• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1136-1140
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• 2008

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'-phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.9-18
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• 2017

Nine bacilli with fibrinolytic activities were isolated from doenjang, a traditional Korean fermented soy food. Among them, RSB34 showed the strongest activity and was identified as Bacillus amyloliquefaciens by 16S rRNA and recA gene sequencing. During growth on LB up to 96 h, RSB34 showed the highest fibrinolytic activity ($83.23mU/{\mu}l$) at 48 h. Three bands of 23, 27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDS-PAGE and 27 and 42 kDa bands by fibrin zymography. The gene encoding the 27 kDa fibrinolytic enzyme AprE34 was cloned by PCR. BLAST analyses confirmed that the gene was a homolog to genes encoding AprE-type proteases. aprE34 was overexpressed in Escherichia coli BL21(DE3) using pET26b(+). Recombinant AprE34 was purified and examined for its properties. The $K_m$ and $V_{max}$ values of recombinant AprE34 were $0.131{\pm}0.026mM$ and $16.551{\pm}0.316{\mu}M/l/min$, respectively, when measured using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. aprE34 was overexpressed in B. subtilis WB600 using pHY300PLK. B. subtilis transformants harboring pHYRSB34 (pHY300PLK with aprE34) showed higher fibrinolytic activity than B. amyloliquefaciens RSB34.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.9
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• pp.1293-1302
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• 2014

The aim of study was to determine the energy requirements for maintenance and growth of forty-one Saanen, intact male kids with initial body weight (BW) of $5.12{\pm}0.19$ kg. The baseline (BL) group consisted of eight kids averaging $5.46{\pm}0.18$ kg BW. An intermediate group consisted of six kids, fed for ad libitum intake, that were slaughtered when they reached an average BW of $12.9{\pm}0.29$ kg. The remaining kids (n = 27) were randomly allocated into nine slaughter groups (blocks) of three animals distributed among three amounts of dry matter intake (DMI; ad libitum and restricted to 70% or 40% of ad libitum intake). Animals in a group were slaughtered when the ad libitum-treatment kid in the group reached 20 kg BW. In a digestibility trial, 21 kids (same animals of the comparative slaughter) were housed in metabolic cages and used in a completely randomized design to evaluate the energetic value of the diet at different feed intake levels. The net energy for maintenance ($NE_m$) was $417kJ/kg^{0.75}$ of empty BW (EBW)/d, while the metabolizable energy for maintenance ($ME_m$) was $657kJ/kg^{0.75}$ of EBW/d. The efficiency of ME use for NE maintenance ($k_m$) was 0.64. Body fat content varied from 59.91 to 92.02 g/kg of EBW while body energy content varied from 6.37 to 7.76 MJ/kg of EBW, respectively, for 5 and 20 kg of EBW. The net energy for growth ($NE_g$) ranged from 7.4 to 9.0 MJ/kg of empty weight gain by day at 5 and 20 kg BW, respectively. This study indicated that the energy requirements in goats were lower than previously published requirements for growing dairy goats.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.952-959
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• 2007

His-His-Leu (HHL), a tripeptide derived from a Korean soybean paste, is an angiotensin-I-converting enzyme (ACE) inhibitor. We report here a method of producing this tripeptide efficiently by expressing tandem multimers of the codons encoding the peptide in E. coli and purifying the HHL after hydrolysis of the peptide multimers. The HHL gene, tandemly multimerized to a 40-mer, was ligated with ubiquitin as a fusion gene (UH40). UH40 was inserted into vector pET29b; the UH40 fusion protein was then produced in E. coli BL21. The recombinant UH40 protein was purified by cation-exchange chromatography with a yield of 17.3mg/l and analyzed by matrixassisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and protein N-terminal sequencing. Leucine aminopeptidase was used to cleave a 405-Da HHL monomer from the UH40 fusion protein and the peptide was purified using reverse-phase high-performance liquid chromatography (HPLC) on a C18 HPLC column, with a final yield of 6.2mg/l. The resulting peptide was confirmed to be HHL with the aid of MALDI-TOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE inhibiting activity. These results suggest that our production method is useful for obtaining a large quantity of recombinant HHL for functional antihypertensive peptide studies.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.3
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• pp.317-325
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• 2017

