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Characterization of Bruton's Tyrosine Kinase Genetic Mutations in One Korean X-linked Agammaglobulinemia Family (반성 열성 범저감마글로불린혈증 1가계 3환자의 Bruton's Tyrosine Kinase 유전자 변이 및 임상 양상)

  • Jo, Eun-Kyeong;Song, Chang-Hwa;Park, Jeong-Kyu;Baek, Young-Jong;Rhu, Hye-Young;Lee, Jae-Ho;Hwang, Tai-Ju;Kook, Hoon
    • Clinical and Experimental Pediatrics
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    • v.45 no.2
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    • pp.183-191
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    • 2002
  • Purpose : X-linked agammaglobulinemia(XLA) is an immunodeficiency caused by abnormalities in Bruton's tyrosine kinase(Btk), and is characterized by a deficiency of peripheral blood B cells. We studied the cytoplasmic expression of Btk protein and analyzed the Btk gene in peripheral blood mononuclear cells from two siblings and one cousin with XLA, as well as additional family members. Methods : Btk protein expression was analyzed by flow cytometry. Isolation of the coding sequence of the Btk gene was performed by amplification using the reverse transcription-polymerase chain reaction(RT-PCR) technique. Sequence alterations were screened by the single-stranded conformation polymorphism(SSCP) method and characterized by standard sequencing protocols. Results : Cytoplasmic expression of Btk protein in monocytes was not detected in three patients with XLA. In addition, Btk protein analysis clearly showed cellular mosaicism in monocytes from four obligate carriers, findings further supported by SSCP. A single base pair mutation(T to C) in Btk-exon three, which encodes the PH domain, was identified in four XLA patients. A diagnostic sequencing analysis was established to detect heterozygotic pattern in 4 carrier females. Furthermore, we found significant clinical heterogeneity in individuals with the same gene mutation. Conclusion : The implicating genetic alteration provided valuable clues to the pathogenesis of XLA in Korea and the flow cytometric analysis was suggested as a useful tool for rapid detection of XLA patients and carriers. The present study has identified a genetic mutation in the Btk coding region and demonstrated heterogeneity in clinical manifestations among patients with the same mutation. A flow cytometric analysis was found to be informative in establishing a deficiency of Btk protein in both patients and carriers and is recommended as a frontline procedure in the molecular diagnosis and work-up of XLA.

Quality Evaluation of Domestic and Foreign Extruded Pellets and Moist Pellet Based on Biochemical Analyses for Juvenile Olive Flounder, Parazichthys Olivaceus (시판용 넙치(치어)사료의 성분 비교분석을 통한 품질평가)

  • 최세민;한경민;왕소길;이승형;배승철
    • Journal of Aquaculture
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    • v.17 no.2
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    • pp.144-150
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    • 2004
  • This experiment was conducted to evaluate the parameters such as nutrient requirements, POY, AnV, Totox, VBN, total plate count, dietary fatty acids and amino acids composition, that are not included in the registered standard composition items required by the Ministry of Agriculture and Forestry, of a moist pellet (MP), three domestic extruded pellets (DEP-1, DEP-2, DEP-3), and two foreign extruded pellets (FEP-1, FEP-2) that are utilized by domestic flounder farms at present. The crude protein was added in excess of the dietary protein requirement in 6 kinds of feeds. When considering the proper PH ratio, it is obvious that protein was added in excess, especially in MP and FEP-2. Crude fat was also added in excess, especially in FEP-1. MP contained a higher dietary phosphorus content than formulated feeds, surpassing the dietary phosphorus requirement and greatly increasing the possibility for causing water pollution. The oxidation of fatty acid and decomposition of protein in MP were higher than in formulated feeds, and may also cause problems on fish farms. Also, it is difficult to store and manage MP, Among the fatty acids, EPA and DHA contents in MP were higher than those in formulated feeds. It is necessary to conduct further studies of EPA and DHA contents in formulated feeds. Lysine content in MP and FEP-2 could meet the dietary lysine requirement of flounder, however, the possibility of insufficient lysine content in the other formulated feeds was high and we considered that extra supplementation was necessary. Therefore, it is necessary to set up quality control standards according to fish species and sizes while considering the specific character of aquatic formulated feeds to restore the confidence of feed companies and aquaculturists to these feeds. This may be an opportunity to make an earlier change from MP to formulated feeds.

