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Isolation of a Promoter Element that is Functional in Bacillus subtilis for Heterologous Gene Expression

  • Maeng, Chang-Jae;Kim, Hyung-Kwoun;Park, Sun-Yang;Koo, Bon-Tag;Oh, Tae-Kwang;Lee, Jung-Kee
    • Journal of Microbiology and Biotechnology
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    • v.11 no.1
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    • pp.85-91
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    • 2001
  • To construct an efficient Bacillus subtilis expression vector, strong promoters were isolated from the chromosomal DNA libraries of Clostridium acetobutylicum ATCC 4259, Thermoactinomyces sp. E79, and Bacillus thermoglucosidasius KCTC 3400. The $P_{C27}$ promoter cloned from the clostridial chromosmal DNA showed a 5-fold higher promoter strength than the $P_{SP02}$ promoter in the expression of the cat gene, and its sequence was estimated as an upstream region of the predicted hypothetical gene (tet-R family bacterial transcription regulator gene) in C. acetobutylicum. As a promoter element, $P_{C27}$ exhibited putative nucleotide sequences that can bind with bacterial RNAP and the 3'end of the 16S rRNA just upstream of the start codon. In addition, the promoter activity of $P_{C27}$ was distinctively repressed in the presence of glucose. Using $P_{C27}$ as the promoter element, a glucose controllable B. subtilis expression vector was constructed and the lipase gene from Staphylococcus haemolyticus KCTC 8957P was expressed in B. subtilis. When compared with the lipase expression by the T7 promoter induced by IPTG in E. coli, the $P_{C27}$ promoter showed about a 1.5-fold higher expression level in B. subtilis than that without induction.

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Time-resolved Fluoroimmunoassay (TR-FIA) Analysis of Fecal Progesterone and Estradiol in Leopard Cats (Prionailurus bengalensis) (삵에서 TR-FIA를 이용한 분변내 Estradiol과 Progesterone의 검사)

  • Kim, Young-Seob;Kim, Ji-Yong;Jung, So-Young;Lee, Bong-Joo;Shin, Nam-Shik
    • Journal of Veterinary Clinics
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    • v.27 no.6
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    • pp.693-697
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    • 2010
  • This study, conducted with four leopard cats (Prionailurus bengalensis), used time-resolved fluoroimmunoassay (TR-FIA) to analyze estradiol and progesterone concentrations in fecal samples. We measured fecal samples taken during estrus period, diestrus period, pregnancy and non-pregnancy period. During estrus (February), the mean minimum estradiol concentration was $4.02{\pm}1.9$ng/g, and the mean maximum was $86.01{\pm}35.2$ng/g (dry fecal weight). During diestrus (November), the mean minimum estradiol concentration was $4.42{\pm}1.32$ng/g and mean maximum was $15.62{\pm}6.48$ng/g (dry fecal weight). Midgestation (April), the mean minimum progesterone concentration was $427{\pm}24.49$ng/g and the mean maximum was $1490{\pm}265.27$ng/g. During non-pregnancy (November), the mean minimum progesterone concentration was $71.25{\pm}29.61$ng/g and the mean maximum was $291.75{\pm}90.30$ng/g. These results suggest that steroid hormone analysis of feces using TR-FIA is a valid method for noninvasively determining ovarian activity associated with estrus and pregnancy in leopard cats. This study will contribute to building breeding management and reproductive plans for endangered species.

Chlorogenic Acid Enhances Glucose Metabolism and Antioxidant System in High-fat Diet and Streptozotocin-induced Diabetic Mice (고지방식이와 스트렙토조토신으로 유도한 당뇨마우스에서 Chlorogenic Acid의 혈당강하 및 항산화 효과)

