• IMMUNE NETWORK
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• v.23 no.3
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• pp.25.1-25.17
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• 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces excessive pro-inflammatory cytokine release and cell death, leading to organ damage and mortality. High-mobility group box 1 (HMGB1) is one of the damage-associated molecular patterns that can be secreted by pro-inflammatory stimuli, including viral infections, and its excessive secretion levels are related to a variety of inflammatory diseases. Here, the aim of the study was to show that SARS-CoV-2 infection induced HMGB1 secretion via active and passive release. Active HMGB1 secretion was mediated by post-translational modifications, such as acetylation, phosphorylation, and oxidation in HEK293E/ACE2-C-GFP and Calu-3 cells during SARS-CoV-2 infection. Passive release of HMGB1 has been linked to various types of cell death; however, we demonstrated for the first time that PANoptosis, which integrates other cell death pathways, including pyroptosis, apoptosis, and necroptosis, is related to passive HMGB1 release during SARS-CoV-2 infection. In addition, cytoplasmic translocation and extracellular secretion or release of HMGB1 were confirmed via immunohistochemistry and immunofluorescence in the lung tissues of humans and angiotensin-converting enzyme 2-overexpressing mice infected with SARS-CoV-2.

• Experimental and Molecular Medicine
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• v.54
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• pp.788-800
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• 2022

Cell-to-cell propagation of α-synuclein is thought to be the underlying mechanism of Parkinson's disease progression. Recent evidence suggests that inflammation plays an important role in the propagation of protein aggregates. However, the mechanism by which inflammation regulates the propagation of aggregates remains unknown. Here, using in vitro cultures, we found that soluble factors secreted from activated microglia promote cell-to-cell propagation of α-synuclein and further showed that among these soluble factors, TNF-α had the most robust stimulatory activity. Treatment of neurons with TNF-α triggered cellular senescence, as shown by transcriptomic analyses demonstrating induction of senescence-associated genes and immunoanalysis of senescence phenotype marker proteins. Interestingly, secretion of α-synuclein was increased in senescent neurons, reflecting acquisition of a senescence-associated secretory phenotype (SASP). Using vacuolin-1, an inhibitor of lysosomal exocytosis, and RNAi against rab27a, we demonstrated that the SASP was mediated by lysosomal exocytosis. Correlative light and electron microscopy and immunoelectron microscopy confirmed that propagating α-synuclein aggregates were present in electron-dense lysosome-like compartments. TNF-α promoted the SASP through stimulation of lysosomal exocytosis, thereby increasing the secretion of α-synuclein. Collectively, these results suggest that TNF-α is the major inflammatory factor that drives cell-to-cell propagation of α-synuclein by promoting the SASP and subsequent secretion of α-synuclein.

• Tuberculosis and Respiratory Diseases
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• v.60 no.4
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• pp.426-436
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• 2006

Background : Particulate matters (PM) when inhaled is known to induce pulmonary diseases including asthma and chronic bronchitis when inhaled. Despite the epidemiological proofevidence, the pathogenesis of PM-related pulmonary diseases is unclearremain poorly understood. Methods : Primary alveolar macrophages were harvested from the SPF and inflammatory rats by bronchioalveolar lavage (BAL). The cultured primary alveolar macrophages were treated with the medium only, PM only ($5{\sim}40{\mu}g/cm^2$), LPS (5ng/ml) only, and PM with LPS for 24 and 48 hours. The level of secreted nitric oxide (NO) was assayed from the cultured medium by using the Griess reaction. The cultured cells were utilized for the western blotting against the inducible nitric oxide synthase (iNOS) proteins. Immunocyto- chemical staining against the iNOS and NT-proteins were performed in cells that cultured in the $Lab-Tek^{(R)}$ chamber slide after treatments. Results : The PM that utilizein this experiments induced NO formation with iNOS expression in the cultured SPF and inflammatory rats alveolar macrophages, by itself. When the cells were co-treated with PM and LPS, there was a statistically significant synergistic effect on NO formation and iNOS expression over the LPS effect. The cells from the sham control showed minimal immunoreactivity for the NT-proteins. Significantly higher quantities of NT-proteins were detected in the PM and PM with LPS co-treated cells than from the sham control. Conclusion : Increased iNOS expression and NO formation with increased NT-proteins formation might be involved in the pathogenesis of PM-induced lung injury.

