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A helicase-primase inhibitor, amenamevir (ASP2151), is the active pharmaceutical ingredient of a drug for the herpes zoster that is caused by reactivation of varicella-zoster virus (VZV). Here we report a new amenamevir-resistant VZV isolated under the selection pressure of amenamevir. The resistant virus has a nonsynonymous mutation K350N in the helicase gene ORF55. A recombinant virus artificially constructed harboring the ORF55 K350N also acquired amenamevir resistance, and thus the single amino-acid substitution in helicase is revealed to be responsible for the resistance. We observed that the drug-resistant virus and the ORF55 K350N recombinant virus have high resistance to amenamevir, as the EC 50 values in a plaque reduction assay were > 100 μM, while the two viruses remained susceptible to the nucleoside analog drug acyclovir. No defect in viral growth was observed for these resistant viruses in a plaque size assay in human malignant melanoma cells. However, defect in plaque formation was observed from resistant virus in human fetal lung fibroblast cells, showing that the growth of the resistant virus is dependent on the cell type. We observed that the single amino-acid substitution in the helicase induces amenamevir resistance, confirming the importance of the helicase in amenamevir's inhibition of virus growth. Our findings highlight the importance of regulating the clinical use of amenamevir to minimize the risk of the emergence of helicase K350N mutation, especially in the long-term use of amenamevir by immunosuppressed patients., (© 2024 Wiley Periodicals LLC.)

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued, enabling the virus to escape from host immunity by changing its spike antigen, while biased toward the receptor-binding domain and N-terminal domain. Here, we isolated a novel pan-SARS-CoV-2 neutralizing antibody (which we named MO11) for even the recent dominators XBB.1.16 and EG.5.1, from a convalescent patient who had received three doses of an original mRNA COVID-19 vaccination. A cryo-electron microscopy analysis of the spike-MO11 complex at 2.3 Å atomic resolution revealed that it recognizes a conserved epitope hidden behind a glycan shield at N331 on subdomain 1 (SD1), holding both the N- and C-terminal segments comprising SD1. Our identification of MO11 unveiled the functional importance of SD1 for the spike's function, and we discuss the potential availability of a novel common epitope among the SARS-CoV-2 variants.IMPORTANCENovel severe acute respiratory syndrome coronavirus 2 variants with immune evasion ability are still repeatedly emerging, nonetheless, a part of immunity developed in responding to the antigen of earlier variants retains efficacy against recent variants irrespective of the numerous mutations. In exploration for the broadly effective antibodies, we identified a cross-neutralizing antibody, named MO11, from the B cells of the convalescent patient. MO11 targets a novel epitope in subdomain 1 (SD1) and was effective against all emerging variants including XBB.1.16 and EG.5.1. The neutralizing activity covering from D614G to EG.5.1 variants was explained by the conservation of the epitope, and it revealed the importance of the subdomain on regulating the function of the antigen for viral infection. Demonstrated identification of the neutralizing antibody that recognizes a conserved epitope implies basal contribution of such group of antibodies for prophylaxis against COVID-19., Competing Interests: The authors declare no conflict of interest.

Background: Next-generation sequencing (NGS) is essential for lung cancer treatment. It is important to collect sufficient tissue specimens, but sometimes we cannot obtain large enough samples for NGS analysis. We investigated the yield of NGS analysis by frozen cytology pellets using an Oncomine Comprehensive Assay or Oncomine Precision Assay., Methods: We retrospectively enrolled patients with lung cancer who underwent bronchoscopy at Kobe University Hospital and were enrolled in the Lung Cancer Genomic Screening Project for Individualized Medicine. We investigated the amount of extracted DNA and RNA and determined the NGS success rates. We also compared the amount of DNA and RNA by bronchoscopy methods. To create the frozen cytology pellets, we first effectively collected the cells and then quickly centrifuged and cryopreserved them., Results: A total of 132 patients were enrolled in this study between May 2016 and December 2022; of them, 75 were subjected to frozen cytology pellet examinations and 57 were subjected to frozen tissue examinations. The amount of DNA and RNA obtained by frozen cytology pellets was nearly equivalent to frozen tissues. Frozen cytology pellets collected by endobronchial ultrasound-guided transbronchial needle aspiration yielded significantly more DNA than those collected by transbronchial biopsy methods. (P < 0.01) In RNA content, cytology pellets were not inferior to frozen tissue. The success rate of NGS analysis with frozen cytology pellet specimens was comparable to the success rate of NGS analysis with frozen tissue specimens., Conclusions: Our study showed that frozen cytology pellets may have equivalent diagnostic value to frozen tissue for NGS analyses. Bronchial cytology specimens are usually used only for cytology, but NGS analysis is possible if enough cells are collected to create pellet specimens. In particular, the frozen cytology pellets obtained by endobronchial ultrasound-guided transbronchial needle aspiration yielded sufficient amounts of DNA., Trial Registration: This was registered with the University Medical Hospital Information Network in Japan (UMINCTR registration no. UMIN000052050)., (© 2024. The Author(s).)