  • Lee, Jin;Seo, Kwon-Il;Kim, Myung-Joo;Lee, Su-Jin;Park, Eun-Mi;Lee, Mi-Kyung
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.41 no.6
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    • pp.774-781
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    • 2012
  • This study investigated dose-response effects of chlorogenic acid (CA) on glucose metabolism and the antioxidant system in streptozotocin (STZ)-induced diabetic mice with a high-fat diet (HFD). Male ICR mice were fed with a HFD (37% calories from fat) for 4 weeks prior to intraperitoneal injection with STZ (100 mg/kg body weight). Diabetic mice were supplemented with two doses of CA (0.02% and 0.05%, wt/wt) for 6 weeks. Both doses of CA significantly improved fasting blood glucose level, glucose tolerance and insulin tolerance without any changes in plasma insulin and C-peptide levels. Plasma leptin concentration was significantly higher in the CA-supplemented groups than in the diabetic control group. Both doses of CA significantly increased hepatic glucokinase activity and decreased glucose-6-phosphatase activity compared to the diabetic control group. The ratio of glucokinase/glucose-6-phosphatase was dose-independently higher in CA-supplemented mice than in diabetic control mice. CA supplementation dose-independently elevated superoxide dismutase and catalase activities, whereas it lowered lipid peroxide levels compared to the diabetic control mice in the liver and erythrocyte. These results suggest that low-dose CA may be used as a hypoglycemic agent in a high-fat diet and STZ-induced diabetic mice.

Gene Expression of Detoxification Enzymes in Tenebrio molitor after Fungicide Captan Exposure (살진균제인 캡탄 처리 후 갈색거저리의 해독효소 유전자 발현)

  • Jang, Ho am;Baek, Hyoung-Seon;Kim, Bo Bae;Kojour, Maryam Ali Mohammadie;Patnaik, Bharat Bhusan;Jo, Yong Hun;Han, Yeon Soo
    • Korean journal of applied entomology
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    • v.61 no.1
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    • pp.155-163
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    • 2022
  • The application of fungicides is indispensable to global food security, and their use has increased in recent times. Fungicides, directly or indirectly, have impacted insects, leading to genetic and molecular-level changes. Various detoxification mechanisms allow insects to eliminate reactive oxygen species (ROS) toxicity induced by agrochemicals including fungicides. In the present study, we analyzed the mRNA expression levels of detoxifying enzymes in Tenebrio molitor larvae following exposure to non-lethal doses (0.2, 2, and 20 ㎍/µL) of a fungicide captan. Transcripts of peroxidases (POXs), catalases (CATs), superoxide dismutases (SODs), and glutathione-s-transferases (GSTs) were screened from the T. molitor transcriptome database. RT-qPCR analysis showed that TmPOX5 mRNA increased significantly 24 h post-captan exposure. A similar increase was noticed for TmSOD4 mRNA 3 h post-captan exposure. Moreover, the expression of TmCAT2 mRNA increased significantly 24 h post-treatment with 2 ㎍/µL captan. TmGST1 and TmGST3 mRNA expression also increased noticeably after captan exposure. Taken together, these results suggest that TmPOX5 and TmSOD4 mRNA can be used as biomarkers or xenobiotics sensors for captan exposure in T. molitor, while other detoxifying enzymes showed differential expression.

Effect of n-3 fatty acid deficiency on fatty acid compositions of nervous system in rats reared by artificial method. (N-3 지방산 결핍이 혈청 및 신경조직의 지방산 조성에 미치는 영향)

  • Lim, Sun-Young
    • Journal of Life Science
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    • v.17 no.5 s.85
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    • pp.634-640
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    • 2007
  • Our previous study suggested that n-3 fatty acid deficiency was associated with significantly reduced spatial learning as assessed by Morris water maze test. Here we investigated an effect of n-3 fatty acid deficiency on rat brain, retina and serum fatty acyl compositions at 15 wks age using a first generational artificial rearing technique. Newborn Rat pups were separated on day 2 and assigned to two artificial rearing groups or a dam-reared control group. Pups were hand fed artificial milk via custom-designed nursing bottles containing either 0.02%(n-3 Deficient) or 3.1% (n-3 Adequate) of total fatty acids as a-linolenic acid(LNA). At day 21, rats were weaned to either n-3 deficient or n-3 adequate pelleted diets and fatty acid compositions of brain, retina and liver were analyzed at 15 wks age. Brain docosahexaenoic acid(DHA) was lower(58% and 61%, P<0.05) in n-3 deficient in comparison to n-3 adequate and dam-reared groups, receptively, while brain docosapentaenoic acid(DPAn-6) was increased in the n-3 deficient group. In retina and serum fatty acid compositions, the decreased precentage of DHA and increased precentage of DPAn-6 were observed. These results suggested that artificial rearing method can be used to produce n-3 fatty acid deficiency in the first generation and that adequate brain DHA levels are required for optimal brain function.

