Production of a Recombinant Laccase from Pichia pastoris and Biodegradation of Chlorpyrifos in a Laccase/Vanillin System

( Hui fang Xie ),( Qi Li ),( Min Min Wang ),( Lin Guo Zhao ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.6

The recombinant strain P. pastoris GS115-lccC was used to produce laccase with high activity. Factors influencing laccase expression, such as pH, methanol concentration, copper concentration, peptone concentration, shaker rotate speed, and medium volume were investigated. Under the optimal conditions, laccase activity reached 12,344 U/L on day 15. The recombinant enzyme was purified by precipitating and dialyzing to electrophoretic homogeneity, and was estimated to have a molecular mass of about 58 kDa. When guaiacol was the substrate, the laccase showed the highest activity at pH 5.0 and was stable when the pH was 4.5~6.0. The optimal temperature for the laccase to oxidize guaiacol was 60℃, but it was not stable at high temperature. The enzyme could remain stable at 30℃ for 5 days. The recombinant laccase was used to degrade chlorpyrifos in several laccase/mediator systems. Among three synthetic mediators (ABTS, HBT, VA) and three natural mediators (vanillin, 2,6-DMP, and guaiacol), vanillin showed the most enhancement on degradation of chlorpyrifos. Both laccase and vanillin were responsible for the degradation of chlorpyrifos. A higher dosage of vanillin may promote a higher level of degradation of chlorpyrifos, and the 2-step addition of vanillin led to 98% chlorpyrifos degradation. The degradation of chlorpyrifos was faster in the L/V system (kobs = 0.151) than that in the buffer solution (kobs = 0.028).

Development of a Molecular Marker for Fruiting Body Pattern in Auricularia auricula-judae

( Fang-jie Yao ),( Li-xin Lu ),( Peng Wang ),( Ming Fang ),( You-min Zhang ),( Ying Chen ),( Wei-tong Zhang ),( Xiang-hui Kong ),( Jia Lu ),( Yoichi Honda ) 한국균학회 2018 Mycobiology Vol.46 No.1

The fruiting body pattern is an important agronomic trait of the edible fungus Auricularia auricula-judae, and an important breeding target. There are two types of fruiting body pattern: the cluster type and the chrysanthemum type. We identified the fruiting body pattern of 26 test strains, and then constructed two different near-isogenic pools. Then, we developed sequence characterized amplified region (SCAR) molecular markers associated with the fruiting body pattern based on sequence-related amplified polymorphism (SRAP) markers. Ten different bands (189-522 bp) were amplified using 153 pairs of SRAP primers. The SCAR marker “SCL-18” consisted of a single 522-bp band amplified from the cluster-type strains, but not the chrysanthemum strains. This SCAR marker was closely associated with the cluster- type fruiting body trait of A. auricula-judae. These results lay the foundation for further research to locate and clone genes controlling the fruiting body pattern of A. auricula-judae.

Involvement of Orai1 in tunicamycin-induced endothelial dysfunction

Hui Yang,Yumei Xue,Sujuan Kuang,Mengzhen Zhang,Jinghui Chen,Lin Liu,Zhixin Shan,Qiuxiong Lin,Xiaohong Li,Min Yang,Hui Zhou,Fang Rao,Chunyu Deng 대한약리학회 2019 The Korean Journal of Physiology & Pharmacology Vol.23 No.2

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca2+ homeostasis. The store-operated calcium (SOC) channel is the primary Ca2+ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca2+ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca2+ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.

Characterization of a Blend-Biosurfactant of Glycolipid and Lipopeptide Produced by Bacillus subtilis TU2 Isolated from Underground Oil-Extraction Wastewater

( Fang Yu Cheng ),( Cheng Tang ),( Huan Yang ),( Hui Min Yu ),( Yu Chen ),( Zhong Yao Shen ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.3

Biosurfactants have versatile properties and potential industrial applications. A new producer, B. subtilis TU2, was isolated from the underground oil-extraction wastewater of Shengli Oilfield, China. Preliminary flask culture showed that the titer of biosurfactant obtained from the broth of TU2 was ~1.5 g/l at 48 h (718 mg/l after purification), with a reduced surface tension of 32.5 mN/m. The critical micelle concentration was measured as 50 mg/l and the surface tension maintained stability in solution with 50 g/l NaCl and 16 g/l CaCl2 after 5 days of incubation at 70oC. FT-IR spectra exhibited the structure information of both glycolipid and lipopeptide. MALDI-TOF-MS analyses confirmed that the biosurfactant produced by B. subtilis TU2 was a blend of glycolipid and lipopeptide, including rhamnolipid, surfactin, and fengycin. The blended biosurfactant showed 86% of oil-washing efficiency and fine emulsification activity on crude oil, suggesting its potential application in enhanced oil recovery.

