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Establishing and validating an HPLC protocol for pralsetinib impurities analysis, coupled with HPLC-MS/MS identification of stress degradation products

  • Rajesh Varma Bhupatiraju;Pavani Peddi;Venkata Swamy Tangeti;Battula Sreenivasa Rao
    • Analytical Science and Technology
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    • v.37 no.5
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    • pp.280-294
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    • 2024
  • This study introduces a novel analytical method for the assessment of pralsetinib impurities and degradation products (DPs), addressing critical gaps in existing methodologies. This research aims to develop a robust HPLC method for impurity analysis, characterize degradation products using LC-MS, and evaluate the environmental impact of the method. The study began by optimizing HPLC conditions with various columns and buffers, ultimately achieving successful separation using an XBridge® RP-C18 column with ethanol as solvent A and 50 mM formic acid at pH 2.9. This setup provided excellent peak resolution and symmetry, essential for reliable stability studies. The developed HPLC method was then adapted for HPLC-MS/MS, enhancing sensitivity and detection efficiency of DPs. Stress degradation studies of pralsetinib under different conditions (acidic, basic, oxidative, thermal, and photolytic) revealed significant degradation under acidic (29.3 %) and basic (21.5 %) conditions, with several DPs identified. Oxidative stress resulted in 19.8 % degradation, while thermal and photolytic conditions caused minimal degradation. HPLC-MS/MS analysis identified structures of five degradation products, providing detailed insights into pralsetinib's stability and degradation pathways. Method validation followed ICH guidelines Q2(R1), confirming method's specificity, selectivity, sensitivity, linearity, accuracy, precision, and robustness. The method exhibited strong linearity with a coefficient of determination (r2) greater than 0.999 for pralsetinib and its impurities. This method advances impurity detection and DPs characterization, ensuring the quality and safety of pralsetinib. Additionally, method's environmental impact was assessed, aligning with sustainable analytical practices. These findings provide essential data on pralsetinib's stability, guiding storage conditions and ensuring its efficacy and safety in pharmaceutical applications.

Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 2015.05a
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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Mechanical Anisotropy of Pocheon Granite under Uniaxial Compression (일축압축하에서 포천화강암의 역학적 이방성)

  • Park Deok-Won
    • The Journal of Engineering Geology
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    • v.15 no.3
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    • pp.337-348
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    • 2005
  • Jurassic granite from Pocheon area were tested to investigate the effect of microcracks on mechanical properties of the granite. Three oriented core specimens were used for uniaxial compressive tests and each core specimen are perpendicular to the axes'R'(rift plane),'c'(grain plane) and'H'(hardway plane), respectively Among vacious elastic constants, the variation of Poisson's ratio as function of the directions was examined. From the related chart between ratio of failure strength and Poisson's ratio, H-specimen shows the highest range in Poisson's ratio and Poisson's ratio decreases in the order of C-specimen and R-specimen. The curve pattern is nearly linear in stage $I\simIII$ but the slope increases abruptly in stage H-3. As shown in the related chart, diverging point of a curve is formed when ratio of failure strength is $0.92\sim0.96$ Stage IV -3 is out of elastic region. The behaviour of rock in the four fracturing stages was analyzed in term of the stress-volumetric strain me. From the stress increment-volumetric strain equations governing the behaviour of rock, characteristic material constants, a, n, Q, m and $\varepsilon_v^{mcf}$, were determined. Among these, inherent microcrack porosity$(a, 10^{-3})$ and compaction exponent(n) in the microcrack closure region(stage I ) show an order of $a^R(3.82)>a^G(3.38)>a^H(2.32)\;and\;n^R(3.69)>n^G(2.79)>n^H(1.99)4, respectively. Especially, critical volumetric microcrack strain($\varepsilon_v^{mcf}$) in the stage W is highest in the H-specimen, normal to the hardway plane. These results indicate a strong correlation between two major sets of microcracks and mechanical properties such as Poisson's ratio and material constants. Correlation of strength anisotropy with microcrack orientation can have important application in rock fracture studies.