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent liver disease in parallel with worldwide epidemic of obesity. Reactive oxygen species (ROS) contributes to the development and progression of NAFLD. Peroxisomes play an important role in fatty acid oxidation and ROS homeostasis, and catalase is an antioxidant exclusively expressed in peroxisome. The present study examined the role of endogenous catalase in early stage of NAFLD. 8-week-old male catalase knock-out (CKO) and age-matched C57BL/6J wild type (WT) mice were fed either a normal diet (ND: 18% of total calories from fat) or a high fat diet (HFD: 60% of total calories from fat) for 2 weeks. CKO mice gained body weight faster than WT mice at early period of HFD feeding. Plasma triglyceride and ALT, fasting plasma insulin, as well as liver lipid accumulation, inflammation (F4/80 staining), and oxidative stress (8-oxo-dG staining and nitrotyrosine level) were significantly increased in CKO but not in WT mice at 2 weeks of HFD feeding. While phosphorylation of Akt (Ser473) and $PGC1{\alpha}$ mRNA expression were decreased in both CKO and WT mice at HFD feeding, $GSK3{\beta}$ phosphorylation and Cox4-il mRNA expression in the liver were decreased only in CKO-HF mice. Taken together, the present data demonstrated that endogenous catalase exerted beneficial effects in protecting liver injury including lipid accumulation and inflammation through maintaining liver redox balance from the early stage of HFD-induced metabolic stress.

• The Journal of Korean Medicine
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• v.29 no.4
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• pp.94-103
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• 2008

Objectives: The aim of this study was to determine whether celastrol, the triterpenoid component of Celastrus orbiculatus, offers neuroprotection against Parkinson's disease (PD) in mice administered 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine(MPTP). Methods: We examined how celastrol affected MPTP-induced neuronal loss of tyrosine hydroxylase (TH)-positive dopaminergic neurons in substantia nigra pars compacta (SNpc) in the midbrain of mice. C57BL/6J mice were divided into four groups: (1) saline-saline, (2) saline-celastrol, (3) MPTP-saline, and (4) MPTP-celastrol. The mice were injected intraperitoneally (i.p.) with four administrations of MPTP (18mg/kg) at 2 h intervals and then i.p. administered celastrol (3mg/kg) two times at 12 h after last celastrol administration. Expression of TH on the SNpc of brain tissues were analyzed at 7 days after the treatments by immunohistochemistry and Western blot. Results: Immunohistochemical analysis using TH antibody showed that celastrol provided significantly protective effects against MPTP-induced loss of TH-positive dopaminergic neurons in the SNpc region of the midbrain of mice. Our Western blot study also showed that celastrol significantly inhibits the MPTP-induced neuronal damage via the up-regulation of TH protein levels in MPTP mice. Conclusions: The present results suggest that it may be possible to use celastrol for the prevention of nigral degenerative disorders including PD, caused by exposure to toxic substances.

• Nutrition Research and Practice
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• v.7 no.5
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• pp.352-358
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• 2013

Korean pine nut oil (PNO) has been reported to have favorable effects on lipid metabolism and appetite control. We investigated whether PNO consumption could influence weight gain, and whether the PNO-induced effect would result in an improvement of immune function in high-fat diet (HFD)-induced obese mice. C57BL/6 mice were fed control diets with 10% energy fat from either PNO or soybean oil (SBO), or HFDs with 45% energy fat from 10% PNO or SBO and 35% lard, 20% PNO or SBO and 25% lard, or 30% PNO or SBO and 15% lard for 12 weeks. The proliferative responses of splenocytes upon stimulation with concanavalin A (Con A) or lipopolysaccharide (LPS), Con A-stimulated production of interleukin (IL)-2 and interferon (IFN)-${\gamma}$, and LPS-stimulated production of IL-6, IL-$1{\beta}$, and prostaglandin $E_2$ ($PGE_2$) by splenocytes were determined. Consumption of HFDs containing PNO resulted in significantly less weight gain (17% less, P < 0.001), and lower weight gain was mainly due to less white adipose tissue (18% less, P = 0.001). The reduction in weight gain did not result in the overall enhancement in splenocyte proliferation. Overall, PNO consumption resulted in a higher production of IL-$1{\beta}$ (P = 0.04). Replacement of SBO with PNO had no effect on the production of IL-2, IFN-${\gamma}$, IL-6, or $PGE_2$ in mice fed with either the control diets or HFDs. In conclusion, consumption of PNO reduced weight gain in mice fed with HFD, but this effect did not result in the overall improvement in immune responses.

