Selenoprotein thioredoxin reductase mediated menadione reduction: catalytic properties & inhibition effects / 生物工程学报

Thioredoxin reductase (TrxR) is one class of the most important antioxidant selenoproteins and is involved in regulating tumor genesis and progression. It has been reported that naphthoquinones can target and inhibit TrxR1 activity therefore produce reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the naphthoquinone compounds to become potential antitumor chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated naphthoquinone reduction and inhibition of TrxR1 by naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of menadione on TrxR1 activity, and eventually identified the interaction between menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated menadione reduction, though the Sec⁴⁹⁸ is capable to catalyze the menadione reduction, indicating that TrxR1-mediated menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys⁴⁹⁸ is mainly responsible for menadione catalysis in comparison to Cys⁴⁹⁷, while the N-terminal Cys⁶⁴ is slightly stronger than Cys⁵⁹ regarding the menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of menadione, suggesting that menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by menadione. Hereby it will be useful for the research and development of naphthoquinone anticancer drugs targeting TrxR1.

Red fluorescence of oral bacteria is affected by blood in the growth medium / 대한구강보건학회지

OBJECTIVES: Dental plaque emits red fluorescence under a visible blue light near the ultra-violet end of the light spectrum. The fluorescence characteristics of each microorganism have been reported in several studies. The aim of this study was to evaluate changes in red fluorescence of oral microorganisms that is affected by blood in the culture media. METHODS: The gram-positive Actinomyces naeslundii (AN, KCTC 5525) and Lactobacillus casei (LC, KCTC 3109) and gram negative Prevotella intermedia (PI, KCTC 3692) that are known to emit red fluorescence were used in this study. Each bacterium was activated in broth and cultivated in different agar media at 37℃ for 7 days. Tryptic soy agar with hemin and vitamin K3 (TSA), TSA with sheep blood (TSAB), basal medium mucin (BMM) medium, and BMM with sheep blood (BMMB) were used in this study. Fluorescence due to bacterial growth was observed under 405-nm wavelength blue light using the quantitative light-induced fluorescence-digital (QLF-D) device. The red, green, and blue fluorescence values of colonies were obtained using image-analysis software and the red to green ratio (R/G value) and red to total RGB ratio (R/RGB value) were calculated for quantitative comparison. RESULTS: The QLF-D images of the AN, LC, and PI colonies showed red fluorescence in all media, but the fluorescence of all bacteria was reduced in TSA and BMM media, compared with in TSAB and BMMB media. Both the R/G and the R/RGB values of all bacteria were significantly reduced in growth media without blood (P<0.001). CONCLUSIONS: Based on this in vitro study, it can be concluded that red fluorescence of oral bacteria can be affected by growth components, especially blood. Blood-containing medium could be a significant factor influencing red fluorescence of oral bacteria. It can be further hypothesized that bleeding in the oral cavity can increase the red fluorescence of dental plaque.

Red fluorescence of oral bacteria interacting with Porphyromonas gingivalis / 대한구강보건학회지

OBJECTIVES: Dental plaque is composed of 700 bacterial species. It is known that some oral microorganisms produce porphyrin, and thus, they emit red fluorescence when illuminated with blue light at a specific wavelength of <410 nm. Porphyromonas gingivalis belongs to the genus Porphyromonas, which is characterized by the production of porphyrin. The aim of this study was to evaluate red fluorescence emission of some oral microorganisms interacting with P. gingivalis. METHODS: Five bacterial strains (P. gingivalis, Streptococcus mutans, Lactobacillus casei, Actinomyces naeslundii, and Fusobacterium nucleatum) were used for this study. Tryptic soy agar medium supplemented with hemin, vitamin K3, and sheep blood was used as a growth medium. The fluorescence emission of bacterial colonies was evaluated under 405 nm-wavelength blue light using a Quantitative Light-induced Fluorescence Digital (QLF-D) camera system. Each bacterium was cultured alone and co-cultured in close proximity with P. gingivalis. The red/green (R/G) ratio of fluorescence image was calculated and the differences of R/G ratio according to each growth condition were compared using the Mann-Whitney test (P<0.05). RESULTS: Single cultured S. mutans, L. casei and A. naeslundii colonies emitted red fluorescence (R/G ratio=2.15±0.06, 4.31±0.17, 5.52±1.29, respectively). Fusobacterium nucleatum colonies emitted green fluorescence (R/G ratio=1.36±0.06). The R/G ratios of A. naeslundii and F. nucleatum were increased when P. gingivalis was co-cultured with each bacterium (P<0.05). In contrast, the R/G ratios of S. mutans and L. casei were decreased when P. gingivalis was co-cultured with each bacterium (P=0.002, 0.003). CONCLUSIONS: This study confirmed that P. gingivalis could affect the red fluorescence of other oral bacteria under 405 nm-wavelength blue light. Our findings concluded that P. gingivalis has an important role for red fluorescence emission of dental biofilm.