Operation of High Performance Elutriation-Type Sludge Fermenter and Feasibility for Its Application (고성능 세정식 슬러지 산발효조의 운전 및 적용성 평가)

  • Ahn, Young-Ho;Speece, R.E.
    • Journal of Korean Society of Environmental Engineers
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    • v.27 no.1
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    • pp.85-92
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    • 2005
  • The performance of a novel fermentation process, adopting a sludge blanket type configuration for higher hydrolysis/acidogenesis of the municipal primary sludge, was investigated under batch and semi-continuous conditions with various pH and temperature conditions. This acid elutriation slurry reactor provided higher system performance with a short HRT (5 days) and higher acidogenic effluent quality under pH 9 and thermophilic ($55^{\circ}C$) conditions. The hydrolysis of the sludge was revealed to be significantly dependent on seasonal effects for sludge characteristics but with little impact on acidogenesis. Based on the rainy season at the optimum conditions, VFA production and recovery fraction ($VFA_{COD}/COD$) were $0.18\;g\;VFA_{COD}\;g^{-1}\;VSS_{COD}$ and 63%. As byproducts, nitrogen and phosphorus releasing were $0.006\;g\;N\;g^{-1}\;VSS_{COD}$ and $0.003\;g\;P\;g^{-1}\;VSS_{COD}$, respectively. For the mass balance in a full-scale plant($Q=158,880\;m^3\;day^{-1}$) based on the rainy season, the VFA and non-VFA(as COD) production were $3,110\;kg\;VFA_{COD}\;day^{-1}$ and $1,800\;kg\;COD\;day^{-1}$, resulting in an increase of organics of $31\;mg\;COD\;L^{-1}$ and $20\;mg\;VFA_{COD}\;L^{-1}$ and nutrients of $0.7\;mg\;N\;L^{-1}$ and $0.3\;mg\;P\;L^{-1}$ in the influent sewage. The economical benefit from this process application was estimated to be about $67 per $1,000m^3$ of sewage except for energy requirements and also, better benefits can be expected during the dry season. Also, the results revealed that the process has various additional advantages such as pathogen-free stabilized solids production, excellent solids control and economical benefits.

Modification of Vermiculite for Use as a Floating Adsorbent for Copper Removal (수중의 구리 제거를 위한 질석흡착제의 표면개질 연구)

  • Lee, Sangmin;Lee, Taeyoon
    • Journal of the Korean GEO-environmental Society
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    • v.17 no.11
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    • pp.5-14
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    • 2016
  • The main objective of this study was to evaluate the removal properties of Cu from existing exfoliated vermiculite (EV) coated with a mixed solution of sulfuric acid and glycerol on the $580^{\circ}C$ in heating, which uses coated with glycerol of copper ions can be removed more effectively. Serial batch kinetic tests and batch sorption tests were conducted to determine the removal characteristics for Cu in aqueous solution. The result of batch kinetic test shows that removal rate, $K_{obs}$ (1/hr), of Cu are 0.579, 0.878, 3.459, and 6.578 for MEV weight 1 g (25 g/L), 2 g (50 g/L), 3 g (75 g/L), 4 g (100 g/L), respectively. In this case the initial pH of the solution was 3.26. The removal experiment according to the concentration, $K_{obs}$ (1/hr), of Cu are 1.96, 0.878, 1.25, and 1.04 for the initial concentration of 3 mg/L, 5 mg/L, 8 mg/L, 10 mg/L, and the initial pH of the solution are 3.46, 3.26, 3.10, 2.96, respectively. Influence of initial pHs on Cu removal were tested under 1g of MEV with 5 mg/L of Cu solution. $K_{obs}$ (1/hr) were increased from 0.263 (pH 3) to 0.525 (pH 5). It leads to the conclusion that the removal rates are inversely proportional to the initial Cu concentration and are increased proportional to the initial pHs. Sorption capacity of MEV was determined by batch sorption tests. The maximum sorption capacity ($Q_{max}$) obtained from Langmuir was 0.761 mg/g, Linear and Freundlich partition coefficients were 0.494 L/g and 0.729 L/g (1/n = 0.476). These results show that the MEV could be used as an excellent adsorbent for copper contained in various types of aqueous solutions.