Objectives: The combination of chemotherapy and immune checkpoint inhibitors (chemo-ICI) has become the new standard of treatment for extensive-stage small cell lung cancer (ES-SCLC). Recently, slight changes in interstitial shadows, defined as interstitial lung abnormalities (ILA), have been identified. In patients with ES-SCLC who received chemo-ICI, there are limited data on the incidence of drug-induced interstitial lung disease (D-ILD) in daily practice and the association between the development of D-ILD and ILA in the baseline computed tomography (CT)., Materials and Methods: A multicenter, retrospective study was conducted to investigate the incidence of D-ILD, the risk factors for developing D-ILD, progression-free survival (PFS), and overall survival (OS) in patients with ES-SCLC who received chemo-ICI between August 2019 and November 2021., Results: This study enrolled 70 patients (median age, 71 years; including 58 men) from nine institutions in Japan. There were 62 patients (89%) treated with carboplatin/etoposide/atezolizumab and 8 patients treated with carboplatin or cisplatin/etoposide/durvalumab. Twenty-nine patients (41.4%) were found to have ILA at baseline CT. Eleven patients (15.7%) developed D-ILD. The proportion of patients with ILA was significantly higher in the group who developed D-ILD than in the group who did not (9/11 (81.8%) vs. 20/59 (33.9%), respectively, P = 0.0057). In addition, the frequency of ground glass attenuation (GGA) and reticulation was higher in patients who developed D-ILD. There was no significant difference in PFS and OS between patients who developed D-ILD and those who did not (median PFS, 8.0 (95% confidence interval (CI), 5.5-9.5) months vs. 5.0 (95% CI, 4.5-5.6) months, respectively, P = 0.11 and median OS, not reached (NR) (95% CI, 8.7-NR) vs. 18.2 (95% CI, 13.2-NR) months, respectively, P = 0.20)., Conclusion: The incidence of D-ILD in patients with ES-SCLC who received chemo-ICI in clinical practice was higher than that in clinical trials. Patients with pre-existing ILA were more likely to develop D-ILD., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Matters requiring disclosure of COI with regard to our presentation are lecture fee by Chugai Pharmaceutical Co Ltd and research expenses from company by AstraZeneca., (© 2023 The Authors. Published by Elsevier Ltd.)

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Among the phospholipase A 2 (PLA 2 ) superfamily, group IVA cytosolic PLA 2 (cPLA 2 α) is currently attracting much attention as a central regulator of arachidonic acid (AA) metabolism linked to eicosanoid biosynthesis. Following cell activation, cPLA 2 α selectively releases AA, a precursor of a variety of eicosanoids, from phospholipids in perinuclear membrane compartments. cPLA 2 α-null mice display various phenotypes that could be largely explained by reduced eicosanoid signaling. In contrast, group IVE cPLA 2 ε, another member of the cPLA 2 family, acts as a Ca 2+ -dependent N-acyltransferase rather than a PLA 2 , thereby regulating the biosynthesis of N-acylethanolamines (NAEs), a unique class of lipid mediators with an anti-inflammatory effect. In response to Ca 2+ signaling, cPLA 2 ε translocates to phosphatidylserine-rich organelle membranes in the endocytic/recycling pathway. In vivo, cPLA 2 ε is induced in keratinocytes of psoriatic skin, and its genetic deletion exacerbates psoriatic inflammation due to a marked reduction of NAE-related lipids. cPLA 2 ε also contributes to NAE generation in several if not all mouse tissues. Thus, the two members of the cPLA 2 family, cPLA 2 α and cPLA 2 ε, catalyze distinct enzymatic reactions to mobilize distinct sets of lipid mediators, thereby differently regulating pathophysiological events in health and disease. Such segregation of the cPLA 2 α-eicosanoid and cPLA 2 ε-NAE pathways represents a new paradigm of research on PLA 2 s and lipid mediators., (Copyright © 2022. Published by Elsevier Inc.)

Psoriasis is an inflammatory disorder characterized by keratinocyte hyper-proliferation and Th17-type immune responses. However, the roles of bioactive lipids and the regulation of their biosynthesis in this chronic skin disease are not fully understood. Herein, we show that group IVE cytosolic phospholipase A 2 (cPLA 2 ε/PLA2G4E) plays a counterregulatory role against psoriatic inflammation by producing the anti-inflammatory lipid N-acylethanolamine (NAE). Lipidomics analysis of mouse skin revealed that NAE species and their precursors (N-acyl-phosphatidylethanolamine and glycerophospho-N-acylethanolamine) were robustly increased in parallel with the ongoing process of imiquimod (IMQ)-induced psoriasis, accompanied by a marked upregulation of cPLA 2 ε in epidermal keratinocytes. Genetic deletion of cPLA 2 ε exacerbated IMQ-induced ear swelling and psoriatic marker expression, with a dramatic reduction of NAE-related lipids in IMQ-treated, and even normal, skin. Stimulation of cultured human keratinocytes with psoriatic cytokines concomitantly increased PLA2G4E expression and NAE production, and supplementation with NAEs significantly attenuated the cytokine-induced upregulation of the psoriatic marker S100A9. Increased expression of cPLA 2 ε was also evident in the epidermis of psoriatic patients. These findings reveal for the first time the in vivo role of cPLA 2 ε, which is highly induced in the keratinocytes of the psoriatic skin, promotes the biosynthesis of NAE-related lipids, and contributes to limiting psoriatic inflammation., (© 2022 Federation of American Societies for Experimental Biology.)

