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Gram-Scale Production of Ginsenoside F1 Using a Recombinant Bacterial β-Glucosidase

  • An, Dong-Shan;Cui, Chang-Hao;Siddiqi, Muhammad Zubair;Yu, Hong Shan;Jin, Feng-Xie;Kim, Song-Gun;Im, Wan-Taek
    • Journal of Microbiology and Biotechnology
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    • v.27 no.9
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    • pp.1559-1565
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    • 2017
  • Naturally occurring ginsenoside F1 (20-O-${\beta}$-$\text\tiny{D}$-glucopyranosyl-20(S)-protopanaxatriol) is rare. Here, we produced gram-scale quantities of ginsenoside F1 from a crude protopanaxatriol saponin mixture comprised mainly of Re and Rg1 through enzyme-mediated biotransformation using recombinant ${\beta}$-glucosidase (BgpA) cloned from a soil bacterium, Terrabacter ginsenosidimutans Gsoil $3082^T$. In a systematic step-by-step process, the concentrations of substrate, enzyme, and NaCl were determined for maximal production of F1. At an optimized NaCl concentration of 200 mM, the protopanaxatriol saponin mixture (25 mg/ml) was incubated with recombinant BgpA (20 mg/ml) for 3 days in a 2.4 L reaction. Following octadecylsilyl silica gel column chromatography, 9.6 g of F1 was obtained from 60 g of substrate mixture at 95% purity, as assessed by chromatography. These results represent the first report of gram-scale F1 production via recombinant enzyme-mediated biotransformation.

Antimicrobial, Antioxidant, and Anticoagulation Activities of Salicornia europaea seeds (함초 씨의 항균, 항산화 및 항혈전 활성)

  • Kim, Mi-Sun;Kim, Deuk Hoi;Sohn, Ho-Yong
    • Microbiology and Biotechnology Letters
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    • v.44 no.4
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    • pp.452-460
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    • 2016
  • This study was designed to develop a functional pharma-food using Salicornia europaea (SE). Tiny seeds from the mature SE were collected, and their biological activities were evaluated. The extraction yield of the seed in hot water was found to be 29.6% and the hot water extract (HWE) contained 25.7 mg/g total polyphenol (TP) and 11.5 mg/g total flavonoid (TF), which are similar to those contained in leaf and stem of SE. Among the subsequent organic solvent fractions, the ethylacetate (EA) fraction exhibited the highest content of TP (158.3 mg/g), TF (136.2 mg/g), and total sugar (228.3 mg/g). The EA fraction exhibited broad-range antibacterial activities against gram-positive bacteria, and the butanol fraction exhibited growth inhibitory effect against only Staphylococcus epidermidis. An antioxidation activity assay of the HWE and its fractions showed the EA fraction to have the highest radical scavenging activity with $RC_{50}$ values of 57.0, 29.0, and $28.9{\mu}g/ml$ against DPPH anion, ABTS cation, and nitrite, respectively. The $RC_{50}$ values of vitamin C against DPPH anion, ABTS cation, and nitrite were 10.7, 4.0, and $18.0{\mu}g/ml$, respectively, indicating that the EA fraction of SE has potent antioxidant compounds. In an anticoagulation assay, the EA fraction exhibited a 15-fold extended thrombin time at 5 mg/ml and activated partial thromboplastin time at 7 mg/ml, which are comparable to the activities of aspirin. The HWE and its fractions had no hemolysis activities against human RBCs at up to 1 mg/ml. These results suggest that the EA fraction from SE has a great potential as a new antibacterial and anticoagulation agent.