MiR-542-5p Inhibits Hyperglycemia and Hyperlipoidemia by Targeting FOXO1 in the Liver

Fang Tian,Hui-Min Ying,Yuan-Yuan Wang,Bo-Ning Cheng,Juan Chen 연세대학교의과대학 2020 Yonsei medical journal Vol.61 No.9

Purpose: This research was designed to investigate how miR-542-5p regulates the progression of hyperglycemia and hyperlipoidemia. Materials and Methods: An in vivo model with diabetic db/db mice and an in vitro model with forskolin/dexamethasone (FSK/DEX)-induced primary hepatocytes and HepG2 cells were employed in the study. Bioinformatics analysis was conducted to identifythe expression of candidate miRNAs in the liver tissues of diabetic and control mice. H&E staining revealed liver morphologyin diabetic and control mice. Pyruvate tolerance tests, insulin tolerance tests, and intraperitoneal glucose tolerance test were utilizedto assess insulin resistance. ELISA was conducted to evaluate blood glucose and insulin levels. Red oil O staining showed lipiddeposition in liver tissues. Luciferase reporter assay was used to depict binding between miR-542-5p and forkhead box O1 (FOXO1). Results: MiR-542-5p expression was under-expressed in the livers of db/db mice. Further in vitro experiments revealed that FSK/DEX, which mimics the effects of glucagon and glucocorticoids, induced cellular glucose production in HepG2 cells and in primaryhepatocytes cells. Notably, these changes were reversed by miR-542-5p. We found that transcription factor FOXO1 is a target ofmiR-542-5p. Further in vivo study indicated that miR-542-5p overexpression decreases FOXO1 expression, thereby reversing increasesin blood glucose, blood lipids, and glucose-related enzymes in diabetic db/db mice. In contrast, anti-miR-542-5p exertedan adverse influence on blood glucose and blood lipid metabolism, and its stimulatory effects were significantly inhibited by sh-FOXO1 in normal control mice. Conclusion: Collectively, our results indicated that miR-542-5p inhibits hyperglycemia and hyperlipoidemia by targeting FOXO1.

RESEARCH : Effects of repeated tuberculin skin testings on immune responses in experimental monkeys

( Fang Ui Min ),( Jing Wang ),( Wen Yuan ),( Hui Wen Kuang ),( Wei Bo Zhao ) 한국동물자원과학회(구 한국축산학회) 2014 한국축산학회지 Vol.56 No.32

Though many alternative methods to tuberculin skin testing (TST) have been established and evaluated in recent years, sensitivities and specificities of most methods could not meet the requirements of golden standards. In this study, we sought to identify whether repeated TSTs could affect the immune responses in experimental monkeys. Nine natural tuberculosis (TB) monkeys receiving repeated TSTs biweekly were used to demonstrate the effect on TST responsiveness. Two healthy monkeys were administrated with repeated TSTs to analyze the immune response profiling. Intrapalpebral reactions in TB infections gradually weakened or presented intermittent positive reactions. The leukocyte counts, cytokine responses, and antibody responses to all antigens except Old tuberculin (OT) and MPT64L showed no specific changes for TB in healthy monkeys. Positive antibody responses to OT and MPT64L emerged during the first half experimental period, which may cause by their cross-reactivity with mycobacterial species. Results showed that repeated TSTs had no significant effects on immune responses in healthy monkeys but a progressive reduction in TST responsiveness in TB infections.

Telbivudine-Induced Myopathy: Clinical Features, Histopathological Characteristics, and Risk Factors

Min-Yu Lan,Hui-Chen Lin,Tsung-Hui Hu,Shu-Fang Chen,Chien-Hung Chen,Yung-Yee Chang,King-Wah Chiu,Tsu-Kung Lin,Shun-Sheng Chen 대한신경과학회 2023 Journal of Clinical Neurology Vol.19 No.1