Background: Surfactant proteins (SP) A and D belong to collectin family proteins, which play important roles in innate immune response in the lung. We previously demonstrated that cigarette smoke (CS) increases the acrolein modification of SP-A, thereby impairing the innate immune abilities of this protein. In this study, we focused on the effects of CS and its component, acrolein, on the innate immunity role of another collectin, SP-D., Methods: To determine whether aldehyde directly affects SP-D, we examined the lungs of mice exposed to CS for 1 week and detected aldehyde-modified SP-D using an aldehyde reactive probe. The structural changes in CS extract (CSE) or acrolein-exposed recombinant human (h)SP-D were determined by western blot, liquid chromatography-electrospray ionization tandem mass spectrometry, and blue native-polyacrylamide gel electrophoresis analyses. Innate immune functions of SP-D were determined by bacteria growth and macrophage phagocytosis., Results: Aldehyde-modified SP-D as well as SP-A was detected in the lungs of mice exposed to CS for 1 week. Exposure of hSP-D to CSE or acrolein induced an increased higher-molecular -weight of hSP-D and acrolein induced modification of five lysine residues in hSP-D. These modifications led to disruption of the multimer structure of SP-D and attenuated its ability to inhibit bacterial growth and activate macrophage phagocytosis., Conclusion: CS induced acrolein modification in SP-D, which in turn induced structural and functional defects in SP-D., General Significance: These results suggest that CS-induced structural and functional defects in SP-D contribute to the dysfunction of innate immune responses in the lung following CS exposure., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)

During the manufacture of cell- and tissue-based products, such as engineered cartilage for autologous chondrocyte implantation, maximizing the number of cells isolated from donor tissue substantially improves the productivity of these products. The method used for agitating tissues with digestive fluid and enzymes can considerably affect both the quality and quantity of isolated cells. This study aimed to investigate the effectiveness of a rotation/revolution-type agitator for chondrocyte isolation following the enzymatic digestion of rat costal cartilage. Cartilage tissue cut into 1 mm 3 -thick sections was equally divided between two groups and placed in 50-mL conical tubes; sections in both groups were digested using 0.1 mg/mL liberase TH (collagenase/thermolysin) at 37 °C for 4 h with either rotation/revolution or conventional orbital agitation method. Compared with using conventional orbital agitator, using the rotation/revolution-type agitator resulted in a significant (>two-fold) increase in the number of isolated cells. In subsequent primary cultures, chondrocytes obtained by rotation/revolution agitation showed superior initial attachment to tissue culture dish on day 1 and 2 compared with those obtained by conventional agitation; however, no differences in cell proliferation or cartilage-related molecule expression patterns were observed between cells derived from either method after 3 days of subculture. These findings suggested that there are no disadvantages to the proposed rotation/revolution agitation method. Rotation/revolution-type agitators are a promising apparatus for preparing chondrocytes for primary cultures and cartilage tissue engineering., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We recently reported that the lectin surfactant protein D (SP-D) suppresses epidermal growth factor receptor (EGFR) signaling by interfering with ligand binding to EGFR through an interaction between the carbohydrate-recognition domain (CRD) of SP-D and N -glycans of EGFR. Here, we report that surfactant protein A (SP-A) also suppresses EGF signaling in A549 human lung adenocarcinoma cells and in CHOK1 cells stably expressing human EGFR and that SP-A inhibits the proliferation and motility of the A549 cells. Results with 125 I-EGF indicated that SP-A interferes with EGF binding to EGFR, and a ligand blot analysis suggested that SP-A binds EGFR in A549 cells. We also found that SP-A directly binds the recombinant extracellular domain of EGFR (soluble EGFR or sEGFR), and this binding, unlike that of SP-D, was not blocked by EDTA, excess mannose, or peptide: N -glycosidase F treatment. We prepared a collagenase-resistant fragment (CRF) of SP-A, consisting of CRD plus the neck domain of SP-A, and observed that CRF directly binds sEGFR but does not suppress EGF-induced phosphorylation of EGFR in or proliferation of A549 cells. These results indicated that SP-A binds EGFR and down-regulates EGF signaling by inhibiting ligand binding to EGFR as well as SP-D. However, unlike for SP-D, SP-A lectin activity and EGFR N -glycans were not involved in the interaction between SP-A and EGFR. Furthermore, our results suggested that oligomerization of SP-A is necessary to suppress the effects of SP-A on EGF signaling., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The extent to which defective innate immune responses contribute to chronic obstructive pulmonary disease (COPD) is not fully understood. Pulmonary surfactant protein A (SP-A) plays an important role in regulating innate immunity in the lungs. In this study, we hypothesised that cigarette smoke (CS) and its component acrolein might influence pulmonary innate immunity by affecting the function of SP-A. Indeed, acrolein-modified SP-A was detected in the lungs of mice exposed to CS for 1 week. To further confirm this finding, recombinant human SP-A (hSP-A) was incubated with CS extract (CSE) or acrolein and then analysed by western blotting and nanoscale liquid chromatography-matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry. These analyses revealed that CSE and acrolein induced hSP-A oligomerisation and that acrolein induced the modification of six residues in hSP-A: His39, His116, Cys155, Lys180, Lys221, and Cys224. These modifications had significant effects on the innate immune functions of hSP-A. CSE- or acrolein-induced modification of hSP-A significantly decreased hSP-A's ability to inhibit bacterial growth and to enhance macrophage phagocytosis. These findings suggest that CS-induced structural and functional defects in SP-A contribute to the dysfunctional innate immune responses observed in the lung during cigarette smoking.