• IMMUNE NETWORK
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• v.1 no.2
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• pp.116-125
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• 2001

Background: Nitric oxide (NO) production has been described as a double-edged sword eliciting both pro- and anti-inflammatory effects in different immune reactions. This work was undertaken to investigate the immunoregulatory role of NO in experimental allergic encephalomyelitis (EAE) and experimental allergic uveitis (EAU). Method: We examined whether molsidomine (MSDM), a NO donor, administration to the myelin basic protein (MBP)- or interphotoreceptor retinoid binding protein (IRBP)-immunized rats could suppress EAE development by shifting toward the Th2 cytokine response. In the EAE experiments, the rats were treated orally with MSDM (10 mg/kg/day) at the early stage (-1~4 days) or throughout the experimental period (-1~15 days). Results: This resulted in significant amelioration of the disease and mild clinical symptoms, while MBP-immunization without MSDM administration showed severe EAE development. A marked reduction in inflammation was also observed in the spinal cord, indicating the crucial role of NO in the pathogenesis of EAE in in vivo. In the EAU experiments, a 24 h pre-treatment with MSDM prior to IRBP immunization resulted in significant inhibition of the disease. Furthermore, MSDM administration for 2 1 days completely reduced the incidence and severity of EAU. To investigate whether MSDM could modulate cytokine switching from Th 1 to Th2, culture supernatants of MBP- or IRBP-stimulated inguinal lymphocytes were analyzed. MSDM treatment enhanced IL-10 secretion but decreased IFN-${\gamma}$. IL-4 was undetectable in all groups. In contrast, the MBP-or IRBP-immunized rats without MSDM secreted high concentrations of IFN-${\gamma}$, but low concentrations of IL-10. Conclusion: In conclusion, NO administation suppresses EAE and EAU by modulating the Th1/Th2 balance during inflammatory immune responses. This work further suggests that NO may be useful in the therapeutic control of autoimmune disease.

• Journal of Korean Neurosurgical Society
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• v.59 no.2
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• pp.106-116
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• 2016

Objective : Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process. Methods : U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot. Results : Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001). Conclusion : Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.

• BMB Reports
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• v.40 no.4
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• pp.459-466
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• 2007

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7$\cdot}$Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.68-72
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• 2010

The ${\beta}$-1,4-glucanase gene of Bacillus licheniformis B1 was expressed in Esherichia coli BL21, and a protein with a mass of 50 kDa that was soluble was overproduced. A protein with a mass of 37 kDa was secreted from B. licheniformis. It seems that the ${\beta}$-1,4-glucanase produced in E. coli contained the leader peptide and unprocessed carboxy-terminal region, but its processing occurred in the carboxyterminal in Bacillus. The optimal temperature of ${\beta}$-1,4-glucanase was $40^{\circ}C$. The enzyme still had 76% maximal activity at $60^{\circ}C$. The optimal pH of the enzyme was 7. The enzyme retained considerable activities over the weak-acidic, neutral, and weak-basic pH range. Acidic fungal cellulases are used in food, detergent, pulp, paper, textile industries. However, studies about neutral and alkaline cellulase are not enough. The cellulase developed in this study may be useful for industrial applications in the fields of biofuel development.