Applicability of Theoretical Adsorption Models for Studies on Adsorption Properties of Adsorbents(III) (흡착제의 흡착특성 규명을 위한 흡착모델의 적용성 평가(III) - 열역학적 특성을 중심으로)

  • Na, Choon-Ki;Jeong, Jin-Hwa;Park, Hyun-Ju
    • Journal of Korean Society of Environmental Engineers
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    • v.34 no.4
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    • pp.260-269
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    • 2012
  • The aim of this study is to evaluate the applicability of adsorption models for understanding the thermodynamic properties of adsorption process. For this study, the adsorption isotherm data of $NO_3$-N ion onto a commercial anion exchange resin obtained at various experimental conditions, i.e. different initial concentrations of adsorbate, different dosages of adsorbent, and different temperatures, were used in calculating the thermodynamic parameters and the adsorption energy of adsorption process. The Gibbs free energy change (${\Delta}G^0$) of adsorption process could be calculated using the Langmuir constant $b_M$ as well as the Sips constant, even though the results were significantly dependant on the experimental conditions. The thermodynamic parameters such as standard enthalpy change (${\Delta}H^0$), standard entropy change (${\Delta}S^0$) and ${\Delta}G^0$ could be calculated by using the experimental data obtained at different temperatures, if the adsorption data well fitted to the Langmuir isotherm model and the plot of ln b versus 1/T gives a straight line. As an alternative, the empirical equilibrium constant(K) defined as $q_e/C_e$ could be used for evaluating the thermodynamic parameters instead of the Langmuir constant. The results from the applications of D-R model and Temkin model to evaluate the adsorption energy suggest that the D-R model is better than Temkin model for describing the experimental data, and the availability of Temkin model is highly limited by the experimental conditions. Although adsorption energies determined using D-R model show significantly different values depending on the experimental conditions, they were sufficient to show that the adsorption of $NO_3$-N onto anion exchange resin is an endothermic process and an ion-exchange process.

Evaluation of Design and Operation Parameters for a Spherical Sulfur Denitrification Reactor Treating High Strength Municipal Wastewater (고농도 도시하수 처리를 위한 입상황 탈질 반응조의 설계 및 운영인자 평가)

  • Kim, Yong-Hak;Chae, Kyu-Jung;Yim, Seong-Keun;Lee, Young-Man;Bae, Woo-Keun
    • Journal of Korean Society of Environmental Engineers
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    • v.32 no.12
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    • pp.1087-1093
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    • 2010
  • Autotrophic denitrification is known as an effective and economical alternative for heterotrophic denitrification using external carbon sources such as methanol. In this study, we evaluated design and operation parameters for a sulfur denitrification reactor (SDR) treating high strength nitrogen wastewater. The SDR was filled with spherical sulfur media in connected to a pilot-scale nutrient removal process (daily flow rate, $Q=18\;m^3/d$) using moving spongy media. Total nitrogen (TN) concentration of the final effluent was below the 7.0 mg TN/L because nitrate was additionally removed through autotrophic denitrificationin without adding alkalinity (initial alkalinity was $169.4{\pm}20.8\;mg$ $CaCO_3$/L). During the test period, 60~80% of nitrogen in the influent was removed even in low temperature (below $15^{\circ}C$). The alkalinity consumption for nitrate removal in SDR was $4.09{\pm}1.29$ g $CaCO_3/g$ ${NO_3}^-$-N, and the residual alkalinity of influent of SDR was higher than that of theoretical requirements for full conversion of nitrate. The consumption of sulfur was 943.8 g S/d and it was 2.4 times higher than theoretical value (400.1 g S/d) due to abrasion and loss of sulfur media in backwash, etc.

Effects of Stress and Personality Characteristics on Sleep (스트레스와 성격요인이 수면에 미치는 영향)

  • Yoon, Ho-Kyoung;Kang, Seung-Gul;Ham, Byung-Joo;Lee, Heon-Jeong;Kwon, Ho-In;Suh, Kwang-Yoon;Kim, Leen
    • Sleep Medicine and Psychophysiology
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    • v.10 no.1
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    • pp.32-38
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    • 2003
  • Objectives: The purpose of this study is to investigate the effects of stress and personality on sleep patterns, and further, to identify potential correlations between stress and personality characteristics. Methods: A total of 174 healthy college students were subjects for this study. Participants filled out the Daily Stress Inventory before sleep and the Modified Pittsburgh Sleep Quality Index after sleep on three consecutive days. They also filled out the 16 Personality Factor Questionnaire (16-PF), BDI (Beck Depression Inventory) and STAI (State-Trait Anxiety Inventory). Results: Minor stresses highly correlated with subjective sleep quality and symptoms of non-restorative sleep. However, total sleep time, sleep latency, awakening frequency, and frequency of dreams were not explained by stress scores. The O (guilty feeling), C (low ego strength) and Q4 (high anxiety) factors of the 16-PF also highly correlated with symptoms of non-restorative sleep and significantly affected sleep patterns. BDI and STAI scores also correlated with the above personality factors and minor stresses. Conclusion: This study showed that minor stresses impaired the restorative effects of sleep. Personality characteristics such as low ego strength, high levels of anxiety, and feelings of guilt were vulnerable to minor stresses. Minor stress, various personality characteristics, different coping patterns, and emotional response are highly correlated with each other and affect sleep patterns.