Report on the Stability of Freeze-dried Standard Solution (동결건조표준액의 안정성에 관한 보고)

  • Park, Jun Mo;Yoo, Hye Jung;Kim, Han Chul;Han, Geul Soon
    • The Korean Journal of Nuclear Medicine Technology
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    • v.16 no.2
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    • pp.139-148
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    • 2012
  • Purpose : Since standard solution is the one that knows its exact concentration, the curve of the dissolution has been determined according to the amount of the solution, compared to the amount of the unknown sample. Therefore, the antigen that makes up standard materials should be made in a pure form. The configuration of the standard substance solution in the kit we use is a freeze-dried material, or made and comes as a liquid. Lyophilized reference material is used after dissolving in usually D.W. (Distilled Water), and if the antigen to use is too sensitive, reagents should be freeze-dried. Furthermore, when freeze-dried reference has to be frozen again after being dissolved, it should be kept under $-20^{\circ}C$ until the expiration date according to the reports. Since it is not expressed in the experiment if it is safe or stable to reuse the solution which was dissolved a few times, thus, this time it is tested and evaluated that the changes of the standard solution by freezing and melting several times, and its results and the effectiveness of it were compared to the solution which was kept in a fridge. Materials and Methods : Among Vitro diagnostic kits on the market made by radioimmunoassay, parathyroid hormone (PTH), adrenocorticotropic hormone (ACTH), luteinizing hormone (LH) are made of freeze-dried standard solution and all composed of the same Lot.NO. These hormones melted in D.W. and were separated into three groups. In the first group, melting and freezing were repeated, and in the second group, The solution only for one time use was put into a test tube after melting and freeze it. The third group was kept in the refrigerator. This experiment has been conducted from January to February in 2012. January to 2012. PH test was employed because ph is prone to changing depending on the change of protein. Each group of the standard solution, cpm (counter per minute), and the patient relative concentration values were compared by date, and Through the correlation coefficient and Paired t-test, the significant level of each group was analyzed. Results : ACTH, PTH, LH pH values were too subtle denaturation rather than numerical changes in the protein. In addition, when the standard solution of ACTH, PTH, LH was refrigerated, after 3 days and 7 days, there was a significant difference observed between the solution being kept in a refrigerator and a freezer within a significance level. Conclusion : Standard solution should be kept in a freezer, and being kept in a fridge, it is recommended to use the solution as soon as possible.

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Studies on the Effect of Cation on the Activity at the 5th Instar Larvae of Bombyx mori (5령유충의 배맥관운동에 대한 양이온의 영향에 관한 연구)

  • 윤종관;사기언
    • Journal of Sericultural and Entomological Science
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    • v.18 no.2
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    • pp.89-93
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    • 1976
  • The physiological saline solution for animals is known as Ringer's solution which is used for keeping the function of cold blooded vertebrate animals. Primaily the saline solution is used for the purpose of perfusion experiment in frogs. Later the saline solution is applied in several kinds of animals including human being with satisfactory results. However, this saline solution was introduced to silkworm and it was found that the result was not as successful as in the case of other animals and human being. Normally, in the case of silkworm, the physiological saline solution is prepared in order to maintain the normal function of separated organs and tissues. To this end, the saline solution is adjusted to contain the certain amount and strength of ions, osmosis pressure and hydrogen concentration. The most of cases, the physiological saline solution should be prepared so that the constituent of the solution be the same with the blood selium and body fluid. The hydrogen concentration in the ion element of the saline solution is adjustable by adding Na$\^$+/, K$\^$+/, Ca$\^$++/, Mg$\^$++/ which are followed by adding of buffer solution such as NaHCO$_3$and NaH$_2$PO$_4$. Determination of optimum concentration of cation in the physiological saline solution, and the optimum mixing rate of more than two kinds of cations are based on the movement of dorsal vessel in the silkworm larvae. The optimum concentration of cations in the solution is prepared by adding NaCl solution which is under zero point. However, this solution was further added with the different concentration of KCl and CaCl$_2$. By dropping the prepared solution on the 5th larvae, the effects of solution was measured. The measurement was done by observation of movement' of dorsal vessel and its time length, and the number of pulses. According to the experiment, it was found that when only NaCl solution was applied, the number of pulses is increased for a moment, and the pulse stopped after one hour or so. When KCl solution was added the time of pulse was prolonged and in the contrast, the number of pulses was slow down. If KCl and CaCl$_2$solutions are added the time of pulse was further prolonged. Even though the adding of KCl and CaCl$_2$are found to be effectible, the correlation between the concentration of solution and the movement of dorsal vessel was not observed. However, it was same in the case of adding Ca$\^$++/ or K$\^$+/. It was found that when Mg$\^$++/ was added to dorsal vessel the number of the pulses was not decreased although the prolonged time pulse was observed.