A Nudix Hydrolase Protein, Ysa1, Regulates Oxidative Stress Response and Antifungal Drug Susceptibility in Cryptococcus neoformans

A nucleoside diphosphate-linked moiety X (Nudix) hydrolase-like gene, YSA1, has been identified as one of the gromwell plant extract-responsive genes in Cryptococcus neoformans. Ysa1 is known to control intracellular concentrations of ADP-ribose or O-acetyl-ADP-ribose, and has diverse biological functions, including the response to oxidative stress in the ascomycete yeast, Saccharomyces cerevisiae. In this study, we characterized the role of YSA1 in the stress response and adaptation of the basidiomycete yeast, C. neoformans. We constructed three independent deletion mutants for YSA1, and analyzed their mutant phenotypes. We found that ysa1 mutants did not show increased sensitivity to reactive oxygen species-producing oxidative damage agents, such as hydrogen peroxide and menadione, but exhibited increased sensitivity to diamide, which is a thiol-specific oxidant. Ysa1 was dispensable for the response to most environmental stresses, such as genotoxic, osmotic, and endoplasmic reticulum stress. In conclusion, modulation of YSA1 may regulate the cellular response and adaptation of C. neoformans to certain oxidative stresses and contribute to the evolution of antifungal drug resistance.

A case of Small Colony Variants (SCVs) of Staphylococcus aureus from Sputum of a Patient with Chronic Renal Failure / 대한임상미생물학회지

Recently, small colony variants (SCVs) of Staphylococcus aureus causing fatal infections are increasing, but rarely reported in Korea. S. aureus, SCVs are slow growing subpopulation that cause persistent and relapsing infections. S. aureus, SCVs are frequently auxotrophic for hemin, menadione, and CO2, and are often disrupted in their electron transport activity. With S. aureus, SCVs virulence is altered by a decrease in -toxin production and susceptibility to various antibiotics, allowing their intracellular survival. We isolated S. aureus, SCVs from the sputum of a 67 year old male with pneumonia, chronic renal failure with hemodialysis and preventive antibiotic therapy. Because S. aureus, SCVs are easily missed or misdiagnosed as normal flora in routine culture due to their atypical growth behavior and biochemical reaction, the correct identification is very important, especially when no bacteria or unusual bacteria are found in patients with persistent or relapsing infections with long term antibiotic therapy.

A Case of Methicillin-Resistant Staphylococcus aureus Small Colony Variants(SCVs) Isolated from Urine of a Patient with Persistent and Relapsing Bladder Stone / 대한임상미생물학회지

Methicillin-resistant Staphylococcus aureus colony variants (SCVs) are frequently auxotrophic for hemin, menadione, thiamine, and CO2 involved in biosynthesis of the electron transport chain element. This phenotype grows slowly, and forms very small, nonhemolytic colonies in routine culture, so it may be led to the misidentification of this organism. We isolated an organism with catalase-positive, gram-positive cocci in cluster from the urine of a 55-years-old woman with persistent and relapsing bladder stone, who had undergone the antibiotic treatment with cefotaxime, ceftizoxime, amikacin, and/or micronomicin, intermittently for three years. The possibility of SCVs should have been ruled out because this organism didn't grow on Mueller-Hinton agar (MHA) for the susceptibility test. It formed small colonies on blood agar plate overnight, and grew only on MHA with supplement of hemin, or with 5% CO2. This organism was coagulase-positive, DNase-positive, manitol-salt positive, and identified as S. aureus with VITEK GPI card. The susceptibility test could be performed after adding hemin(1mg/mL) into bacterial suspension and showed susceptibility against vancomycin, teicoplanin, and rifampin. Because these phenotypes can be misidentifide as other non-pathogenic organisms due to their atypical characteristics, we should consider SCVs in case of small, nonhemolytic colonies with catalase-positive, gram-positive cocci in cluster, showing no growth on MHA. In addition, infections caused by SCVs are recently recognized in relation to persistent and relapsing infection, so they could be isolated from the patients with long-term antibiotic therapy.

