• Asian Pacific Journal of Cancer Prevention
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• v.16 no.16
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• pp.7179-7188
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• 2015

Cancer is a major health obstacle around the world, with hepatocellular carcinoma (HCC) and colorectal cancer (CRC) as major causes of morbidity and mortality. Nowadays, there isgrowing interest in the therapeutic use of natural products for HCC and CRC, owing to the anticancer activity of their bioactive constituents. Boswellia serrata oleo gum resin has long been used in Ayurvedic and traditional Chinese medicine to alleviate a variety of health problems such as inflammatory and arthritic diseases. The current study aimed to identify and explore the in vitro anticancer effect of B. Serrata bioactive constituents on HepG2 and HCT 116 cell lines. Phytochemical analysis of volatile oils of B. Serrata oleo gum resin was carried out using gas chromatography-mass spectrometry (GC/MS). Oleo-gum-resin of B. Serrata was then successively extracted with petroleum ether (extract 1) and methanol (extract 2). Gas-liquid chromatography (GLC) analysis of the lipoidal matter was also performed. In addition, a methanol extract of B. Serrata oleo gum resin was phytochemically studied using column chromatography (CC) and thin layer chromatography (TLC) to obtain four fractions (I, II, III and IV). Sephadex columns were used to isolate ${\beta}$-boswellic acid and identification of the pure compound was done using UV, mass spectra, $^1H$ NMR and $^{13}C$ NMR analysis. Total extracts, fractions and volatile oils of B. Serrata oleo-gum resin were subsequently applied to HCC cells (HepG2 cell line) and CRC cells (HCT 116 cell line) to assess their cytotoxic effects. GLC analysis of the lipoidal matter resulted in identification of tricosane (75.32%) as a major compound with the presence of cholesterol, stigmasterol and ${\beta}$-sitosterol. Twenty two fatty acids were identified of which saturated fatty acids represented 25.6% and unsaturated fatty acids 74.4% of the total saponifiable fraction. GC/MS analysis of three chromatographic fractions (I,II and III) of B. Serrata oleo gum resin revealed the presence of pent-2-ene-1,4-dione, 2-methyl- levulinic acid methyl ester, 3,5- dimethyl- 1-hexane, methyl-1-methylpentadecanoate, 1,1- dimethoxy cyclohexane, 1-methoxy-4-(1-propenyl)benzene and 17a-hydroxy-17a-cyano, preg-4-en-3-one. GC/MS analysis of volatile oils of B. Serrata oleo gum resin revealed the presence of sabinene (19.11%), terpinen-4-ol (14.64%) and terpinyl acetate (13.01%) as major constituents. The anti-cancer effect of two extracts (1 and 2) and four fractions (I, II, III and IV) as well as volatile oils of B. Serrata oleo gum resin on HepG2 and HCT 116 cell lines was investigated using SRB assay. Regarding HepG2 cell line, extracts 1 and 2 elicited the most pronounced cytotoxic activity with $IC_{50}$ values equal 1.58 and $5.82{\mu}g/mL$ at 48 h, respectively which were comparable to doxorubicin with an $IC_{50}$ equal $4.68{\mu}g/mL$ at 48 h. With respect to HCT 116 cells, extracts 1 and 2 exhibited the most obvious cytotoxic effect; with $IC_{50}$ values equal 0.12 and $6.59{\mu}g/mL$ at 48 h, respectively which were comparable to 5-fluorouracil with an $IC_{50}$ equal $3.43{\mu}g/mL$ at 48 h. In conclusion, total extracts, fractions and volatile oils of B. Serrata oleo gum resin proved their usefulness as cytotoxic mediators against HepG2 and HCT 116 cell lines with different potentiality (extracts > fractions > volatile oil). In the two studied cell lines the cytotoxic acivity of each of extract 1 and 2 was comparable to doxorubicin and 5-fluorouracil, respectively. Extensive in vivo research is warranted to explore the precise molecular mechanisms of these bioactive natural products in cytotoxicity against HCC and CRC cells.

