• Asian Pacific Journal of Cancer Prevention
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• v.17 no.2
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• pp.615-618
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• 2016

Ethyl acetate extracts obtained from culture of endophytic fungi Aspergillus sp isolated from Piper crocatum Ruiz & Pav, have been shown to possess cytotoxic activity against T47D breast cancer cells. Investigations were here conducted to determine bioactive compounds responsible for the activity. Bioassay guided fractionation was employed to obtain active compounds. Structure elucidation was performed based on analysis of LC-MS, $^1H$-NMR, $^{13}C$-NMR, COSY, DEPT, HMQC, HMBC data. Cytotoxity assays were conducted in 96 well plates against T47D and Vero cell lines. Bioassay guided isolation and chemical investigation led to the isolation of pyrophen, a 4-methoxy-6-(1'-acetamido-2'-phenylethyl)-2H-pyran-2-one. Further analysis of its activity against T47D and Vero cells showed an ability to inhibit the growth of T47D cells with IC50 values of $9.2{\mu}g/mL$ but less cytotoxicity to Vero cells with an $IC_{50}$ of $109{\mu}g/mL$. This compound at a concentration of 400 ng/mL induced S-phase arrest in T47D cells.

• Nutrition Research and Practice
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• v.9 no.4
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• pp.358-363
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• 2015

BACKGROUND/OBJECTIVES: Previous studies have indicated that when compared to young mice, old mice have lower global DNA methylation and higher p16 promoter methylation in colonic mucosa, which is a common finding in colon cancer. It is also known that a Western-style diet (WSD) high in fat and calories, and low in calcium, vitamin D, fiber, methionine and choline (based on the AIN 76A diet) is tumorigenic in colons of mice. Because DNA methylation is modifiable by diet, we investigate whether a WSD disrupts DNA methylation patterns, creating a tumorigenic environment. SUBJECTVIES/METHODS: We investigated the effects of a WSD and aging on global and p16 promoter DNA methylation in the colon. Two month old male C57BL/6 mice were fed either a WSD or a control diet (AIN76A) for 6, 12 or 17 months. Global DNA methylation, p16 promoter methylation and p16 expression were determined by LC/MS, methyl-specific PCR and real time RT-PCR, respectively. RESULTS: The WSD group demonstrated significantly decreased global DNA methylation compared with the control at 17 months (4.05 vs 4.31%, P = 0.019). While both diets did not change global DNA methylation over time, mice fed the WSD had lower global methylation relative to controls when comparing all animals (4.13 vs 4.30%, P = 0.0005). There was an increase in p16 promoter methylation from 6 to 17 months in both diet groups (P < 0.05) but no differences were observed between diet groups. Expression of p16 increased with age in both control and WSD groups. CONCLUSIONS: In this model a WSD reduces global DNA methylation, whereas aging itself has no affect. Although the epigenetic effect of aging was not strong enough to alter global DNA methylation, changes in promoter-specific methylation and gene expression occurred with aging regardless of diet, demonstrating the complexity of epigenetic patterns.

• Food Industry
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• s.99
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• pp.32-37
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• 1989

양질의 국산 생강엑기스 제조기술 개발을 위한 본 연구결과를 요약하면 다음과 같다. 1) 본 연구에서 시료로 사용한 생강은 전라북도 봉동산과 충청남도 서산산이며, 이들 건강은 수분이 약$10\%$, 회분 $8\~9\%$, 조지방 $4\~5\%$이다. 2) 생강엑기스의 유효성분들은 건강입자에 내포되어 있는 상태에서 추출 속도는 반응층을 통한 확산모델로 설명된다. 침출 효율을 개선하기위해서는 다음과 같은 조건이 필수적이다. 3) 건강의 입자는 $10\~20{\mu}m$정도의 전분입자가 될수록 많이 노출되도록 160목을 통과하는 작은 입자로 분쇄하면 추출효율은 최대화 할 수 있다. - 추출온도는 엑기스의 주요성분의 손실이 무시되는 최대온도, $40^{\circ}C$가 최적이다. - 160목, $40^{\circ}C$에서 추출시간 3-4시간이 최적이다. - 이같은 조건에서의 엑기스 회수율은 약 $8\%$이다. 4) 엑기스내의 비자극성 성분은 회분 $0.5\~0.8\%$, 조지방 $1.2\~1.8\%$, 조단백 $2.8\~3.5\%$이고, 유리당은 거의 침출되지 않는다. 엑기스내의 주요 지방산은 Linoleic acid가 가장 많이 함유되어 있다. 5) 기계건조보다 일광건조에 의한 건강에서 추출된 엑기스의 품질이 양호하며, 외국산 고급 엑기스와 품질면에서 대등한 것이다. 6) 위와 같은 결과는 TLC로 분리하고 분리된 각 Spot를 HPLS로 분석, IR, NMR, LC/MS를 사용하여 주요성분을 확인 및 정량화하였다. 이로부터 엑기스내의 주요성분은 gingerol이 약 0.38, Shogaol이 약 0.027, 그리고 Paradol이 0.03의 농도분율을 가지고 있음을 알았다. 7) 기계건조 건강으로부터 얻은 엑기스는 상온 $\~100^{\circ}C$ 범위에서 휘발 및 열분해에 의한 무게감량이 양건강에 비해 약 2.7배나 높다. 그러므로 생강엑기스를 사용하여 제조되는 생강차 제조시 열풍건조($60^{\circ}C$, 30분)는 품질에 지대한 영향을 미친다는 것을 발견하였다. 8) 생강엑기스 제조는 건강 재배방법 저장기간과 방법, 건조방법이 건강특성을 좌우한다. 9) 본 연구에서 제시된 열분석(DSC와 TGA)방법을 도입한다면 신속하고 경제적으로 생강 엑기스 품질을 평가하는 데에 큰 기여가 있을 것으로 생각된다. 10) 양호한 생강차를 만들 수 있다고 선정된 엑기스는 수입 엑기스와 함께 양건강의 제품이다.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1237-1244
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• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1224-1229
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• 2012