Surfactant protein A (SP-A) is a multifunctional host defense collectin that was first identified as a component of pulmonary surfactant. Although SP-A is also expressed in various tissues, including the urinary tract, its innate immune functions in nonpulmonary tissues are poorly understood. In this study, we demonstrated that adherence of uropathogenic Escherichia coli (UPEC) to the bladder was enhanced in SP-A-deficient mice, which suggests that SP-A plays an important role in innate immunity against UPEC. To understand the innate immune functions of SP-A in detail, we performed in vitro experiments. SP-A directly bound to UPEC in a Ca 2+ -dependent manner, but it did not agglutinate UPEC. Our results suggest that a bouquet-like arrangement seems unsuitable to agglutinate UPEC. Meanwhile, SP-A inhibited growth of UPEC in human urine. Furthermore, the binding of SP-A to UPEC decreased the adherence of bacteria to urothelial cells. These results indicate that direct action of SP-A on UPEC is important in host defense against UPEC. Additionally, adhesion of UPEC to urothelial cells was decreased when the cells were preincubated with SP-A. Adhesion of UPEC to urothelial cells is achieved via interaction between FimH, an adhesin located at bacterial pili, and uroplakin Ia, a glycoprotein expressed on the urothelium. SP-A directly bound to uroplakin Ia and competed with FimH for uroplakin Ia binding. These results lead us to conclude that SP-A plays important roles in host defense against UPEC., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Human β-defensin 3 (hBD3) is known to be involved in mast cell activation. However, molecular mechanisms underlying the regulation of hBD3-induced mast cell activation have been poorly understood. We previously reported that SP-A and SP-A-derived peptide 01 (SAP01) regulate the function of hBD3. In this study, we focused on the effects of SP-A and SAP01 on the activation of mast cells induced by hBD3. SAP01 directly bound to hBD3. Mast cell-mediated vascular permeability and edema in hBD3 administered rat ears were decreased when injected with SP-A or SAP01. Compatible with the results in rat ear model, both SP-A and SAP01 inhibited hBD3-induced chemotaxis of mast cells in vitro. Direct interaction between SP-A or SAP01 and hBD3 seemed to be responsible for the inhibitory effects on chemotaxis. Furthermore, SAP01 attenuated hBD3-induced accumulation of mast cells and eosinophils in tracheas of the OVA-sensitized inflammatory model. SP-A might contribute to the regulation of inflammatory responses mediated by mast cells during infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In the bone, collagen fibrils form a lamellar structure called the "twisted plywood-like model." Because of this unique structure, bone can withstand various mechanical stresses. However, the formation of this structure has not been elucidated because of the difficulty of observing the collagen fibril production of the osteoblasts via currently available methods. This is because the formation occurs in the very limited space between the osteoblast layer and bone matrix. In this study, we used ultra-high-voltage electron microscopy (UHVEM) to observe collagen fibril production three-dimensionally. UHVEM has 3-MV acceleration voltage and enables us to use thicker sections. We observed collagen fibrils that were beneath the cell membrane of osteoblasts elongated to the outside of the cell. We also observed that osteoblasts produced collagen fibrils with polarity. By using AVIZO software, we observed collagen fibrils produced by osteoblasts along the contour of the osteoblasts toward the bone matrix area. Immediately after being released from the cell, the fibrils run randomly and sparsely. But as they recede from the osteoblast, the fibrils began to run parallel to the definite direction and became thick, and we observed a periodical stripe at that area. Furthermore, we also observed membrane structures wrapped around filamentous structures inside the osteoblasts. The filamentous structures had densities similar to the collagen fibrils and a columnar form and diameter. Our results suggested that collagen fibrils run parallel and thickly, which may be related to the lateral movement of the osteoblasts. UHVEM is a powerful tool for observing collagen fibril production.

An enzyme, O -acetylserine(thiol)lyase (OASTL), also known as O -acetylserine sulfhydrylase or cysteine synthase (CSase), catalyses the incorporation of sulfide into O -acetylserine and produces cysteine. We previously identified a cDNA encoding an OASTL-like protein from Spinacia oleracea , ( SoCSaseLP ), but a recombinant SoCSaseLP produced in Escherichia coli did not show OASTL activity. The exon-intron structure of the SoCSaseLP gene shared conserved structures with other spinach OASTL genes. The SoCSaseLP and a Beta vulgaris homologue protein, KMT13462, comprise a unique clade in the phylogenetic tree of the OASTL family. Interestingly, when the SoCSaseLP gene was expressed in tobacco plants, total OASTL activity in tobacco leaves was reduced. This reduction in total OASTL activity was most likely caused by interference by SoCSaseLP with cytosolic OASTL. To investigate the possible interaction of SoCSaseLP with a spinach cytosolic OASTL isoform SoCSaseA, a pull-down assay was carried out. The recombinant glutathione S -transferase (GST)-SoCSaseLP fusion protein was expressed in E. coli together with the histidine-tagged SoCSaseA protein, and the protein extract was subjected to glutathione affinity chromatography. The histidine-tagged SoCSaseA was co-purified with the GST-SoCSaseLP fusion protein, indicating the binding of SoCSaseLP to SoCSaseA. Consistent with this interaction, the OASTL activity of the co-purified SoCSaseA was reduced compared with the activity of SoCSaseA that was purified on its own. These results strongly suggest that SoCSaseLP negatively regulates the activity of other cytosolic OASTL family members by direct interaction.