Background and Purpose Oral nucleos(t)ide analogs (NAs) are the mainstay treatment for chronic hepatitis B (CHB). Myotoxicity is an important extrahepatic effect related to NA treatment. Telbivudine is the NA for CHB that is frequently associated with muscle-related side effects. The risk factors for telbivudine-induced myopathy (TIM) are not yet clear. Methods This study characterized the clinical, magnetic resonance images (MRI), and pathological features of 12 TIM cases. A group of telbivudine-tolerant (TT) patients with CHB who received regular telbivudine treatment during the same period without the occurrence of myopathy was collected. Demographic and clinical factors were compared between the patients with TIM and the TT controls. Factors independently associated with TIM were identified using logistic regression analysis. Results The patients with TIM (males/females: 7/5, mean age: 57 years) developed myopathy after using telbivudine for a median period of 19.5 months. Muscle histopathology revealed abnormal proliferation, subsarcolemmal or sarcoplasmic accumulations, and ultrastructural defects of mitochondria. When compared with TT cases, patients with TIM had a lower estimated glomerular filtration rate and were more frequently positive for hepatitis B e antigen (HBeAg). Conclusions Mitochondrial abnormalities are characteristic histopathological features, and impaired renal function and HBeAg positivity are risk factors for TIM. Telbivudine-induced mitochondrial dysfunction and immune activation related to mitochondrial damage and HBeAg serostatus changes may underlie TIM. Constant clinical surveillance of myopathy during telbivudine treatment is needed due to the significant latency of its development. Dose adjustment for impaired renal function does not eliminate the risk of TIM occurrence.

Prevalence, Risk Factors, and Psychological Effects of Primary Nocturnal Enuresis in Chinese Young Adults

Hui Jie Hu,Zhen Wei Zhang,Yu Liang,Yan Yan Luo,Qi Feng Dou,Cui Ping Song,Hui Min Zhang,Ying Zhao,Guang Run Tian,Ke Zhang,Qiu Fang Mao,Jing Gui Song,Soren Rittig,Jian Guo Wen 대한배뇨장애요실금학회 2021 International Neurourology Journal Vol.25 No.1

Purpose: This study aimed to investigate the prevalence, risk factors, and effects of primary nocturnal enuresis (PNE) on physical and mental health in young adults in mainland China. Methods: An anonymous questionnaire was used to collect information including the sociodemographic characteristics, history of PNE, family history, daytime voiding symptoms, Pittsburgh Sleep Quality Index (PSQI) scores, Self-Esteem Scale (SES), and Self-Rating Depression Scale (SDS). A total of 22,500 university students from 23 provinces and 368 cities in mainland China were included. Results: In total, 21,082 questionnaires were collected, and 20,345 of them qualified for statistical analysis. The PNE prevalence was 1.17%, and the distribution of monosymptomatic nocturnal enuresis (MNE) and nonmonosymptomatic nocturnal enuresis (NMNE) was 66.1% and 33.9%, respectively. In total, 28% of respondents with PNE reported bedwetting daily, 31.6% between 1 and 7 times weekly, and 40.4% between 1 and 4 times monthly; 80% of PNE cases had no history of treatment. The prevalence of PNE in patients with a family history, frequency, urgency, urinary incontinence, and recurrent urinary tract infections was significantly higher than in those without these conditions (P<0.001). PNE was significantly correlated with the PSQI total score (sleep quality) (P=0.011). The SES score was lower and the SDS was higher (P<0.001) in the PNE group than in those without PNE. Conclusions: In mainland China, the PNE prevalence among young adults was found to be high, and PNE had significant effects on physical and mental health. Risk factors included a family history, daytime voiding symptoms, and lack of treatment.

Cloning and Characterization of a β-Glucosidase from Marine Microbial Metagenome with Excellent Glucose Tolerance

( Wei Fang ),( Bao Lin Sun ),( Yu Zhi Hong ),( Ze Min Fang ),( Juan Juan Liu ),( Hui Peng ),( Xue Cheng Zhang ),( Ya Zhong Xiao ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.9

Involvement of Orai1 in tunicamycin-induced endothelial dysfunction

Yang, Hui,Xue, Yumei,Kuang, Sujuan,Zhang, Mengzhen,Chen, Jinghui,Liu, Lin,Shan, Zhixin,Lin, Qiuxiong,Li, Xiaohong,Yang, Min,Zhou, Hui,Rao, Fang,Deng, Chunyu The Korean Society of Pharmacology 2019 The Korean Journal of Physiology & Pharmacology Vol.23 No.2

Endoplasmic reticulum (ER) stress is mediated by disturbance of $Ca^{2+}$ homeostasis. The store-operated calcium (SOC) channel is the primary $Ca^{2+}$ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on $Ca^{2+}$ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular $Ca^{2+}$ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.