Characterization of a Glutamate Decarboxylase (GAD) from Enterococcus avium M5 Isolated from Jeotgal, a Korean Fermented Seafood

  • Lee, Kang Wook;Shim, Jae Min;Yao, Zhuang;Kim, Jeong A;Kim, Hyun-Jin;Kim, Jeong Hwan
    • Journal of Microbiology and Biotechnology
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    • v.27 no.7
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    • pp.1216-1222
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    • 2017
  • To develop starters for the production of functional foods or materials, lactic acid bacteria producing ${\gamma}-aminobutyric$ acid (GABA) were screened from jeotgals, Korean fermented seafoods. One isolate producing a high amount of GABA from monosodium $\text\tiny{L}$-glutamate (MSG) was identified as Enterococcus avium by 16S rRNA gene sequencing. E. avium M5 produced $18.47{\pm}1.26mg/ml$ GABA when incubated for 48 h at $37^{\circ}C$ in MRS broth with MSG (3% (w/v)). A gadB gene encoding glutamate decarboxylase (GAD) was cloned and overexpressed in E. coli BL21 (DE3) using the pET26b (+) expression vector. Recombinant GAD was purified through a Ni-NTA column and the size was estimated to be 53 kDa by SDS-PAGE. Maximum GAD activity was observed at pH 4.5 and $55^{\circ}C$and the activity was dependent on pyridoxal 5'-phosphate. The $K_m$ and $V_{max}$ values of GAD were $3.26{\pm}0.21mM$ and $0.0120{\pm}0.0001mM/min$, respectively, when MSG was used as a substrate. Enterococcus avium M5 secretes a lot of GABA when grown on MRS with MSG, and the strain is useful for the production of fermented foods containing a high amount of GABA.

Comparison of Based on Histogram Equalization Techniques by Using Normalization in Thoracic Computed Tomography (흉부 컴퓨터 단층 촬영에서 정규화를 사용한 다양한 히스토그램 평준화 기법을 비교)

  • Lee, Young-Jun;Min, Jung-Whan
    • Journal of radiological science and technology
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    • v.44 no.5
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    • pp.473-480
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    • 2021
  • This study was purpose to method that applies for improving the image quality in CT and X-ray scan, especially in the lung region. Also, we researched the parameters of the image before and after applying for Histogram Equalization (HE) such as mean, median values in the histogram. These techniques are mainly used for all type of medical images such as for Chest X-ray, Low-Dose Computed Tomography (CT). These are also used to intensify tiny anatomies like vessels, lung nodules, airways and pulmonary fissures. The proposed techniques consist of two main steps using the MATLAB software (R2021a). First, the technique should apply for the process of normalization for improving the basic image more correctly. In the next, the technique actively rearranges the intensity of the image contrast. Second, the Contrast Limited Adaptive Histogram Equalization (CLAHE) method was used for enhancing small details, textures and local contrast of the image. As a result, this paper shows the modern and improved techniques of HE and some advantages of the technique on the traditional HE. Therefore, this paper concludes that various techniques related to the HE can be helpful for many processes, especially image pre-processing for Machine Learning (ML), Deep Learning (DL).

Effect of Imipramine on the Contractility of Single Cells Isolated from Canine Detrusor (Imipramine이 배뇨근 세포의 수축성에 미치는 직접작용)

