• Toxicological Research
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• 제32권2호
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• pp.115-121
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• 2016

This study was conducted to examine the cellular regulatory mechanisms of sulfasalazine (SSZ) in rabbit articular chondrocytes treated with sodium nitroprusside (SNP). Cell phenotype was determined, and the MTT assay, Western blot analysis and immunofluorescence staining of type II collagen was performed in control, SNP-treated and SNP plus SSZ ($50{\sim}200{\mu}g/mL$) rabbit articular chondrocytes. Cellular proliferation was decreased significantly in the SNP-treated group compared with that in the control (p < 0.01). SSZ treatment clearly increased the SNP-reduced proliferation levels in a concentration-dependent manner (p < 0.01). SNP treatment induced significant dedifferentiation and inflammation compared with control chondrocytes (p < 0.01). Type II collagen expression levels increased in a concentration-dependent manner in response to SSZ treatment but were unaltered in SNP-treated chondrocytes (p < 0.05 and < 0.01, respectively). Cylooxygenase-2 (COX-2) expression increased in a concentration-dependent manner in response to SSZ treatment but was unaltered in SNP-treated chondrocytes (p < 0.05). Immunofluorescence staining showed that SSZ treatment increased type II collagen expression compared with that in SNP-treated chondrocytes. Furthermore, phosphorylated extracellular regulated kinase (pERK) expression levels were decreased significantly in the SNP-treated group compared with those in control chondrocytes (p < 0.01). Expression levels of pERK increased in a concentration-dependent manner by SSZ but were unaltered in SNP-treated chondrocytes. pp38 kinase expression levels increased in a concentration-dependent manner by SSZ but were unaltered in control chondrocytes (p < 0.01). In summary, SSZ significantly inhibited nitric oxide-induced cell death and dedifferentiation, and regulated extracellular regulated kinases 1 and 2 and p38 kinase in rabbit articular chondrocytes.

• Asian Pacific Journal of Cancer Prevention
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• 제13권9호
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• pp.4807-4814
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• 2012

Purpose: The chemoresistance of human hepatocellular carcinoma (HCC) to cytotoxic drugs, especially intrinsic or acquired multidrug resistance (MDR), still remains a major challenge in the management of HCC. In the present study, possible mechanisms involved in MDR of HCC were identified using a 5-fluorouracil (5-FU)-resistant human HCC cell line. Methods: BEL-7402/5-FU cells were established through continuous culturing parental BEL-7402 cells, imitating the pattern of chemotherapy clinically. Growth curves and chemosensitivity to cytotoxic drugs were determined by MTT assay. Doubling times, colony formation and adherence rates were calculated after cell counting. Morphological alteration, karyotype morphology, and untrastructure were assessed under optical and electron microscopes. The distribution in the cell cycle and drug efflux pump activity were measured by flow cytometry. Furthermore, expression of potential genes involved in MDR of BEL-7402/5-FU cells were detected by immunocytochemistry. Results: Compared to its parental cells, BEL-7402/5-FU cells had a prolonged doubling time, a lower mitotic index, colony efficiency and adhesive ability, and a decreased drug efflux pump activity. The resistant cells tended to grow in clusters and apparent changes of ultrastructures occurred. BEL-7402/5-FU cells presented with an increased proportion in S and G2/M phases with a concomitant decrease in G0/G1 phase. The MDR phenotype of BEL-7402/5-FU might be partly attributed to increased drug efflux pump activity via multidrug resistance protein 1 (MRP1), overexpression of thymidylate synthase (TS), resistance to apoptosis by augmentation of the Bcl-xl/Bax ratio, and intracellular adhesion medicated by E-cadherin (E-cad). P-glycoprotein (P-gp) might play a limited role in the MDR of BEL-7402/5-FU. Conclusion: Increased activity or expression of MRP1, Bcl-xl, TS, and E-cad appear to be involved in the MDR mechanism of BEL-7402/5-FU.

• Journal of Plant Biotechnology
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• 제41권2호
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• pp.81-88
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• 2014