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.46-50
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• 2013

Some rhizobacteria producing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase can make plant to continue growth under the stress conditions through lowering the level of phytohormone, ethylene which inhibits the plant growth and accelerates plant aging. In this study, some rhizobacteria producing ACC deaminase have been isolated from the rhizosphere of plants grown at sand beaches, and identified as Escherichia hermannii m-2, Enterobacter asburiae m-4, Pseudomonas thivervalensis BD2-26 and Pseudomonas brassicacearum subsp. neoaurantiaca BD3-35 through sequencing of 16S rRNA genes. Strain BD3-35 showed the highest activity of ACC deaminase among the isolates, 20.26 ${\alpha}$-ketobutyrate ${\mu}M/mg$ protein/h. Strains BD3-35 and BD2-26 secreted a phytohormone cytokinin, and strains m-4 and m-2 could produce auxin and abscisic acid, respectively. When these bacteria were applied to the 7-day old tomato plant under drought stress for 7 days, strains BD3-35, m-2, and m-4 increased the length of tomato root by 14, 15, and 35%, respectively, and strains m-2, BD2-26 and BD3-35 increased the dry weight of tomato plant by 22, 33, and 68%, respectively compared to the uninoculated control tomatoes. Therefore, these rhizobacteria may be utilized as a microbial fertilizer for the plants under drought stress.

• BMB Reports
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• v.38 no.1
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• pp.77-81
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• 2005

The monocyte chemotactic protein-3 (MCP3), on chromosome 17q11.2-q12, is a secreted chemokine, which attracts macrophages during inflammation and metastasis. In an effort to discover additional polymorphism(s) in genes whose variant(s) have been implicated in asthma, we scrutinized the genetic polymorphisms in MCP3 to evaluate it as a potential candidate gene for asthma host genetic study. By direct DNA sequencing in twenty-four individuals, we identified four sequence variants within the 3 kb full genome including 1,000bp promoter region of MCP3; one in promoter region (-420T>C), three in intron (+136C>G, +563C>T, +984G>A) respectively. The frequencies of those four SNPs were 0.020 (-420T>C), 0.038 (+136C>G), 0.080 (+563C>T), 0.035 (+984G>A), respectively, in Korean population (n = 598). Haplotypes, their frequencies and linkage disequilibrium coefficients (|D'|) between SNP pairs were estimated. The associations with the risk of asthma, skin-test reactivity and total serum IgE levels were analyzed. Using statistical analyses for association of MCP3 polymorphisms with asthma development and asthma-related phenotypes, no significant signals were detected. In conclusion, we identified four genetic polymorphisms in the important MCP3 gene, but no significant associations of MCP3 variants with asthma phenotypes were detected. MCP3 variation/haplotype information identified in this study will provide valuable information for future association studies of other allergic diseases.

• Biomolecules & Therapeutics
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• v.28 no.3
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• pp.259-266
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• 2020

The present research work primarily investigated whether spinosin has the potential of improving the pathogenesis of Alzheimer's disease (AD) driven by β-amyloid (Aβ) overproduction through impacting the procession of amyloid precursor protein (APP). Wild type mouse Neuro-2a cells (N2a/WT) and N2a stably expressing human APP695 (N2a/APP695) cells were treated with spinosin for 24 h. The levels of APP protein and secreted enzymes closely related to APP procession were examined by western blot analysis. Oxidative stress related proteins, such as nuclear factor-erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were detected by immunofluorescence assay and western blot analysis, respectively. The intracellular reactive oxygen species (ROS) level was analyzed by flow cytometry, the levels of Aβ_1-42 were determined by ELISA kit, and Thioflavin T (ThT) assay was used to detect the effect of spinosin on Aβ_1-42 aggregation. The results showed that ROS induced the expression of ADAM10 and reduced the expression of BACE1, while spinosin inhibited ROS production by activating Nrf2 and up-regulating the expression of HO-1. Additionally, spinosin reduced Aβ_1-42 production by impacting the procession of APP. In addition, spinosin inhibited the aggregation of Aβ_1-42. In conclusion, spinosin reduced Aβ_1-42 production by activating the Nrf2/HO-1 pathway in N2a/WT and N2a/APP695 cells. Therefore, spinosin is expected to be a promising treatment of AD.