• Journal of agricultural medicine and community health
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• v.13 no.1
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• pp.41-59
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• 1988

In many of the helminthic infections, diagnosis is accomplished by the demonstration of the eggs or, occasionally the adult worms or their parts. Diagnosis can be made by the identification of larval stage obtained from stool or surgically extracted materials too. However some kinds of parasitic disease can not be diagnosed by above mentioned procedure alone. Brain cysticercosis, ectopic paragonimiasis, Capillaria hepatica infection in liver is a good example. In such a case, immunologic method would be helpful for the decision of physician. In this paper, immunologic tools such as indirect hemagglutination test, indirect fluorescent antibody test, circumoval precipitation test, ELISA, western blot were applied for the diagnosis of Clonorchis sinenisis, Cysticercosis and C. hepatica a infection and their efficacy was evaluated. The results obtained were as follows ; 1) In the diagnosis of clonorchiasis, ELISA revealed sensitiveity of 83.3%, but cross reaction against antibody of Paragonimus westermani and Taenia species were observed. For the identification of cross reaction and species specific band of Ag-Ab reaction, western blot was applied. 59Kd relative molecular weight and 21Kd band were identified as a Clonorchis sinensis specific band. OD values of ELISA performed with sera of 18 months after praziquantel treatment decreased to half level compared to that of before treatment. Negative conversion rate of ELISA after 18 months of treatment was 60%. 2) In the diagnosis of cysticercosis, IFAT disclosed 95.8%(23/24) of sensitivity and reaction was most strongly occurred in inner membrane. ELISA revealed 90.0% (36/40) of sensitivity, but cross reaction was observed in both technique. In western blot, 91, 63 and 21Kd Mw bands were identified as a strongly positive band. Among them 63Kd band showed positive reaction against almost all sera of cysticercosis patient. 3) Circumoval precipitation, ELISA, IFAT, showed 85.0% of sensitivity in the diagnosis of C. hepatica infection in rat. The antigenic localities were inner membrane of sectioned egg antigen on the prectipitates around the mucoid plugs which were induced by circumoval precipitation reaction. Sera from rats infected with 2000eggs were collected periodically to observe the changing patterns of antibody titers by IFAT and ELISA, which showed that high titers were detected at weeks 3 and 5, then gradually declined through weeks 9until to negatively converted at weeks 13.

• Childhood Kidney Diseases
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• v.3 no.2
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• pp.209-216
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• 1999

Purpose : Nephrogenic diabetes insipidus (NDI) is a rare X-linked disorder associated with renal tubule resistance to arginine vasopressin (AVP). The hypothesis that the defect underlying NDI might be a dysfunctional renal AVPR2 has recently been proven by the identification of mutations in the AVPR2 gene in NDT patients. To investigate the association of mutations in th AVPR2 gene with NDI, we analyzed the AVPR2 gene located on the X chromosome. Methods : We have analyzed the AVPR2 gene in a kindred with X-linked NDI. The proband and proband's mother were analyzed by polymerase chain reaction-single strand conformational polymorphism(PCR-SSCP) and DNA sequencing of the AVPR2 gene. We also have used restriction enzyme analysis of genomic PCR product to evaluate the AVPR2 gene. Results : C to T transition at codon 202, predictive of an exchange of tryptophan 202 by cysteine(R202C) in the third extracellular domain was identified. This mutation causes a loss of Hae III site within the gene. Conclusion : We found a R202C missense mutation in the AVPR2 gene causing X-linked NDI, and now direct mutational analysis is available for carrier screening and early diagnosis.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.1
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• pp.240-246
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• 2020

This study was conducted to develop specific Single Nucleotide Polymorphism (SNP) markers to identify the genetic characteristics and breed of White Hanwoo (WH) using a molecular biological method. SNP genotyping was performed with an Illumina Bovine HD 777K SNP chip using DNA extracted from 48 Hanwoo and 22 WH. The minor allele frequency (MAF) difference of each SNP was calculated and the statistical significance (P-value) of the MAF difference was calculated through Fisher's Exact test (Genotype). SNPs with 100% difference in the MAF difference were selected based on marker selection criteria. The nine SNP markers with genetic differences were selected. The selected markers have different alleles as being Hanwoo- and WH- specific. Therefore, based on these results, it can be concluded that the Hanwoo and WH varieties can be clearly distinguished by using these SNPs. So, the patent of the WH breed identification markers was registered. WH is a breed that shows the characteristics of a Korean native species that is separate from the native Hanwoo. It is expected that genetic characteristics research on the WH can be used to identify the breed and as a knowledge base for enhancing the value of breeding stock.