Relationship between Pain Reaction and Electrical Stimulation of Peripheral Nerve with Special Reference of Stimulatory Parameters (말초신경 자극시 자극의 강도, 빈도 및 기간의 변화가 동통반응에 미치는 영향)

  • Paik, Kwang-Sea;Leem, Joong-Woo;Kim, In-Kyo;Lee, Seung-Il;Kang, Doo-Hee
    • The Korean Journal of Physiology
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    • v.19 no.2
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    • pp.227-232
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    • 1985
  • Previously, we had reported that the electrical stimulation of peripheral nerve with stimlatory parameters of 20 V strength and 2 Hz frequency for 60 min resulted in reducing the pain reaction. The present study was performed to evaluate if the pain reaction was affected by the peripheral nerve stimulation with different stimulatory parameters in the decerebrated cat. The flexion reflex was used as an index of the pain reaction. The reflex was elicited by stimulating the sural nerve (stimulus strength of 20 $V\;\times\;0.5$msec) and recorded as a compound action potential from the motor nerve innervated to the posterior biceps femoris muscle. The common perneal nerve was selected as a peripheral nerve on which the electrical stimulation of various intensities and frequencies was applied. The results are summarized as follows : 1) The peripheral nerve stimulation with 100 mV strength, regardless of frequencies, did not affect the pain reaction induced by the sural nerve stimulation. 2) When the stimulus of 1V intensity and slow frequency (2 Hz) was applied to the peripheral nerve for 30 min or 60 min, the pain reaction was significantly reduced comparing to the control. However, this reduced pain reaction by the peripheral nerve stimulation was not reversed by the injection of naloxone (0.02 mg/kg) 3) High frequency stimulus (60 Hz) of 1V intensity for 30 or 60 min did not show any effects of affecting the pain reaction. These results suggest that the stimulus of relatively high intensity (at least 1V) and low frequency (2 Hz) is needed to elicite the analgesic effect by the peripheral nerve stimulation. By the 1V stimulus, $A\delta$ nerve fiber is activated. Therefore, an $A\delta$ or smaller nerve fibers must be activated for showing analgesia by the peripheral nerve stimulation. However, the mechanism of analgesia by the $A\delta$ nerve activation alone was not related to the endogeneous morphine system since the reduced pain reaction by the $A\delta$ fiber activation alone was not reversed by the treatment of naloxone.

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In vivo Study of the Renal Protective Effects of Capsosiphon fulvescens against Streptozotocin-induced Oxidative Stress (스트렙토조토신 유발 당뇨 쥐의 산화스트레스에 대한 매생이 추출물의 신장 보호 효과)

  • Nam, Mi-Hyun;Koo, Yun-Chang;Hong, Chung-Oui;Yang, Sung-Yong;Kim, Se-Wook;Jung, Hye-Lim;Lee, Hwa;Kim, Ji-Yeon;Han, Ah-Ram;Son, Won-Rak;Pyo, Min-Cheol;Lee, Kwang-Won
    • Korean Journal of Food Science and Technology
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    • v.46 no.5
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    • pp.641-647
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    • 2014
  • In this study, we evaluated the effect of Capsosiphon fulvescens extract (CFE) and its active compound, pheophorbide A (PhA), on diabetic kidney failure. Diabetes mellitus (DM) was induced by a single intraperitoneal injection of streptozotocin (STZ; 40 mg/kg body weight (BW)). After a week, the rats were orally administered CFE (4 and 20 mg/kg BW) or PhA (0.2 mg/kg BW) once a day for 9 weeks. After scarification, renal tissue samples were collected for biochemical and histochemical analyses. Our study showed that the treatment with CFE and PhA significantly decreased lipid peroxidation level and the activities of glutathione peroxidase and glutathione-S-transferase (p<0.05), but it increased glutathione level and the activities of glutathione reductase, superoxide dismutase, and catalase in the renal tissues (p<0.05). The CFE- and PhA-treated rats with DM showed improved histochemical appearance and decreased abnormal glycogen accumulation. Therefore, we suggest that PhA-containing CFE could exert renal protective effects against STZ-induced oxidative stress.