Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades

Mismatch-repair (MMR) systems promote genomic stability by correction of DNA replication errors. Thus, MMR proteins—prokaryotic MutS and MutL homodimers or their MutSα and MutLα heterodimer homologs, plus accessory proteins—specifically couple mismatch recognition to nascent-DNA excision. In vivo excision-initiation signals—specific nicks in some prokaryotes, perhaps growing 3′ ends or Okazaki-fragment 5′ ends in eukaryotes—are efficiently mimicked in vitro by nicks or gaps in exogenous DNA substrates. In some models for recognition–excision coupling, MutSα bound to mismatches is induced by ATP hydrolysis, or simply by binding of ATP, to slide along DNA to excision-initiation sites, perhaps in association with MutLα and accessory proteins. In other models, MutSα·MutLα complexes remain fixed at mismatches and contact distant excision sites by DNA looping. To challenge the hypothesis that recognition complexes remain fixed, we placed biotin–streptavidin blockades between mismatches and pre-existing nicks. In human nuclear extracts, mismatch efficiently provoked the initiation of excision despite the intervening barriers, as predicted. However, excision progress and therefore mismatch correction were prevented

The Effect of Flavored E-cigarettes on Murine Allergic Airways Disease

© 2019, The Author(s). Flavored e-cigarettes are preferred by the majority of users yet their potential toxicity is unknown. Therefore our aim was to determine the effect of selected flavored e-cigarettes, with or without nicotine, on allergic airways disease in mice. Balb/c mice were challenged with PBS or house dust mite (HDM) (Days 0, 7, 14–18) and exposed to room air or e-cigarette aerosol for 30 min twice daily, 6 days/week from Days 0–18 (n = 8–12/group). Mice were exposed to Room Air, vehicle control (50%VG/%50PG), Black Licorice, Kola, Banana Pudding or Cinnacide without or with 12 mg/mL nicotine. Mice were assessed at 72 hours after the final HDM challenge. Compared to mice challenged with HDM and exposed to Room Air, nicotine-free Cinnacide reduced airway inflammation (p = 0.045) and increased peripheral airway hyperresponsiveness (p = 0.02), nicotine-free Banana Pudding increased soluble lung collagen (p = 0.049), with a trend towards increased airway inflammation with nicotine-free Black Licorice exposure (p = 0.089). In contrast, all e-cigarettes containing nicotine suppressed airway inflammation (p < 0.001 for all) but did not alter airway hyperresponsiveness or airway remodeling. Flavored e-cigarettes without nicotine had significant but heterogeneous effects on features of allergic airways disease. This suggests that some flavored e-cigarettes may alter asthma pathophysiology even when used without nicotine

Absence of c-Jun NH<inf>2</inf>-terminal kinase 1 protects against house dust mite-induced pulmonary remodeling but not airway hyperresponsiveness and inflammation

Chronic allergic asthma leads to airway remodeling and subepithelial fibrosis via mechanisms not fully understood. Airway remodeling is amplified by profibrotic mediators, such as transforming growth factor-β1 (TGF-β1), which plays a cardinal role in various models of fibrosis. We recently have identified a critical role for c-Jun-NH2-terminal-kinase (JNK) 1 in augmenting the profibrotic effects of TGF-β1, linked to epithelial-to-mesenchymal transition of airway epithelial cells. To examine the role of JNK1 in house dust mite (HDM)-induced airway remodeling, we induced allergic airway inflammation in wild-type (WT) and JNK1-/- mice by intranasal administration of HDM extract. WT and JNK1-/- mice were sensitized with intranasal aspirations of HDM extract for 15 days over 3 wk. HDM caused similar increases in airway hyperresponsiveness, mucus metaplasia, and airway inflammation in WT and JNK1-/- mice. In addition, the profibrotic cytokine TGF-β1 and phosphorylation of Smad3 were equally increased in WT and JNK1-/- mice. In contrast, increases in collagen content in lung tissue induced by HDM were significantly attenuated in JNK1-/- mice compared with WT controls. Furthermore HDM-induced increases of α-smooth muscle actin (α-SMA) protein and mRNA expression as well as the mesenchymal markers high-mobility group AT-hook 2 and collagen1A1 in WT mice were attenuated in JNK1-/- mice. The let-7 family of microRNAs has previously been linked to fibrosis. HDM exposure in WT mice and primary lung epithelial cells resulted in striking decreases in let-7g miRNA that were not observed in mice or primary lung epithelial cells lacking JNK1-/- mice. Overexpression of let-7g in lung epithelial cells reversed the HDM-induced increases in α-SMA. Collectively, these findings demonstrate an important requirement for JNK1 in promoting HDM-induced fibrotic airway remodeling. © 2014 the American Physiological Society