Induction of Thioredoxin by Oxidative Stress and Overexpression of Thioredoxin in Lung Cancer Tissue / 결핵

BACKGROUND: Reactive oxygen species are involved in multi -stage process of carcinogenesis. The most of cancer cell lines and cancer cells in tumor tissue produce reactive oxygen species and on the other hand, the activities of catalase, Mn - and CuZn-superoxide dismutase in tumor cells are usually low. These persistent oxidative stress in tumor tissue facilitates tumor invasion and metastasis. 12- kDa thioredoxin, which regulate the intracellular redox potential with glutathione and glutaredoxin is involved in cell activation, proliferation, differentiation and re dox- mediated apoptosis. It is also purified as 14 -kDa and 10- kDa eosinophilic cytotoxic enhancing factor(ECEF) from human histiocytic cell(U937) and 10 -kDa ECEF has more than 20 times eosinophilic stimulation activity than 14 - kDa ECEF. It has been reported that adult T-cell leukemia, squamous cell carcinoma of uterine cervix, and hepatocellular carcinoma show increased amounts of human thioredoxin and thioredoxin mRNA is increased in lung cancer. In this study, we investigated the expression of conventional antioxidant enzymes such as catalase, CuZn-SOD, and glutathione peroxidase and thioredoxin in lung cancer tissue compared to adjacent normal lung tissue and the induction of thioredoxin in macrophage cells after treatment of oxidative stress and endotoxin . METHODS: We measured the amount of conventional antioxidant enzymes such as catalase, CuZn-SOD, and glutathione peroxidase and thioredoxin in lung cancer tissue compared to adjacent normal lung tissue by immunoblot analysis and the induction of thioredo xin in mouse monocyte - macrophage cells(RAW 264.7) by treatment of 5 microM menadione and 1 microgram/ml endotoxin. RESULTS: On immunoblot analysis, the expression of 12 -kDa thioredoxin was increased in lung cancer tissue compared to paired normal lung tissue. but th e expression of catalase and CuZn-SOD were decreased in lung cancer tissue compared to paired normal tissue and the expression of glutathione peroxidase in lung cancer was variable. The expression of truncated thioredoxin was also increased in lung cancer. When mouse monocyte - macrophage cells were treated with 5 microM menadione and 1 microG/ml endotoxin, the expression of thioredoxin was peaked at 12 hrs and sustained to 48 hrs. CONCLUSION: In contrast with other conventional antioxidants, the expression of 12-kDa and truncated thioredoxin in lung cancer were increased and it is closely associated with persistent oxidative stress in tumor microenvironment. Considering especially the biological functions of truncated thioredoxin, the increased amount of truncated thioredoxin has significant role in tumor growth through cell proliferation.

Altered expression of Peroxiredoxin and Thioredoxin in septic animal model / 결핵

BACKGROUND: In sepsis, excessive generation of reactive oxygen species plays key roles in the pathogenesis of acute lung injury. The serum antioxidants such as catalase and MnSOD are elevated in sepsis and considered as predictors of acute respiratory distress syndrome(ARDS) and prognostic factors of sepsis. Peroxiredoxin(Prx) has recently been known as an unique and major intracellular antioxidant. In this study, we evaluated the expression of Prx I and Prx II in mouse monocyte-macrophage cells(RAW267.7) after treatment of oxidative stress and endotoxin and measured the amount of Prx I, Prx II and thioredoxin(Trx) in peritoneal and bronchoalveolar lavage fluid of septic animal model. METHODS: Using immunoblot analysis with specific antibodies against Prx I, Prx II and Trx, we evaluated the distribution of Prx I and Prx II in human neutrophil, alveolar macrophage and red blood cell. We evaluated the expression of Prx I and Prx II in mouse monocyte-macrophage cells after treatment of 5 micro M menadione and 1 micro gram/ml lipopolysaccharide(LPS) and measured the amount of Prx I, Prx II and Trx in peritoneal lavage fluid of intraperitoneal septic animals(septic animal model induced with intraperitoneal 6 micro gram/kg LPS injection) and those in bronchoalveolar lavage fluid of intraperitoneal septic animals and intravenous septic animals(septic animal model induced with intravenous 5 micro gram/kg LPS injection) and compared with the severity of lung inflammation. RESULTS: The distribution of Prx I and Prx II were so different among human neutrophil, alveolar macrophage and red blood cell. The expression of Prx I in mouse monocyte-macrophage cells was increased after treatment of 5 micro M menadione and 1 micro gram/ml lipopolysaccharide but that of Prx II was not increased. The amount of Prx I, Prx II and Trx are increased in peritoneal lavage fluid of intraperitoneal septic animals but were not increased in bronchoalveolar lavage fluid of intraperitoneal and intravenous septic animals regardless of the severity of lung inflammation. CONCLUSION: As intracellular antioxidant, the expression of Prx I is increased in mouse monocyte-macrophage cells after treatment of oxidative stress and endotoxin. The amount of Prx I, Prx II and Trx are increased in local inflammatory site but not increased in injured lung of septic animal model.