• Horticultural Science & Technology
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• v.30 no.4
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• pp.463-470
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• 2012

As the advanced seeding technology through use of plug tray for good cultivation of seeds was propagated along with the expansion and development of horticultural industry, the use of bed soils as growing medium has recently been increased. In this study, the effects of the four clay minerals such as illite, phyllite, zeolite, and bentonite on the early growth of red pepper in the bed soil were investigated. Furthermore, proteome analysis for the leaf and stem samples of red pepper treated with only illite was performed. Of the seedling cultured, the healthy and regular size seeds were selected and cultivated in the pots, after they were treated with four clay minerals. The experiment was performed during the whole six weeks in the glasshouse of the Chungbuk National University. The growth lengths, fresh and dry weights of red pepper were significantly higher in the treatments of illite, phyllite, zeolite, and bentonite than in the control. In addition, the uptake of $K^+$, $Ca^{2+}$, and $Mg^{2+}$ were higher in the treatment of illite, phyllite, zeolite, and bentonite than in the control. The 2-DE patterns for the red pepper by the applications of illite, phyllite, zeolite, and bentonite were similar to each other. Therefore, compared to the samples of control, the proteome analysis for the samples of red pepper treated by only illite were performed. Proteome analysis for red pepper showed that plastid fructose-1, 6-bisphosphate aldolase class 1, aldolase, and glyceraldehydes 3-phosphate dehydrogenase, all of which were involved in the energy metabolism, were highly expressed in leaf tissue by illite treatment. In stem tissue, NAD-dependent formate dehydrogenase involved in energy metabolism, potassium transport protein, and GIA/RGA-like gibberellins response modulator were highly expressed. Based on the results obtained from the proteome analysis, it appears that the proteins specifically and differentially expressed on the illite treatment may be involved in the enhanced growth of red pepper. The identification of some proteins involved in the response of vegetable crops to the treatment of clay mineral can provide new insights that can lead to a better elucidation and understanding of mechanism on their molecular basis.

• Korean Journal of Microbiology
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• v.52 no.2
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• pp.192-201
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• 2016

Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84-90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at $40^{\circ}C$ and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at $10^{\circ}C$ and $20^{\circ}C$, respectively, compared to its maximal activity at $40^{\circ}C$.

• Nuclear Medicine and Molecular Imaging
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• v.41 no.6
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• pp.546-552
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• 2007

Purpose: In the breast cancer patient, lymphatic mapping and sentinel lymph node biopsy are the most important procedure for axillary lymph node staging. We aimed to compare the three radiocolloids [$^{99m}Tc$-antimony trisulfide colloid (ASC), $^{99m}Tc$-tin colloid (TC), and $^{99m}Tc$-human serum albumin (HSA)] for sentinel lymph node mapping. Subjects and Methods: Totally, 397 patients with clinically N0 stage were enrolled. $^{99m}Tc$-ASC was injected in 202 out of 397 patients, $^{99m}Tc$-TC was injected in 120 patients, and $^{99m}Tc$-HSA was injected in the remaining 75 patients. The sentinel lymph nodes were localized by lymphoscintigraphy and selected using intraoperative gamma probe. All sentinel lymph nodes were investigated by intraoperative pathologic consultation. The axillary lymph nodes which were harvested by the lymph node dissection were also investigated. Results: The patients of each group showed similar clinical characteristics. There were no significant differences (p>0.05) in the identification rate of sentinel lymph nodes (IR), false negative rate (FNR), and negative predictive value (NPV). The axillary lymphadenectomy revealed axillary lymph node metastases in those three groups (ASC-33.2%, TC-31.7%, HSA-22.7%). The IR, FNR, and NPV were not significantly different among those groups. Conclusion: Those three $^{99m}Tc$-labeled radiocolloids showed equivalent results in sentinel lymph node mapping of breast cancer.

• The Korean Journal of Mycology
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• v.26 no.3 s.86
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• pp.373-379
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• 1998

Rhizoctonia solani [Thanatephorus cucumeris (frank) Donk], one of the major soil-borne plant pathogens with world-wide distribution, can cause great damages on various crops. In Korea, sheath blight on rice caused by this pathogen is the major concern, and active studies on this pathogen have been performed. However, most of these studies were concerned with pathogenicity of the isolates instead of molecular analyses of different AGs of R. solani. Therefore, in this study, thirty isolates of Rhizoctonia solani collected from various sources were used for the analyses of genetic relationships among themselves and for the rapid anastomosis grouping with RAPD method. As a result, thirty isolates of known and unknown AGs were grouped into five subgroups and each group included AG-1, AG-2, AG-3, AG-4, and AG-5. RS-1 isolate was found to be closely related to AG-5. Isolates RS-4, RS-14, RS-17, and RS-16 were found to be closely related to AG-2-2(III B). Isolate RS-13 was closely related to AG-4, isolates RS-8 and RS-10 were closely related to AG-1(I B), and isolates RS-7 and RS-21 were closely related to AG-2-2(IV). Isolate RS-19 was closely related to AG-1(I C), and isolates RS-3, RS-5, RS-18, RS-6, and RS-15 were found to be closely related to AG-1.