Emerging mechanisms of glutathione-dependent chemistry in biology and disease

Glutathione has traditionally been considered as an antioxidant that protects cells against oxidative stress. Hence, the loss of reduced glutathione and formation of glutathione disulfide is considered a classical parameter of oxidative stress that is increased in diseases. Recent studies have emerged that demonstrate that glutathione plays a more direct role in biological and pathophysiological processes through covalent modification to reactive cysteines within proteins, a process known as S-glutathionylation. The formation of an S-glutathionylated moiety within the protein can lead to structural and functional modifications. Activation, inactivation, loss of function, and gain of function have all been attributed to S-glutathionylation. In pathophysiological settings, S-glutathionylation is tightly regulated. This perspective offers a concise overview of the emerging field of protein thiol redox modifications. We will also cover newly developed methodology to detect S-glutathionylation in situ, which will enable further discovery into the role of S-glutathionylation in biology and disease. © 2013 Wiley Periodicals, Inc

The Duffy antigen receptor for chemokines regulates asthma pathophysiology

© 2017 John Wiley & Sons Ltd Background: The Duffy antigen receptor for chemokines (DARC) is an atypical receptor that regulates pro-inflammatory cytokines. However, the role of DARC in asthma pathophysiology is unknown. Objective: To determine the role of DARC in allergic airways disease in mice, and the association between DARC single nucleotide polymorphisms (SNPs) and clinical outcomes in patients with asthma. Methods: Mice with targeted disruption of the Darc gene (Darc∆E2) or WT mice were challenged over 3 weeks with house dust mite (HDM) antigen. Allergic airways disease was assessed 24 hours and 7 days following the final challenge. Additionally, associations between DARC SNPs and clinical outcomes were analysed in a cohort of poorly controlled asthmatics. Results: Total airway inflammation following HDM did not differ between Darc∆E2 and WT mice. At 24 hours, Darc∆E2 mice had increased airway hyperresponsiveness; however, at 7 days airway hyperresponsiveness had completely resolved in Darc∆E2 but persisted in WT mice. In poorly controlled asthmatics, DARC SNPs were associated with worse asthma control at randomization and subsequent increased risk of healthcare utilization (odds ratio 3.13(1.37-7.27), P=.0062). Conclusions and Clinical Relevance: Our animal model and human patient data suggest a novel role for DARC in the temporal regulation in asthma pathophysiology and symptoms

Reducing protein oxidation reverses lung fibrosis

© 2018, The Author(s). Idiopathic pulmonary fibrosis is characterized by excessive deposition of collagen in the lung, leading to chronically impaired gas exchange and death 1–3 . Oxidative stress is believed to be critical in this disease pathogenesis 4–6 , although the exact mechanisms remain enigmatic. Protein S-glutathionylation (PSSG) is a post-translational modification of proteins that can be reversed by glutaredoxin-1 (GLRX) 7 . It remains unknown whether GLRX and PSSG play a role in lung fibrosis. Here, we explored the impact of GLRX and PSSG status on the pathogenesis of pulmonary fibrosis, using lung tissues from subjects with idiopathic pulmonary fibrosis, transgenic mouse models and direct administration of recombinant Glrx to airways of mice with existing fibrosis. We demonstrate that GLRX enzymatic activity was strongly decreased in fibrotic lungs, in accordance with increases in PSSG. Mice lacking Glrx were far more susceptible to bleomycin- or adenovirus encoding active transforming growth factor beta-1 (AdTGFB1)-induced pulmonary fibrosis, whereas transgenic overexpression of Glrx in the lung epithelium attenuated fibrosis. We furthermore show that endogenous GLRX was inactivated through an oxidative mechanism and that direct administration of the Glrx protein into airways augmented Glrx activity and reversed increases in collagen in mice with TGFB1- or bleomycin-induced fibrosis, even when administered to fibrotic, aged animals. Collectively, these findings suggest the therapeutic potential of exogenous GLRX in treating lung fibrosis