It has been well documented that activation of the ErbB3-PI3K-Akt pathway is implicated in tumor survival and progression. We previously demonstrated that the single N-glycan deletion mutant of soluble ErbB3 protein (sErbB3 N418Q) attenuates heregulin β1-induced ErbB3 signaling. The active PI3K-Akt pathway augments the nuclear accumulation of hypoxia inducible factor (HIF)-1α, which activates the transcription of many target genes and drives cancer progression. In this study, we focused on the effects of sErbB3 N418Q mutant on nuclear accumulation of HIF-1α. Pretreatment with the sErbB3 N418Q mutant suppressed heregulin β1-induced HIF-1α activation in MCF7 cells. Similar results were also obtained in other breast cancer cell lines, T47D and BT474. Interestingly, these suppressive effects were not observed with the sErbB3 wild type. In addition, pretreatment with the sErbB3 N418Q mutant suppressed the cell migration of MCF7 cells induced by heregulin β1. Furthermore, incubation with heregulin β1 also induced the nuclear accumulation of Nrf2, and this effect was also reduced by the sErbB3 N418Q mutant, but not the sErbB3 wild type. These findings indicated that the sErbB3 N418Q mutant suppressed malignant formation of cancer cells by blocking of the HIF-1α and Nrf2 pathways., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Heregulin signaling is involved in various tumor proliferations and invasions; thus, receptors of heregulin are targets for the cancer therapy. In this study we examined the suppressing effects of extracellular domains of ErbB2, ErbB3, and ErbB4 (soluble ErbB (sErbB)) on heregulin β signaling in human breast cancer cell line MCF7. It was found that sErbB3 suppresses ligand-induced activation of ErbB receptors, PI3K/Akt and Ras/Erk pathways most effectively; sErbB2 scarcely suppresses ligand-induced signaling, and sErbB4 suppresses receptor activation at ∼10% efficiency of sErbB3. It was revealed that sErbB3 does not decrease the effective ligands but decreases the effective receptors. By using small interfering RNA (siRNA) for ErbB receptors, we determined that sErbB3 suppresses the heregulin β signaling by interfering ErbB3-containing heterodimers including ErbB2/ErbB3. By introducing the mutation of N418Q to sErbB3, the signaling-inhibitory effects were increased by 2-3-fold. Moreover, the sErbB3 N418Q mutant enhanced anticancer effects of lapatinib more effectively than the wild type. We also determined the structures of N-glycan on Asn-418. Results suggested that the N-glycan-deleted mutant of sErbB3 suppresses heregulin signaling via ErbB3-containing heterodimers more effectively than the wild type. Thus, we demonstrated that the sErbB3 N418Q mutant is a potent inhibitor for heregulin β signaling.

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using (13)C6-glucose and (13)C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with (13)C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a (13)C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using (13)C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.

We previously demonstrated that Siglec-15, a member of the Siglec family of glycan-recognition proteins, is expressed on a subset of macrophages and preferentially recognizes the sialyl-Tn (sTn) antigen, a tumor-associated glycan structure. In this study, we report on the biological significance of the Siglec-15-mediated interaction between monocytes/macrophages and cancer cells. Siglec-15 is expressed on tumor-associated macrophages (TAMs) in various human tumor tissues. We further demonstrated that its expression is substantially elevated in macrophage colony-stimulating factor-induced M2-like macrophages, which produced more transforming growth factor-β (TGF-β) in response to sTn-positive cells than to negative cells. We designed a co-culture model of THP-1 (human monocytic leukemia) cells and H157 (human lung carcinoma) cells mimicking the interaction between monocytes/macrophages and cancer cells that recapitulated the enhanced TGF-β production in Siglec-15 expressing THP-1 cells by the cellular interaction with sTn expressing H157 cells. The enhanced TGF-β production required a direct interaction between the two cell lines through sialic acids. Siglec-15 associates with adaptor protein DNAX activation protein of 12 kDa (DAP12) at the binding determinant Lys(274) in the transmembrane domain and transduces a signal to spleen tyrosine kinase (Syk). The enhanced TGF-β secretion was significantly attenuated by Syk inhibitor treatment of THP-1 cells or by substitution of the Siglec-15 Lys(274) to Ala, which disrupts the molecular interaction between Siglec15 and DAP12. These findings indicate that Siglec-15 recognizes the tumoral sTn antigen and transduces a signal for enhanced TGF-β secretion in TAMs and further suggest that Siglec-15 on macrophages may contribute to tumor progression by the TGF-β-mediated modulation of intratumoral microenvironments.