• 한국균학회소식:학술대회논문집
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• 2015.05a
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• pp.53-53
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• 2015

Agrobacterium tumefaciens-mediated transformation(ATMT) of Flammulina velutipes was used to produce a diverse number of transformants to discover the functions of gene that is vital for its variation color, spore pattern and cellulolytic activity. Futhermore, the transformant pool will be used as a good genetic resource for studying gene functions. Agrobacterium-mediated transformation was conducted in order to generate intentional mutants of F. velutipes strain KACC42777. Then Agrobacterium tumefaciens AGL-1 harboring pBGgHg was transformed into F. velutipes. This method is use to determine the functional gene of F. velutipes. Inverse PCR was used to insert T-DNA into the tagged chromosomal DNA segments and conducting sequence analysis of the F. velutipes. But this experiment had trouble in diverse morphological mutants because of dikaryotic nature of mushroom. It needed to make monokaryotic fruiting varients which introduced genes of compatible mating types. In this study, next generation sequencing data was generated from 28 strains of Flammulina velutipes with different phenotypes using Illumina Hiseq platform. Filtered short reads were initially aligned to the reference genome (KACC42780) to construct a SNP matrix. And then we built a phylogenetic tree based on the validated SNPs. The inferred tree represented that white- and brown- fruitbody forming strains were generally separated although three brown strains, 4103, 4028, and 4195, were grouped with white ones. This topological relationship was consistently reappeared even when we used randomly selected SNPs. Group I containing 4062, 4148, and 4195 strains and group II containing 4188, 4190, and 4194 strains formed early-divergent lineages with robust nodal supports, suggesting that they are independent groups from the members in main clades. To elucidate the distinction between white-fruitbody forming strains isolated from Korea and Japan, phylogenetic analysis was performed using their SNP data with group I members as outgroup. However, no significant genetic variation was noticed in this study. A total of 28 strains of Flammulina velutipes were analyzed to identify the genomic regions responsible for producing white-fruiting body. NGS data was yielded by using Illumina Hiseq platform. Short reads were filtered by quality score and read length were mapped on the reference genome (KACC42780). Between the white- and brown fruitbody forming strains. There is a high possibility that SNPs can be detected among the white strains as homozygous because white phenotype is recessive in F. velutipes. Thus, we constructed SNP matrix within 8 white strains. SNPs discovered between mono3 and mono19, the parental monokaryotic strains of 4210 strain (white), were excluded from the candidate. If the genotypes of SNPs detected between white and brown strains were identical with those in mono3 and mono19 strains, they were included in candidate as a priority. As a result, if more than 5 candidates SNPs were localized in single gene, we regarded as they are possibly related to the white color. In F. velutipes genome, chr01, chr04, chr07,chr11 regions were identified to be associated with white fruitbody forming. White and Brown Fruitbody strains can be used as an identification marker for F. veluipes. We can develop some molecular markers to identify colored strains and discriminate national white varieties against Japanese ones.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.3
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• pp.189-198
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• 2006

Objective: We previously described that Diva is highly expressed in matured metaphase II (MII) oocytes compared to immature germinal vesicle (GV) oocytes in mouse. We report here that the expression of Diva transcript as well as protein is oocyte-specific. To elucidate its physiological role in oocyte, the binding partner(s) of Diva has been identified by using immunoprecipitation (IP) followed by Mass Spectrometry. Methods: NIH/3T3 cells were transiently transfected for 24 h with either empty vector for control or FLAG-tagged mouse Diva construct, and IP was performed with anti-FLAG antibody. The immuno-isolated complexes were resolved by SDS-PAGE on a 12% gel followed by Coomassie Blue staining. For in-gel digestion, 15 bands of interest were excised manually and digested with trypsin. All mass spectra were acquired at a positive reflector mode by a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Proteins were identified by searching the NCBI nonredundant database using MASCOT Peptide Mass Fingerprint software (Matrixscience, London). Results: Diva-associated complexes were formed in FLAG-tagged mouse Diva-overexpressed NIH/3T3 cells via IP using anti-FLAG-conjugated beads. Among the excised 15 bands, actin and actin-binding proteins such as tropomyosin, tropomodulin 3, and ${\alpha}$-actinin were identified. Binding between Diva and actin or tropomyosin was confirmed by IP followed by Western blot analysis. Both bindings were also detected endogenously in mouse ovaries, indicating that Diva works with actin and tropomyosin. Conclusions: This is the first report that immuno-isolated Diva-associated complexes are related to actin filament of the cytoskeletal system. When we consider the association of Diva with actin and tropomyosin, oocyte-specific Diva may play a role in modulating the cytoskeletal system during oocyte maturation.