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Development of Optimal Nutrient Solution for Tomato Substrate Culture in Closed System (토마토의 순환식 고형배지재배에 적합한 배양액 개발)

  • 최은영;이용범;김재영
    • Journal of Bio-Environment Control
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    • v.7 no.1
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    • pp.43-54
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    • 1998
  • This experiment was conducted to develop optimal nutrient solution for tomato plants, according to the plant growth stages in closed system. Perlite substrate was supplied with 1/2 and 1 strength of the solution of National Horticultural Research Station in Japan. Plants grew better and the nutrient_contents in the leaves were also proper in 1 strength. Based on these results, optimal nutrient solution in perlite was composed by n/w of 1 strength according to the plant growth rates : N 13.5, P 3.3, K 7.0, Ca 7.0, Mg 3.5 me.L$^{-1}$ in seedling stage, N 14.2, P 3.3, K 8.0, Ca 7.5, Mg 4.0 me.L$^{-1}$ in vegetative stage and N 10.0, P 3.0, K 7.0, Ca 6.0, Mg 3.0 me.L$^{-1}$ in reproductive stage. To examine the suitability of the nutrient solution developed in this experiment, tomato plants were grown in rockwool and supplied with two different composition and concentration of nutrient solution composed by n/w of 1 strength in perlite (SCUT) and by Research Station for Greenhouse Vegetable and Floriculture on the Netherlands (PBG). PH and EC in SCUT were changed little in 1 strength but a significant change of PH was shown in 1/2 strength. Later, drained solutions in rockwool were also analyzed according to the Plant growth stages. Low concentrations of N and P in root zone were shown in early growth stage but N was increased in reproductive stage, while, K, Ca, Mg concentration was consistent through the whole growth stage. Considering these results, we found that the rebalance of N and P was needed, that is, reduction of N concentration in reproductive stage and increasing of P concentration in vegetative stage.

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Radiation-induced Apoptosis is Differentially Modulated by PTK Inhibitors in K562 Cells (K562 백혈병 세포주에서 방사선에 의해 유도되는 Apoptosis에 미치는 PTK Inhibitors의 영향)

  • Lee Hyung Sik;Moon Chang Woo;Hur Won Joo;Jeong Su Jin;Jeong Min Ho;Lee Jeong Hyeon;Lim Young kin;Park Heon Joo
    • Radiation Oncology Journal
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    • v.18 no.1
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    • pp.51-58
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    • 2000
  • Purpose :The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induced apoptosis in Ph-positive KS62 leukemia cell line was investigated. Materials and Methods :K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X-ray irradiation and drug treatment, cultures were initiated at 2×106 cells/mL. The cells were irradiated with 10 Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37$^{\circ}C$ for 0$\~$48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells through the cell cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bel-2, bel-X$_{L}$ and bax protein levels. Results :Treatment with 10 Gy X-irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electro-phoresis and TUNEL assay. Genistein failed to enhance the ability of X-irradiation to induce DNA fragmentation. Enhancement of apoptosis by HMA was not attributable to downregulation of the bel-2 or bel-X$_{L}$ anti-apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage of cells in G2/M phase decreased to 30$\~$40$\%$ at 48 h. On the other hand, cells exposed to 10 Gy X-irradiation alone or maintained with genistein did not show marked cell cycle redistribution. Conclusion : We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X-irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p210$^{bcr/abl}$ failed to enhance the radiation induced apoptosis in KS62 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is attributable to bel-2 family. It is plausible that the relationship between cell cycle delays and cell death is essential for drug development based on molecular targeting designed to modify radiation-induced apoptosis.

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Determination of Heterocyclic Amines in Roasted Fish and Shellfish by Liquid Chromatography-Electrospray Ionization/Mass Spectrometry (Liquid chromatography-mass spectrometry를 이용한 가열 조리된 어패류에서의 heterocyclic amines 함량 분석)

  • Lee, Jae-Hwan;Back, Yoo-Mi;Lee, Kwang-Geun;Shin, Han-Seung
    • Korean Journal of Food Science and Technology
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    • v.41 no.3
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    • pp.326-333
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    • 2009
  • Heterocyclic aromatic amines (HCAs) are mutagenic and carcinogenic substances that are formed during the heating of protein-rich foods. HCAs are generally found at low amounts in a complex matrix, which requires sophisticated analysis. In this study, HCAs were extracted from lyophilized fish and shellfish samples using solid-phase extraction (SPE) and determined by liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI-MS). The HCA recoveries in the fish and shellfish ranged from 15.7 to 74.7% with standard deviations from 0.2 to 7.63%. And HCA concentrations ranged from 0.8 to 1,117.7 $ng/g^{-1}$ in cooked food samples. 1-methyl-9H-pyrido[3,4-b]indole (Harman), 9H-pyrido[3,4-b]indole (Norharman), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were the most abundant HCAs formed in the muscle of fried mackerel, at levels of 1,117.7, 926.6, and 133.7 ng/g, respectively. 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-aminodipiryrido[1,2-a:3,2-d]imidazole(Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole(A${\alpha}$C), 2-amino-3methyl-9H-pyrido [1,2-a:3,2-d]imidazole(MeA${\alpha}$C), 2-amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline (TriMeIQx), 2-amino-3,7,8-trimethylimidazo [4,5-f]quinoxaline(7,8-DiMeIQx), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were only detected by small quantities ranged from 1.5 to 98.6 ng/g. Overall, this study provides useful information on HCA levels in fish and shellfish products consumed in Korea.