식물생명공학 연구에 있어서 모델 식물인 Arabidopsis thaliana (애기장대)와 아주 유사한 염생 식물인 salt cress (Thellungiella halophila 또는 Thellungiella parvula)는 고염 스트레스에 대하여 강한 내성을 가지고 있다. 본 연구에서는 고염에 저항성을 가지는 유용유전자를 선별하기 위하여, 200 mM NaCl을 처리한 T. halophila 또는 T. parvula 식물로부터 mRNA를 분리하여 cDNA library를 작성하였다. Full length cDNA library를 Agrobacteria-methods 형질전환 방법에 필요한 binary vector pool을 작성하고 Arabidopsis에 형질전환 시켰다. 형질전환되어진 Arabidopsis를 항생제 Basta 선별을 통하여 형질전환체 pool을 구축하였다(305,400 종자). 이와 같은 방법을 통해 구축 되어진 pool중에서 168,500 종자를 이용하여 고염 스트레스 조건하에서 종자 발아율과 뿌리 생장 촉진되는 현상이 나타나는 내염성 형질전환체를 1차 선별하였다(7,157 개체; 4.24%). 1차 선별된 형질전환체 중에서 1,551개체를 이용하여 2차 선별을 수행하여 내염성 형질전환체 165 개체를 확보하였다(10.6%). 선별된 형질전환체 중 대부분 개체는 고염 스트레스에 대응하여 종자 발아 및 뿌리 생장 모두 야생형보다 촉진된 표현형을 보였다. 그 중 몇 몇의 형질전환체는 야생형에 비하여 특이적인 기관 발달 현상이 나타났다. 예를 들면, 꽃과 줄기의 발달이 야생형과는 다른 표현형을 보이는 형질전환체가 선별되었다. 이렇게 스트레스에 내성을 가지는 형질전환체로부터 유전자 분리 방법을 통하여 해당 유용유전자를 확보할 수가 있다. 본 연구에서는 향후 halopyte 식물체를 이용하여 고염 뿐만 아니라 다양한 환경스트레스(건조, 냉해, 고열, 호르몬 등) 신호전달에 관여하는 유용유전자를 보다 쉽게 확보하는 방법을 제시함으로서, 환경재해극복에 관여하는 신호전달 기작을 보다 쉽게 이해할 수 있을 것으로 사료된다.

• 대한유전성대사질환학회지
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• 제4권1호
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• pp.5-12
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• 2004

Purpose Phenylketonuria is an inborn error of metabolism, which is inherited as an autosomal recessive trait. PKU is resulting from deficiency of phenylalanine hydroxylase. PAH gene spans about 90 kb on chromosome 12q and comprises 13 exons. In order to define the genetic basis of PKU and the frequencies and distribution of PAH mutations in the Korean population, we analyzed PAH gene in independent 80 patients with PKU. Methods All 13 exons including exon-intron boundaries and 2 kb of 5' upstream region of the PAH gene were analyzed by PCR-direct sequencing methods. Results PAH gene analysis revealed 39 different mutations including 10 novel mutations. The novel mutations consisted of 9 missense mutations (P69S, G103S, N207D, T278S, P281A, L293M, G332V, S391I and A447P) and a novel splice site variant (IVS10-3C>G). R243Q, IVS4-1G>A, and E6-96A>G were the most relevant mutations and they accounted in the whole for 38% of the mutant alleles identified in this study. We also observed that. $BH_4$ responsibility was. associated with genotype of R241C, R53H and R408Q. Conc1ustion Our present study with 80 participants extends the previous results to more comprehensive understanding of PAH allele distribution and frequency in Koreans. Although Korean mutation profile of PAH is similar to those of the nearest oriental populations (Japanese, Chinese, and Taiwanese), several different characteristic features are revealed. The characterization of the genotype-phenotype relationship was also performed. Our data would be very useful information for diagnosis, genetic counseling and planning of dietary and therapeutic strategies in Korean PAH patients.

• IMMUNE NETWORK
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• 제10권1호
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• pp.15-25
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• 2010

Background: Rat mast cells were regarded as a good model for mast cell function in immune response. Methods: Rat bone marrow mast cells (BMMC) were prepared both by recombinant rat IL-3 (rrIL-3) and by recombinant mouse stem cell factor (rmSCF), and investigated for both proliferation and differentiation in time course. Rat BMMC was induced by culture of rat bone marrow cells (BMCs) in the presence of both rrIL-3 (5 ng/ml) and rmSCF (5 ng/ml). Culture media were changed 2 times per week with the cell number condition of $5{\times}10^4/ml$ in 6 well plate. Proliferation was analyzed by cell number and cell counting kit-8 (CCK-8) and differentiation was by rat mast cell protease (RMCP) II and histamine. Results: Cell proliferation rates reached a maximum at 8 or 11 days of culture and decreased thereafter. However, both RMCP II production and histamine synthesis peaked after 11 days of culture. By real time RT-PCR, the level of histidine decarboxylase mRNA was more than 500 times higher on culture day 11 than on culture day 5. By transmission electron microscopy, the cells were heterogeneous in size and contained cytoplasmic granules. Using gated flow cytometry, we showed that cultured BMCs expressed high levels of $Fc{\varepsilon}RI$ and the mast cell antigen, ganglioside, on culture day 11. Conclusion: These results indicate that rat BMMCs were generated by culturing BMCs in the presence of rrII-3 and rmSCF and that the BMMCs have the characteristics of mucosal mast cells.