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Comparison of Behavioral Response between Intranasal and Submucosal Midazolam Adminstration (소아 진정 치료 시 구강 점막 하와 비점막 Midazolam 투여의 행동 반응 비교)

  • Kim, Yun-Hee;Jung, Sang-Hyuk;Baek, Kwang-Woo
    • Journal of the korean academy of Pediatric Dentistry
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    • v.35 no.3
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    • pp.427-436
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    • 2008
  • Purpose. The objective of this study was to evaluate the behavioral response and assess the effectiveness of additional intranasal (IN) and submucosal (SM) administration of midazolam during pediatric sedation for dental procedure. Material and methods. Thirty-three cases of healthy (ASAⅠ), uncooperative children aged from 24 to 72 month old at pediatric dental clinic of Ewha Womans University Hospital were selected for this study. Children received oral chloral hydrate 50 mg/kg with hydroxyzine 1.0 mg/kg. After waiting for 45 minutes, midazolam 0.2 mg/kg was administrated via IN route and via SM route randomly maintaining 50% of $N_2O$. A pulse oximeter and a capnograph were used for measuring vital signs ($SpO_2$, PR, RR, $EtCO_2$) throughout the sedation. Behavioral response was evaluated as Quiet (Q), Crying (C), Movement (M) or Struggling (S) in every 2 minutes for 40 minutes. Results. There were also no statistically significant differences in vital signs of the two groups. The behavioral response for the first ten minutes during sedation was a statistically significant difference (P < 0.05) between the two groups. After the first ten minutes, it was revealed that there was no significant difference. Conclusion. This study demonstrated that the addition of IN midazolam to the combination of oral chloral hydrate with hydroxyzine and nitrous oxide/oxygen inhalation is as safe and effective as that of SM midazolam in pediatric sedation for dental procedure.

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Analysis of Telomere Length and Telomerase Activity of Tissues in Korean Native Chicken (한국 재래닭의 주령별 각 조직의 텔로미어 함량과 텔로머레이스 활성도 분석)

  • Jung G.S.;Cho E.J.;Choi D.S.;Lee M.J.;Park C.;Jeon I.S.;Sohn S.H.
    • Korean Journal of Poultry Science
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    • v.33 no.2
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    • pp.97-103
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    • 2006
  • Telomeres are essential for chromosome stability and are related with cell senescence, apoptosis and cancer. Even though telomere length and telomerase activity have been studied extensively, very little is known to analyze the telomere dynamics in chicken cells. This study was carried out to analyze the telomere distribution and telomerase activity of Korean Native Chicken cells along with aging. The cells were collected from brain, heart, liver, kidney and germinal tissues during physiological stages. Telomere distribution was analyzed by Quantitative-Fluorescence in situ Hybridization (Q-FISH) techniques using the chicken telomeric DNA probe. Telomerase activity was performed by Telomeric Repeat Amplification Protocol (TRAP) assay. In results, the telomeres of chicken were found at the ends of all chromosomes with the interstitial telomeres on chromosomes 1, 2 and 3. The amount of telomeres on chicken cells was decreased along with aging in most tissues. Furthermore, the telomere quantity was significantly different among tissues. The relative amount of telomeres in proliferous cells such as testis cells had much more than those of liver, brain, heart, blood and kidney cells. The telomerase activity was down-regulated in cells of brain, heart and liver tissues. Whereas gonadal cells showed a constitutive activity of telomerase during all stage of life. In conclusions, the telomere quantity and telomerase activity in chicken are closely relate to cell proliferation and tissue specificity during developmental stages and aging. There is also closely correlated between the amounts of telomeric DNA and telomerase activity in chicken tissues.