Puerarin (P), an isoflavone derived from kudzu roots, has strong biological activities, but its bioavailability is often limited by its low water solubility. To increase its solubility, P was glucosylated by three dextransucrases from Leuconostoc or Streptococcus species. Leuconostoc lactis EG001 dextransucrase exhibited the highest productivity of puerarin glucosides (P-Gs) among the three tested enzymes, and it primarily produced two P-Gs with a 53% yield. Their structures were identified as ${\alpha}$-$_D$-glucosyl-($1{\rightarrow}6$)-P (P-G) by using LC-MS or $^1H$- or $^{13}C$-NMR spectroscopies and ${\alpha}$-$_D$-isomaltosyl-($1{\rightarrow}6$)-P (P-IG2) by using specific enzymatic hydrolysis, and their solubilities were 15- and 202-fold higher than that of P, respectively. P-G and P-IG2 are easily applicable in the food and pharmaceutical industries as alternative functional materials.

• Journal of Microbiology and Biotechnology
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• v.20 no.9
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• pp.1359-1366
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• 2010

A search of the Streptomyces avermitilis genome reveals that its closest homologs are several O-methyltransferases. Among them, one gene (viz., saomt5) was cloned into the pET-15b expression vector by polymerase chain reaction using sequence-specific oligonucleotide primers. Biochemical characterization with the recombinant protein showed that SaOMT5 was S-adenosyl-L-methionine-dependent Omethyltransferase. Several compounds were tested as substrates of SaOMT5. As a result, SaOMT5 catalyzed O-methylation of flavonoids such as 6,7-dihydroxyflavone, 2',3'-dihydroxyflavone, 3',4'-dihydroxyflavone, quercetin, and 7,8-dihydroxyflavone, and phenolic compounds such as caffeic acid and caffeoyl Co-A. These reaction products were analyzed by TLC, HPLC, LC/MS, and NMR spectroscopy. In addition, SaOMT5 could convert phenolic compounds containing ortho-dihydroxy groups into O-methylated compounds, and 6,7-dihydroxyflavone was known to be the best substrate. SaOMT5 converted 6,7-dihydroxyflavone into 6-hydroxy-7-methoxyflavone and 7-hydroxy-6-methoxyflavone, and caffeic acid into ferulic acid and isoferulic acid, respectively. Moreover, SaOMT5 turned out to be a $Mg^{2+}$-dependent OMT, and the effect of $Mg^{2+}$ ion on its activity was five times greater than those of $Ca^{2+}$, $Fe^{2+}$, and $Cu^{2+}$ ions, EDTA, and metal-free medium.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.403-409
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• 2010

Saccharomyces cerevisiae Hsp30 is a plasma membrane heat shock protein that is induced by various environmental stress conditions. However, the functional role of Hsp30 during diverse environmental stressors is not presently known. To gain insight into its function during thermal stress, we have constructed and characterized a ${\Delta}hsp30$ strain during heat stress. $BY4741{\Delta}hsp30$ cells were found to be more sensitive compared with BY4741 cells, when exposed to a lethal heat stress at $50^{\circ}C$. When budding yeast is exposed to either heat shock or weak organic acid, it inhibits Pma1p activity. In this study, we measured the levels of Pma1p in mutant and Wt cells both during optimal temperature and heat shock temperature. We observed that $BY4741{\Delta}hsp30$ cells showed constitutive reduction of Pma1p. To gain further insights into the role of Hsp30 during heat stress, we compared the total protein profile by 2D gel electrophoresis followed by identification of differentially expressed spots by LC-MS. We observed that contrary to that expected from thermal-stress-induced changes in gene expression, the ${\Delta}hsp30$ mutant maintained elevated levels of Pdc1p, Trx1p, and Nbp35p and reduced levels of Atp2p and Sod1p during heat shock. In conclusion, Hsp30 is necessary during lethal heat stress, for the maintenance of Pma1p and a set of thermal stress response functions.