• Fisheries and Aquatic Sciences
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• 제26권1호
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• pp.48-57
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• 2023

One of the ways to increase the production for aquaculture is through the cultivation of monosexuals by ensuring genital reversal from which energy for reproduction is diverted towards growth. Masculinization has been identified as one of the most prominent techniques, where sex development was directed from female to male. This approach only altered the phenotype and not the genotype. The red claw crayfish (Cherax quadricarinatus) was a relatively new commercial commodity, and the males were known to grow faster than females. Hence, it was proposed to use monocultures comprising an all-male population to increase yield using steroid hormone, synthetic 17α-methyltestosterone. However, this technique generated residues that detrimentally affect human health, the environment, and cultivated organisms. Therefore, finding new safe natural steroid sources was essential, and one of which is exploring of natural hormones extracted from the viscera of sea cucumbers (Holothuria scabra Jaeger). This study focused on the determination of male formation and testosterone levels among juvenile crayfish, after immersing in sea cucumber steroid extract (SCSE). A completely random design with factorial was used with two variables, encompassing the varied doses (0, 2, 4 mg/L, 2 mg/L 17α-methyl testosterone as control group) and immersion times of 18 and 30 h. The result showed the dose-dependent ability of SCSE increase the male genital formation and promote the testosterone level of juvenile crayfish. In addition, the testosterone was influenced by dose and immersion duration time, with the highest level of testosterone observed in treatments of 4 mg/L SCSE with 30 h immersion was 0.248 ng/mL, while the male percentage was 77%. In conclusion, the combination of dose and immersion time significantly affected growth and testosterone levels.

• Korean Journal of Radiology
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• 제22권6호
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• pp.890-900
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• 2021

Objective: To identify the association between morphological and functional characteristics of the bicuspid aortic valve (BAV) and bicuspid aortopathy and to identify the determinants of aortic dilatation using transthoracic echocardiography (TTE) and cardiac computed tomography (CCT). Materials and Methods: This study included 312 subjects (mean [SD] age, 52.7 [14.3] years; 227 males [72.8%]) who underwent TTE and CCT. The BAVs were classified by anterior-posterior (BAV-AP) or right-left (BAV-RL) orientation of the cusps and divided according to the presence (raphe+) or absence of a raphe (raphe-) based on the CCT and intraoperative findings. The dimensions of the sinus of Valsalva and the proximal ascending aorta were measured by CCT. We assessed the determinants of aortic root and proximal ascending aortic dilatation (size index > 2.1 cm/m²) by Univariable and multivariable logistic regression analyses. Results: Of the 312 patients, BAV-AP was present in 188 patients (60.3%), and 185 patients (59.3%) were raphe+. Moderate-to-severe aortic stenosis (AS) was the most common hemodynamic abnormality (54.8%). The most common type of aortopathy was the combined dilated root and mid-ascending aortic phenotype (62.5%). On multivariable analysis, age and AS severity were significantly associated with aortic root dilatation (p < 0.05), and age, sex, and AS severity were significantly associated with ascending aortic dilatation (p < 0.05). However, the orientation of the cusps, presence of a raphe, and severity of aortic regurgitation were not associated with aortic root and ascending aortic dilatation. Conclusion: BAV morphological characteristics were not determinants of aortic dilatation. Age, sex, and AS severity were predictors of bicuspid aortopathy. Therefore, age, sex, and AS severity, rather than valve morphology, need to be considered when planning treatment for BAV patients.

• Asian-Australasian Journal of Animal Sciences
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• 제17권5호
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• pp.598-602
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• 2004

The Tongcheng pig breed is a famous Chinese indigenous breed. The Ministry of Agriculture of China has filed it as 1 of 19 national key conservation breeds selected from more than 100 Chinese indigenous pig breeds in 2000. In order to improve the reproductive performance, it has been intensively selected to increase the litter size for about 10 years. The population randomly sampled from conservation nucleus of eight families in the Tongcheng pigs was genotyped for identification of their estrogen receptor locus polymorphisms with the PCR-RFLPs method. Only AB heterozygotes and BB homozygotes were detected, and $X^2$ test demonstrated that the locus was in disequilibrium at a significant level (p<0.05). In the present paper, the litter sizes in different parities were regarded as different traits. Holistic status of other unspecific and unidentified genes was estimated by using the statistical methods. Coefficients of kurtosis and skewness showed that the litter size still presented segregating characteristic in the 2nd, 5th, 7th, 8th and 9th parities. Analysis of homogeneity of variance between families confirmed the results for the 5th, 7th and 8th parities. The heritability of litter size for the 1st to 10th parities was estimated with paternal half-sib model and individual estimated breeding values (EBVs) were evaluated by a single trait animal model as well. We found that the averages of EBVs for litter size in each parity did not differ significantly between genotypes, despite the significant difference for original phenotype records in the 3rd, 4th and 5th parities (p<0.05 or p<0.01). The results may be explained by the deduction that the polymorphisms of ESR locus are no longer the important genetic base of litter size variation when the frequency of allele B accumulated in the experience of selection procedure, and further conferring that there exist special genes associated with litter size in the recent Tongcheng pigs population can be made.