Background: Protectin D1 (PD1) is an anti-inflammatory and proresolving lipid mediator biosynthesized from the omega-3 fatty acid docosahexaenoic acid (DHA). Exogenous PD1 conferred protection against eosinophilic inflammation in animals with experimental asthma, although its endogenous cellular source and functions in human airways are of interest., Objective: We sought to investigate the synthesizing capacity of PD1 in eosinophils from healthy subjects and patients with severe asthma and its direct effects on eosinophil functions., Methods: Human eosinophil-derived metabolites of arachidonic acid and DHA were analyzed with liquid chromatography-tandem mass spectrometry-based lipidomic analysis. The biological activities of PD1 on the function of human eosinophils, including chemotaxis, adhesion molecule expressions, degranulation, superoxide anion generation, or survival, were examined., Results: We identified PD1 as one of the main anti-inflammatory and proresolving molecules synthesized in human eosinophils. PD1, in nanomolar concentrations, suppressed the chemotaxis induced by CCL11/eotaxin-1 or 5-oxo-eicosatetraenoic acid and modulated the expression of the adhesion molecules CD11b and L-selectin, although it had no effects on the degranulation, superoxide anion generation, or survival of the eosinophils. Compared with the cells harvested from healthy subjects, we observed a prominent decrease in the biosynthesis of PD1 by eosinophils from patients with severe asthma, even in presence of DHA., Conclusion: These observations are a first indication that activated human eosinophils represent a major source of PD1, which can act as a self-resolving machinery in eosinophilic inflammation, whereas the production of PD1 is impaired in patients with severe asthma., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Efferocytosis is believed to be a key regulator for lung inflammation in chronic obstructive pulmonary disease. In this study we pharmacologically inhibited efferocytosis with annexin V and attempted to determine its impact on the progression of pulmonary emphysema in mouse. We first demonstrated in vitro and in vivo efferocytosis experiments using annexin V, an inhibitor for phosphatidylserine-mediated efferocytosis. We then inhibited efferocytosis in porcine pancreatic elastase (PPE)-treated mice. PPE-treated mice were instilled annexin V intranasally starting from day 8 until day 20. Mean linear intercept (Lm) was measured, and cell apoptosis was assessed in lung specimen obtained on day 21. Cell profile, apoptosis, and mRNA expression of matrix metalloproteinases (MMPs) and growth factors were evaluated in bronchoalveolar lavage (BAL) cells on day 15. Annexin V attenuated macrophage efferocytosis both in vitro and in vivo. PPE-treated mice had a significant higher Lm, and annexin V further increased that by 32%. More number of macrophages was found in BAL fluid in this group. Interestingly, cell apoptosis was not increased by annexin V treatment both in lung specimens and BAL fluid, but macrophages from mice treated with both PPE and annexin V expressed higher MMP-2 mRNA levels and had a trend for higher MMP-12 mRNA expression. mRNA expression of keratinocyte growth factor tended to be downregulated. We showed that inhibited efferocytosis with annexin V worsened elastase-induced pulmonary emphysema in mice, which was, at least partly, attributed to a lack of phenotypic change in macrophages toward anti-inflammatory one.

Cigarette smoke (CS) induces oxidative stress, which disables macrophage function. In this study, we examined whether Resolvin E1 (RvE1), a pro-resolving mediator known to enhance macrophage functions, attenuates the damage of macrophages by CS extract (CSE) induced oxidative stress. RvE1 blocked p47phox translocation to plasma membrane induced by CSE in a macrophage cell line, RAW264.7 cells, resulting in suppression of superoxide production. Furthermore, pretreatment of RAW264.7 cells with RvE1 restored the phagocytic activity and reduced cell death induced by treatment of CSE. These results suggest that RvE1 plays important roles in preserving macrophage function under CS-induced oxidative stress.

Cinnamaldehyde treatment inhibited the browning of cut lettuce during cold storage. In this study, to clarify the mechanism of inhibitory action of cinnamaldehyde against the browning and to show its microbiological merit, its effect on the browning of cut lettuce was compared to that of mild heat treatment. Both cinnamaldehyde and mild heat treatments inhibited the induction of phenylalanine ammonia-lyase (PAL) activity because of cutting. As a result, the biosynthesis of polyphenols, which are substrates of polyphenol oxidase, was inhibited. This reduction of polyphenol synthesis caused the inhibition of the browning. Cinnamaldehyde treatment repressed the induction of PAL mRNA, while mild heat treatment did not repress its induction. The increase in microbes in cut lettuce treated with cinnamaldehyde was less than that treated with mild heat after 12 days.

With the recent increasing use of nanoparticles, there is concern that they may become an environmental risk factor as airborne particles. However, the impact of these particles on susceptible subjects with predisposing lung disease have not been sufficiently elucidated. In the present study, we investigated the effects of nanoparticles on pulmonary inflammatory and fibrotic changes induced by intratracheal bleomycin (BLM) challenge in mice. Mice were intratracheally administered either vehicle, 14-nm carbon black nanoparticles (CBNPs), BLM or BLM plus CBNP. First, we assessed lung collagen content, lung compliance and fibrotic changes in histopathology on day 21 after instillation. Then, to elucidate how CBNP contributes to the development of BLM-induced fibrosis, we collected bronchoalveolar lavage (BAL) fluid on days 2, 7, 14 and 21 and determined the total and differential cell counts and concentrations of two proinflammatory cytokines (keratinocyte chemoattractant [KC] and interleukin [IL]-6) and two fibrogenic mediators (CC chemokine ligand 2 [CCL2] and transforming growth factor-β(1) [TGF-β(1)]). Expression of nitrotyrosine, an indicator of oxidant injury, was also evaluated on days 7 and 21. CBNP, when combined with BLM, significantly enhanced BLM-induced increase in lung collagen content, decrease in lung compliance, and fibrotic changes in histopathology. CBNP significantly augmented BLM-induced increase in the numbers of inflammatory cells in BAL fluid on days 2 and 7 and levels of KC and IL-6 on day 2. In addition, CBNP administered in combination with BLM significantly elevated the levels of CCL2 on days 2, 7 and 14, and TGF-β(1) on day 14 in BAL fluid as compared with BLM alone. Nitrotyrosine expression was also increased by BLM plus CBNP compared with BLM alone. In contrast, CBNP did not exert any significant effect on these parameters by itself. These results indicate that CBNP can exaggerate BLM-induced inflammatory and fibrotic changes in the lung, suggesting the potential impact of nanoparticles on lung inflammation and fibrosis.