• Journal of Acupuncture Research
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• v.15 no.2
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• pp.157-171
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• 1998

In order to study the effect of direct moxibustion on depressed immune response and thyroid hormone in rats exposed to cold stress, Sprague-Dawley male rats were put in the horizontal refrigerator by $-10^{\circ}C$ for 14 days and thereafter $-18^{\circ}C$ for 11 days(control group). Sample I group was treated by daily direct moxibustion to bilateral Shinsu(BL23) from the 22nd day to the 25th day for 4 days under the same condition with the control group. Sample II group was treated by daily direct moxibustion to bilateral non-acupoints near the root of the tail from the 22nd day to the 25th day for 4 days under the same condition with the control group. And RBC, WBC, CD4+ T cell count, T3, T4 and TSH were measured. The results were as follows; 1. RBC increased with statistical significance in the sample I and sample II groups compared with the control group. 2. WBC increased with statistical significance in the sample I group, but there was not any significance in the sample II group compared with the control group. 3. Lymphocyte increased with statistical significance both in the sample I and sample II groups compared with the control group. 4. CD4+ T cell count increased with statistical significance both in the sample I and sample II groups compared with the control group. 5. T3, T4 and TSH increased respectively with statistical significance in the sample I and sample II groups compared with the control group.

• The Journal of Korean Medicine
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• v.32 no.3
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• pp.25-34
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• 2011

Objectives: We performed simultaneous determination of five constituents by HPLC in Samul-tang (SMT). Additionally, we investigated the immune-stimulatory potential of SMT on specific cellular and humoral immune responses in ovalbumin (OVA)-immunized mice. Methods: Reverse-phase chromatography using a Gemini C18 column operating at $40^{\circ}C$, and photodiode array (PDA) detection at 190-400 nm, were used for quantification of the five components of SMT. Mobile phase using a gradient flow consisted of two solvent systems. Solvent A was 1.0% (v/v) aqueous acetic acid and solvent B was acetonitrile with 1.0% (v/v) acetic acid. C57BL/6 mice were immunized intraperitoneally with OVA/alum ($100{\mu}g/200{\mu}g$) on days 1, 8, and 15. The extract of SMT (1000 mg/kg) was given to mice orally for 21 days (from day 1 to day 21). At day 22, OVA-, lipopolysaccharide (LPS)- and concanavalin A (Con A)-stimulated splenocyte proliferation and OVA-specific and total antibodies were measured in plasma. Results: Calibration curves were acquired with $r^2$>0.9999, and the relative standard deviation (RSD, %) for intra- and inter-day precision were both less than 3.5%. The recovery was in the range of 95.69-115.12%, with an RSD less than 6.0%. The contents of five components in SMT were 1.08-15.30 mg/g. SMT significantly enhanced Con A-induced splenocyte proliferation in OVA-immunized mice (p<0.01). Also, SMT significantly enhanced OVAspecific IgG, IgG1 and total IgM levels in plasma compared with the OVA-immunized group. Conclusions: The established method will be applied for the quantification of major components and immunestimulating activity in OVA-immunized mouse model of SMT.

• The Journal of Pediatrics of Korean Medicine
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• v.18 no.2
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• pp.31-48
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• 2004

Objective : KagamJwagwiEum(KJE) has been used for the purpose of prevention and treatment of bronchial asthma and allergic asthma in Korea. To investigate the biological effect of KJE, the author examined cytotoxicity and inflammatory cytokines secretion with human leukemic mast cell line, HMC-1. Methods: HMC-1 was stimulated with phorbol 12-myristate 13-acetate(PMA) and calcium ionophore A23187. KJE by itself had no effect on viability of HMC-l. The effects of KJE on the secretion of tumor necrosis $factor-alpha(TNF-{\alpha})$, interleukin(IL)-6 and IL-8 from HMC-1 were evaluated with enzyme-linked immunosorbent assay. Results : KJE inhibited PMA plus A23187-induced $TNF-{\alpha}$ and IL-6 secretion. But KJE had no effect IL-8 secretion: KJE had immunoregulatory effects on cytokines, increased secretion of NO and $TNF-{\alpha}$ but did not effect IL-12 secretion when the cells were primed and trigged with $IFN-{\gamma}$ in the peritoneal macrophages of C57BL/6 mice. Conclusions : Taken together, these results suggest that KJE inhibit the production of inflammatory cytokines in HMC-1 cells and activate macrophages.