• Journal of Acupuncture Research
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• v.34 no.4
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• pp.180-184
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• 2017

Background: Recently, Cervi Parvum Cornu pharmacopuncture has been widely used. But no studies on the indicator materials for Cervi Parvum Cornu pharmacopuncture have been conducted. The aim of this study was to select indicator materials that would aid in the uniform preparation of standardized Cervi Parvum Cornu pharmacopuncture. Methods: Three lots of Cervi Parvum Cornu pharmacopuncture were analysed. Each lot was prepared using the same methods and materials. Chondroitin sulfate, alanine, and leucine were selected as the indicator materials for Cervi Parvum Cornu. For standardization, chondroitin sulfate analysis was performed using the colorimetric method, while alanine and leucine were analyzed using liquid chromatography-mass spectrometry (LC-MS). Results: Analysis of the three lots of Cervi Parvum Cornu pharmacopuncture found chondroitin sulfate levels of $108.9{\pm}17.3ug/ml$, $118.8{\pm}5.0ug/ml$ and $112.3{\pm}11.9ug/ml$. Alanine levels were $44.9{\pm}2.8ug/ml$, $44.6{\pm}0.3ug/ml$, and $43.9{\pm}0.2ug/ml$. Leucine levels were $29.6{\pm}0.7ug/ml$, $29.0{\pm}0.1ug/ml$, and $29.4{\pm}0.1ug/ml$. Conclusion: These results suggest that chondroitin sulfate, alanine, and leucine may be useful for the standardization of Cervi Parvum Cornu pharmacopuncture.

• Journal of Ecology and Environment
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• v.44 no.2
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• pp.72-80
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• 2020

Background: Antibiotics are widely used to treat animals from infections. After fertilizing, antibacterials can remain in the soil while adversely affecting the soil microorganisms. The concentration of oxytetracycline (OTC) in the soil and its effect on the soil microbial community was assessed. To assess the impact of OTC on the soil microbial community, it was added to the soil at concentrations of 50, 150, and 300 mg kg^-1 and incubated for 35 days. Results: The concentration of OTC added to the soil decreased from 150 to 7.6 mg kg^-1 during 30 days of incubation, as revealed by LC-MS. The deviations from the control values in the level of substrate-induced respiration on the 5th day of the experiment were, on average, 26, 68, and 90%, with OTC concentrations at 50, 150, and 300 mg kg^-1, respectively. In samples with 150 and 300 mg kg^-1 of OTC, the number of bacteria from the 3rd to 14th day was 2-3 orders of magnitude lower than in the control. The addition of OTC did not affect the fungal counts in samples except on the 7th and 14th days for the 150 and 300 mg kg^-1 contaminated samples. Genes tet(M) and tet(X) were found in samples containing 50, 150, and 300 mg kg^-1 OTC, with no significant differences in the number of copies of tet(M) and tet(X) genes from the OTC concentration. Conclusions: Our results showed that even after a decrease in antibiotic availability, its influence on the soil microbial community remains.

• KSBB Journal
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• v.32 no.3
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• pp.193-198
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• 2017

Sialylation is important in producing therapeutic proteins such as antibody, cytokine and fusion protein. Thus, enhancement of sialylation is usually performed in CHO cell cultures. ${\alpha}2,6$-Sialyltransferase (ST), which plays a key role in the attachment of ${\alpha}2,6-sialic$ acid, is present in human cells but not in Chinese hamster ovary (CHO) cells. Overexpression of ${\alpha}2,6-ST$ can be used for enhancing the degree of sialylation and achieving human-like glycosylation. In this study, we constructed CHO cells producing human cytotoxic T-lymphocyte antigen4-immunoglobulin (hCTLA4-Ig) as well as ${\alpha}2,6-ST$. Transfected CHO cells were selected using G418 and stable cell line was established. Profiles of viable cell density and hCTLA4-Ig titer in an overexpressed cell line were similar to those of a wild-type cell line. It was confirmed that the total amount of sialic acid was increased and ${\alpha}2,6-sialic$ acid was attached to the terminal residues of N-glycan of hCTLA4-Ig by ESI-LC-MS. Compared to 100% of ${\alpha}2,3-sialic$ acid in wild type cells, 70.9% of total sialylated N-glycans were composed of ${\alpha}2,6-sialic$ acid in transfected cells. In conclusion, overexpression of ${\alpha}2,6-ST$ in CHO cells led to the increase of both the amount of total sialylated N-glycan and the content of ${\alpha}2,6-sialic$ acid, which is more resemble to human-like structure of glycosylation.