Whereas pneumonia is the most common cause of death and disability worldwide, most cases of pneumonia spontaneously resolve. Mechanisms that promote pneumonia resolution remain to be determined. Resolvin E1 (RvE1) is an endogenous mediator that displays proresolving actions in sterile inflammation. In this study, we developed a new model of aspiration pneumonia to evaluate the effect of RvE1 on acute lung injury caused by acid aspiration and subsequent bacterial challenge. Mice received hydrochloric acid into the left lung followed by the enteric pathogen Escherichia coli. I.v. administration of RvE1 (approximately 0.005 mg/kg) prior to acid injury selectively decreased lung neutrophil accumulation by 55% and enhanced clearance of E. coli. RvE1 significantly decreased lung tissue levels of several proinflammatory chemokines and cytokines, including IL-1beta, IL-6, HMGB-1, MIP-1alpha, MIP-1beta, keratinocyte-derived chemokine, and MCP-1, in a manner independent of the anti-inflammatory mediators IL-10 and lipoxin A4. In addition, animals treated with RvE1 had a marked improvement in survival. These findings in experimental aspiration pneumonia have uncovered protective roles for RvE1 in pathogen-mediated inflammation that are both anti-inflammatory for neutrophils and protective for host defense, suggesting that RvE1 represents the first candidate for a novel therapeutic strategy for acute lung injury and pneumonia that harnesses natural resolution mechanisms.

Oxidative stress has been implicated as a cause of various diseases such as anaemia. We found that the SOD1 [Cu,Zn-SOD (superoxide dismutase)] gene deficiency causes anaemia, the production of autoantibodies against RBCs (red blood cells) and renal damage. In the present study, to further understand the role of oxidative stress in the autoimmune response triggered by SOD1 deficiency, we generated mice that had the hSOD1 (human SOD1) transgene under regulation of the GATA-1 promoter, and bred the transgene onto the SOD1(-/-) background (SOD1(-/-);hSOD1(tg/+)). The lifespan of RBCs, levels of intracellular reactive oxygen species, and RBC content in SOD1(-/-);hSOD1(tg/+) mice, were approximately equivalent to those of SOD1(+/+) mice. The production of antibodies against lipid peroxidation products, 4-hydroxy-2-nonenal and acrolein, as well as autoantibodies against RBCs and carbonic anhydrase II were elevated in the SOD1(-/-) mice, but were suppressed in the SOD1(-/-);hSOD1(tg/+) mice. Renal function, as judged by blood urea nitrogen, was improved in the transgenic mice. These results rule out the involvement of a defective immune system in the autoimmune response of SOD1-deficient mice, because SOD1(-/-);hSOD1(tg/+) mice carry the hSOD1 protein only in RBCs. Metabolomic analysis indicated a shift in glucose metabolism to the pentose phosphate pathway and a decrease in the energy charge potential of RBCs in SOD1-deficient mice. We conclude that the increase in reactive oxygen species due to SOD1 deficiency accelerates RBC destruction by affecting carbon metabolism and increasing oxidative modification of lipids and proteins. The resulting oxidation products are antigenic and, consequently, trigger autoantibody production, leading to autoimmune responses.

High-mobility group box 1 (HMGB1) is a nuclear protein that has been found to be a critical mediator of lethality in endotoxemia and sepsis. During the systemic inflammatory response, circulating levels of HMGB1 are increased, but in a delayed fashion compared with early inflammatory mediators. To counteract the inflammatory response of endotoxemia, a secondary anti-inflammatory response ensues in an attempt to prevent inflammation-induced tissue injury. One such cytoprotective gene that is induced during endotoxemia is heme oxygenase (HO)-1. HO-1, and its products of heme metabolism, possess anti-inflammatory and antioxidant properties to counter the damaging effects of endotoxemia. In the present study, we wanted to determine whether tissue and circulating levels of HMGB1 are increased further in the absence of HO-1 during endotoxemia, and whether this increase may contribute to the pathobiology of endotoxemia. Lung inflammation, HMGB1 protein levels, and expression of HMGB1 in inflammatory cells were increased in HO-1(-/-) mice compared with HO-1+/+ mice. After the administration of LPS, tissue levels of HMGB1 were not increased further in HO-1(-/-) mice; however, circulating levels of HMGB1 were higher when compared with HO-1+/+ mice. HO-1(-/-) mice treated with a carbon monoxide-releasing molecule or biliverdin showed a reduction in plasma HMGB1, which was associated with a marked improvement in survival. HO-1(-/-) mice given HMGB1-neutralizing antibody showed improvement in survival compared with control antibody. These data suggest that exaggerated circulating levels of HMGB1 contribute to endotoxin-induced mortality in the absence of HO-1.