• 대한한방내과학회지
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• 제28권3호
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• pp.453-463
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• 2007

Backgrounds : Glutathione-s-transferase (GST) is a kind of phase II metabolism enzyme and plays an important role in the detoxification of various toxic chemicals. It was reported that the genetic polymorphism of GSTM1 and GSTT1 genes may be responsible for asthma development and susceptibility to allergy. Traditional oriental medicine uses a unique diagnostic technique. differentiation-syndrome. to analyze signs and symptoms of patients synthetically. Through differentiation-syndrome. asthma patients can be divided into two groups: the deficiency syndrome group (DSG) and the excess syndrome group (ESG). Objectives : The purpose of this study was to investigate the possible association of GST gene polymorphism with clinical phenotype by differentiation-syndrome of bronchial asthma patients. Materials and Methods : One hundred and ten participants were evaluated by pulmonary function test. Patients with 53 DSG and 31 ESG by differentiation-syndrome were assessed for genetic analysis. GSTM1 and GSTT1 deletion polymorphism was performed by polymerase chain reaction (PCR). Results : GSTM1 gene deletion was detected in 43.4% of individuals in the DSG and in 38.71 % in the ESG. The distribution of GSTM1 polymorphism between DSG and ESG was not significantly different [$x^2$=0.1767, p=0.6742; OR(95% CI)=1.2139(0.4915-2.9979)]. The proportion of GSTT1 null genotypes was 41.51% in the DGS and 45.16% in the ESG. The distribution of GSTT1 polymorphism between DSG and ESG was also not significantly different [$x^2$=0.1065, p=0.7442; OR(95% CI)=0.8618(0.3525-2.1065)]. In the combined analysis of GSTM1 and GSTT1 genes, the frequency of both null type of GSTM1/GSTT1 genes was not significantly different from both positive type of GSTM1/GSTT1 genes[$x^2$=0.0768, p=0.7817; OR(95% CI)=1.2000(0.3303-4.3602)] Conclusions : These results indicate that polymorphism of the GST gene might not be associated with the symptomatic classification of DSG and ESG by differentiation-syndrome in Korean asthmatics.

• Asian-Australasian Journal of Animal Sciences
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• 제25권7호
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• pp.1021-1028
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• 2012

Peripheral blood mononuclear cells (PBMCs) discriminate microbial pathogens and induce T-cell responses of appropriate effector phenotype accordingly. Toll-like receptors (TLRs), in part, mediate this microbial recognition and differentiation while the development of T-cell effector functions critically depends on the release of Th1- or Th2- type cytokines. In the present study, buffalo PBMCs were stimulated under in vitro culture conditions by Bacillus subtilis cell wall petidoglycan, a TLR2 ligand, in a dose- and time- dependent manner. The expression of TLR2 as well as the subsequent differential induction of the Th1 and Th2 type cytokines was measured. Stimulation was analyzed across five doses of peptidoglycan ($10{\mu}g/ml$, $20{\mu}g/ml$, $30{\mu}g/ml$, $40{\mu}g/ml$ and $50{\mu}g/ml$) for 3 h, 12 h, 24 h and 36 h incubation periods. We observed the induction of TLR2 expression in a dose- and time-dependent manner and the peptidoglycan induced tolerance beyond $30{\mu}g/ml$ dose at all incubation periods. The correlation between peptidoglycan stimulation and TLR2 induction was found positive at all doses and for all incubation periods. Increased production of all the cytokines was observed at low doses for 3 h incubation, but the expression of IL-4 was relatively higher than IL-12 at the higher antigen doses, indicating tailoring towards Th2 response. At 12 h incubation, there was a pronounced decrease in IL-4 and IL-10 expression relative to IL-12 in a dose- dependent manner, indicating skewing to Th1 polarization. The expression of IL-12 was highest for all doses across all the incubation intervals at 24 h incubation, indicating Th1 polarization. The relative expression of TNF-${\alpha}$ and IFN-${\gamma}$ was also higher while that of IL-4 and IL-10 showed a decrease. For 36 h incubation, at low doses, relative increase in the expression of IL-4 and IL-10 was observed which decreased at higher doses, as did the expression of all other cytokines. The exhaustion of cytokine production at 36 h indicated that PBMCs became refractory to further stimulation. It can be concluded from this study that the cytokine response to sPGN initially was of Th2 type which skews, more pronouncedly, to Th1 type with time till the cells become refractory to further stimulation.