  • Huh, Chan-Wook;Lee, Kwang-Youn;Kim, Won-Joon
    • Journal of Yeungnam Medical Science
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    • v.11 no.2
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    • pp.293-302
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    • 1994
  • The objective of this study was to establish a good methodology to isolate single smooth muscle cells that are alive and respond properly to pharmacological agents. Canine urinary bladders were employed as the source of single cells, and acetylcholine, atropine and imipramine were used as indicators of pharmacological responsiveness. Imipramine, an antidepressant drug exhibited the anticholinergic and calcium antagonizing properties on rat detrusor muscle. To establish a control value for a further experiment to elucidate the mechanism of action of imipramine on detrusor muscle, we measured the concentration-response of single cells to acetylcholine in the presesnce of imipramine by length of the cells and compared the result with the response in the presence of atropine. Tiny chops of smooth muscle taken from anesthetized canine urinary bladder were incubated in collagenase solution at $36^{\circ}C$ for 17-20 minutes. The collagenase solution included collagenase 1.2 mg/ml, soybean tryspin inhibitor 0.08 mg/ml, bovine serum albumin 2% in 10 ml Krebs-Henseleit buffer solution aerated with a consistent breeze of 95/5% $O_2/CO_2$, to maintain the pH at 7.4. After washing with plain K-H solution on 450 mesh, cells were dissociated from the digested tissue for 12-15 minutes. Cell suspension was transfered in 5 ml test tubes and acetylcholine was added for the final concentration to be $10^{-14}M{\sim}10^{-9}M$. To find the optimal time to fix the cells to determine the contractile responses, 1% acrolein was added 5, 10, 20, 30, 60 and 120 seconds after the administration of ACh. The length of cells fixed by acrolein were measured by microscaler via CCTV camera on phaes-contrast microscope. The average length of 50 cells from a slide glass was taken as the value of a sample at the very concentration point. Single cells were isolated from canine detrusor. The length of untreated cells varied from 82 ${\mu}m$ to 94 ${\mu}m$. The maximal response to actylcholine $10^{-9}M$ was accomplished within 5 seconds of exposure, and the shortening was $19{\pm}3$%. Atropine reduced the contraction of the cells concentration-dependently. Imipramine which exerts a cholinergic blocking action on some smooth muscles also reduced the contraction concentration-dependently and by a similar pattern as atropine. These findings document that imipramine may exerts a cholinergic blocking activity in the single smooth muscle cells isolated from canine urinary bladder.

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Estimation of Humidifier Disinfectant Amounts Inhaled into the Respiratory System (가습기 살균제 피해자 호흡기로 흡입된 가습기 살균제 양 추정 - 호흡기 외부(external) 및 내부 노출(internal exposure) 추정 방법과 사례 -)

  • Park, Dong-Uk;Ryu, Seung-Hun;Lim, Heung-Kyu;Kim, Sun-Kyung;Roh, Hyun-Suk;Cha, Won-Seok;Park, Dooyong
    • Journal of Environmental Health Sciences
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    • v.42 no.3
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    • pp.141-146
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    • 2016
  • In South Korea, many cases of humidifier disinfectant-associated lung injury (HDLI) have been reported among people who used humidifier products containing humidifier disinfectant (HD). The objectives of this study are to estimate both airborne HD concentration in the room where HD was used and the amount of humidifier disinfectant absorbed into the respiratory system. Information and data on the HDs were collected using a structured questionnaire and home environmental investigations include the volume of HD (ml) and hours used per day, concentration of disinfectants contained in the HD brand (${\mu}g/ml$), volume of the room ($m^3$), assumed ventilation rate ($m^3/hr$) and breathing rate assumed ($m^3/hr$). We used children aged under five years old as a sample and estimated both airborne HD concentrations and amount of HD absorbed into the respiratory system. The estimated airborne concentration of HD in the room ranged from 68 to $369{\mu}g/m^3$ for PHMG (polyhexamethylene guanidine phosphate) and from 16 to $239{\mu}g/m^3$ for PGH (oligo (2-(2-ethoxy) ethoxyethyl guanidine). The amount of HD absorbed in the respiratory system per day was estimated to range from 227 to $1,225{\mu}g$ for PHMG and from 53 to $794{\mu}g$ for PGH. In conclusion, a great amount of HD was likely absorbed into respiratory system, likely beyond the level of the capacity of the immune system to remove the HD absorbed.

Analysis of the Entrance Surface Dose (ESD) and Contrast Usage of Trance Femoral Cerebral Angiography (TFCA) and Cerebral Computed Tomographic Angiography (CCTA) for Cerebrovascular Disease Examining (뇌혈관 질환 검사를 위한 뇌혈관 조영술(TFCA)과 뇌혈관 전산화 단층 촬영 검사(CCTA)의 입사표면선량(ESD) 및 조영제 사용량 분석)

