biotinylated short RNA was prepared by in vitro transcription, purified by C18 reverse-phase column chromatography, and subjected to a capping reaction involving three recombinant capping enzymes. This capped RNA was shown to be an efficient substrate for the cap-snatching assay. Cap-snatching activity was then measured with the novel pull-down assay developed in this study, which is based on the streptavidin-biotin interaction. A known inhibitor for the cap-snatching reaction was evaluated by the pull-down assay, demonstrating the efficacy of the established screening system.