Nitric oxide synthase (NOS)2, an inducible enzyme that produces NO during inflammation, is transcriptionally regulated. Our goal was to determine whether high mobility group (HMG)A1 contributes to human (h)NOS2 gene regulation. Using a small molecule inhibitor of HMGA1 binding to DNA, or a dominant-negative form of HMGA1, we blunted the induction of hNOS2 by pro-inflammatory stimuli. Binding of HMGA1 in the region -3506 to -3375 of the hNOS2 promoter, a region not previously known to be involved in hNOS2 regulation, contributed to the induction of hNOS2 promoter in conjunction with upstream enhancer regions. We demonstrate a previously unknown role for HMGA1 in the regulation of hNOS2.

Objective: Acrolein, a known toxin in tobacco smoke, might be involved in atherogenesis. This study examined the effect of acrolein on expression of cyclooxygenase-2 (COX-2) and prostaglandin (PG) production in endothelial cells., Methods and Results: Cyclooxygenase (COX)-2 induction by acrolein and signal pathways were measured using Western blots, Northern blots, immunofluorescence, ELISA, gene silencing, and promoter assay. Colocalization of COX2 and acrolein-adduct was determined by immunohistochemistry. Here we report that the levels of COX-2 mRNA and protein are increased in human umbilical vein endothelial cells (HUVECs) after acrolein exposure. COX-2 was found to colocalize with acrolein-lysine adducts in human atherosclerotic lesions. Inhibition of p38 MAPK activity abolished the induction of COX-2 protein and PGE2 accumulation by acrolein, while suppression of extracellular signal-regulated kinase (ERK) and JNK activity had no effect on the induction of COX-2 expression in experiments using inhibitors and siRNA. Furthermore, rottlerin, an inhibitor of protein kinase Cdelta (PKCdelta), abrogated the upregulation of COX-2 at both protein and mRNA levels., Conclusion: These results provide that acrolein may play a role in progression of atherosclerosis and new information on the signaling pathways involved in COX-2 upregulation in response to acrolein and provide evidence that PKCdelta and p38 MAPK are required for transcriptional activation of COX-2.

The first aim of the present study was to investigate the feasibility of using fewer beam angles to improve delivery efficiency for the treatment of oropharyngeal cancer (OPC) with inverse-planned intensity-modulated radiation therapy (IP-IMRT). A secondary aim was to evaluate whether the simplified IP-IMRT plans could reduce the indirect radiation dose. The treatment plans for 5 consecutive OPC patients previously treated with a forward-planned IMRT (FP-IMRT) technique were selected as benchmarks for this study. The initial treatment goal for these patients was to deliver 70 Gy to > or = 95% of the planning gross tumor volume (PTV-70) and 59.4 Gy to > or = 95% of the planning clinical tumor volume (PTV-59.4) simultaneously. Each case was re-planned using IP-IMRT with multiple beam-angle arrangements, including four complex IP-IMRT plans using 7 or more beam angles, and one simple IMRT plan using 5 beam angles. The complex IP-IMRT plans and simple IP-IMRT plans were compared to each other and to the FPIMRT plans by analyzing the dose coverage of the target volumes, the plan homogeneity, the dose-volume histograms of critical structures, and the treatment delivery parameters including delivery time and the total number of monitor units (MUs). When comparing the plans, we found no significant difference between the complex IP-IMRT, simple IP-IMRT, and FP-IMRT plans for tumor target coverage (PTV-70: p = 0.56; PTV-59.4: p = 0.20). The plan homogeneity, measured by the mean percentage isodose, did not significantly differ between the IP-IMRT and FP-IMRT plans (p = 0.08), although we observed a trend toward greater inhomogeneity of dose in the simple IP-IMRT plans. All IP-IMRT plans either met or exceeded the quality of the FP-IMRT plans in terms of dose to adjacent critical structures, including the parotids, spinal cord, and brainstem. As compared with the complex IP-IMRT plans, the simple IP-IMRT plans significantly reduced the mean treatment time (maximum probability for four pairwise comparisons: p = 0.0003). In conclusion, our study demonstrates that, as compared with complex IP-IMRT, simple IP-IMRT can significantly improve treatment delivery efficiency while maintaining similar target coverage and sparing of critical structures. However, the improved efficiency does not significantly reduce the total number of MUs nor the indirect radiation dose.

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.

Although more than 100 mutations have been identified in the copper/zinc superoxide dismutase (Cu/Zn-SOD) in familial amyotrophic lateral sclerosis (FALS), the mechanism responsible for FALS remains unclear. The finding of the present study shows that FALS-causing mutant Cu/Zn-SOD proteins (FALS mutant SODs), but not wild-type SOD, are barely detected by three monoclonal antibodies (mAbs) in Western blot analyses. The enzyme-linked immunosorbent assay for denatured FALS mutant SODs by dithiothreitol, SDS, or heat treatment also showed a lowered immunoreactivity against the mAbs compared with wild-type SOD. Because all the epitopes of these mAbs are mapped within the Greek key loop (residues 102-115 in human Cu/Zn-SOD), these data suggest that different conformational changes occur in the loop between wild-type and FALS mutant SODs during the unfolding process. Circular dichroism measurements revealed that the FALS mutant SODs are sensitive to denaturation by dithiothreitol, SDS, or heat treatment, but these results do not completely explain the different recognition by the mAbs between wild-type and FALS mutant SODs under the denatured conditions. The study on the conformational changes in local areas monitoring with mAbs may provide a new insight into the etiology of FALS.