  • Seo, Young-Hyun;Hong, Cheon-Gi;Song, Jong-Nam
    • Journal of the Korean Society of Radiology
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    • v.13 no.4
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    • pp.495-502
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    • 2019
  • A typical cerebrovascular disease among cerebrovascular diseases is vascular diseases such as cerebral infarction, cerebral hemorrhage, cerebral aneurysm, cerebrovascular stenosis. If the disease occurs and causes cerebral damage, it may be difficult to recover completely. So that, Must continue to perform health care through examination early. In particular, Because most cerebrovascular disease examining use radiation equipment and Thus this study was to find out how to select about the optimal examining method and X-ray dose decrease method among different examining method though comparison and analysis for the entrance surface dose (ESD) on cerebrovascular examining with Trance Femoral Cerebral angiography (TFCA) and Cerebral Computed Tomographic Angiography (CCTA). Also, want to find out how to select about the optimal examining method for worried patient that contrast medium side effect though measuring and evaluating for contrast usage. Data were collected from 70 patients (43 males and 27 females) who underwent CCTA at Yeosoo region hospital from June 2018 to December 2018 and 61 patients (34 males and 27 females) who underwent TFCA at Pyeongtaek region hospital from June 2018 to November 2018. ESD analysis method collected retrospective data though M-view and PACS PLUS program, Used contrast usage measuring method did reality measuring method. In the analysis using SPSS, the ESD of TFCA was $245.74{\pm}71.91$, which was $32.05{\pm}7.74$ lower than the dose of $277.79{\pm}79.65$ of CCTA ESD, and statistically significant at t = 3.249, p = 0.017 (p<0.05). As a result of the comparison of the total amount of contrast agent, the mean contrast agent used in TFCA was $55.05{\pm}17.68ml$, which was about 14.95 smaller than the amount of contrast agent used in CCTA, and statistically significant t = -4.548, p<0.001. In conclusion, the ESD of TFCA was statistically significantly lower than that of CCTA, and also the used contrast usage was significantly tiny than that of CCTA. Therefore, Select the method to increase the utilization of TFCA for cerebral disease examining, we can consider X-ray dose decrease method at the same time as to decrease side effect of contrast medium.

Histology of Injected Autologous Auricular Cartilage in the Paralyzed Canine Vocal Fold at Two Year (실험적 성대마비 개에서 자가이개연골의 성대근육내 주입 후 조직학적 변화 : 2년 후 결과)

  • Lee Byung-Joo;Lee Jin-Choon;Chon Kyong-Myong;Goh Eui-Kyung;Roh Hwan-Jung;Lee Chang-Hun;Wang Soo-Geun
    • Journal of the Korean Society of Laryngology, Phoniatrics and Logopedics
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    • v.16 no.2
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    • pp.113-117
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    • 2005
  • Background and Objective : Vocal fold augmentation by injectable material under direct visual control is an easy and simple operation. However, when autologous fat or bovine collagen is used, the resoiption creates a problem. And autologous fascia is debating about absorption now days. We previously reported on the one year results of injected autologous auricular cartilage for volumetric augmentation in paralyzed canine vocal cord. This study evaluates the long-term histomorphologic results of injected autologous auricular cartilage for the augmentation of the paralyzed canine vocal fold at two year. Material and Methods . A prospective trial of autologous cartilage augmentation of vocal cord in animal model. Three dogs were operated upon. A piece of auricular cartilage was harvested from the ear and minced into tiny chips with a scalpel. Fat was harvested from inguinal area and minced with a scalpel. The minced cartilage and fat-paste (0.2ml) was injected using a pressure syringe into the paralyzed thyroarytenoid muscle using direct laryngoscopy. Three animals were sacrificed at 2 years. Each subject underwent laryngectomy and serial coronal sections of paraffin blocks from the posterior vocal fold were made. Results There was no significant complication perioperatively and during follow-up. The injected cartilage which appeared to have lost viability existed in the vocalis muscles until 24 months. Fibrotic change was exhibited in the surrounding injected cartilage. Conclusion : The autologous auricular cartilage graft is well tolerated and may be very effective material for volumetric augmentation on paralyzed vocal cord.

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