The Cell Cycle Dependence and Radiation-induced Apoptosis in SCK Mammary Adenocarcinoma Cell Line (SCK선암 세포주에서 방사선에 의한 Apoptosis와 세포 주기)

  • Lee Hyung Sik;Park Hong Kyu;Hur Won Joo;Seo Su Yeong;Lee Sang Hwa;Jung Min Ho;Park Heon Joo;Song Chang Won
    • Radiation Oncology Journal
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    • v.16 no.2
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    • pp.91-98
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    • 1998
  • Purpose : The relationship between environmental PH on the radiation induced-apoptosis in SCK mammary adenocarcinoma cells and cell cycle dependence was investigated. Material and Methods : Mammary adenocarcinoma cells of A/J mice(SCK cells) in exponential growth phase were irradiated with a $l37^Cs$ irradiator at room temperature. The cells were irradiated 1 hour after the media was replaced with fresh media at a different pHs. After incubation at $37^{\circ}C$ for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and flow cytometry. The progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. Bssults : The induction of apoptosis by irradiation in pH 6.6 medium was markedly less than that in pH 7.5 medium. When the cells were irradiated and maintained in pH 7.5 medium, the percentage of cells in $G_2/M$ phase rapidly increased to about $70\%$ at 12 h after an exposure to 120y and returned to control level by 36 h. The percentage of cells in G1 phase decreased as the percentage of cells in $G_2/M$ increased. On the other hand, in pH 6.6 medium the percentage of cells in G2/M phases gradually increased to about $45\%$ at 24 h after 12Gy irradiation and then slowly recessed and consequently, as much as $30-35\%$ of the cells were still in the Ga/M phase 48 h after irradiation. The percentage of cells in G1 phase then increased as the Ga/M arrest began to recede. The radiation-induced Ga/M arrest in PH 0.0 medium lasted markedly longer than that in pH 7.5 medium. Conclusion : Radiation-induced apoptosis in SCK tumor cells are reversely suppressed in an acidic environment. Radiation-induced Ga/M arrest is prolonged in an acidic environment indicating that the suppression of radiation-induced apoptosis and prolongation of radiation-induced Ga/M arrest in an acidic environment are related.

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Effects of Chitosan and Organic Acid Salts on the Shelf-life and Pectin Fraction of Kimchi during Fermentation (키토산과 유기산염 첨가가 배추김치의 저장성과 펙틴분획에 미치는 영향)

  • 이지선;이혜준
    • The Korean Journal of Food And Nutrition
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    • v.13 no.4
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    • pp.319-327
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    • 2000
  • This study was conducted to prolong the edible period of Kimchi by adding chitosan (0.25, 0.5%) and sodium salts of various organic acids(0.01~0.04M citrate, malate, lactate) . The edible period was estimated by measuring changes in pH. titratable acidity(TA), PH/TA ratio, ascorbic acid content and pectin fraction during Kimchi fermentation at 2$0^{\circ}C$. The results were compared by estimating the maturity of Kimchi fermentation. Kimchi with the chitosan showed higher pH and titratable acidity throughout the fermentation period than that without chitosan. The pH decreased during the fermentation in the order of control, 0.25% chitosan, 0.5% chitosan, 0.5% chitosan+Na-citrate, 0.5% chitosan+Na-malate and 0.5% chitosan+Na-lactate. But the titratable acidity increased in the order of control, 0.5% chitosan+Na-malate, 0.25% chitosan. 0.5% chitosan+Na-citrate, 0.5% chitosan and 0.5% chitosan+Na-lactate. The PH/TA ratio decreased in the order of control, 0.25% chitosan, 0.5% chitosan+Na-malate, 0.5% chitosan, 0.5% chitosan+Na-citrate and 0.5% chitosan+Na-lactate. Ascorbic acid content in Kimchi was the highest at the 3rd day and then decreased during fermentation. Ascorbic acid content in Kimchi containing 0.5% chitosan and organic acid salts was higher than others. Alcohol insoluble solids( AIS ) in Kimchi decreased during fermentation in the order of control, 0.25% chitosan, 0.5% chitosan, 0.5% chitosan+Na-palate. 7.5% chitosan+Na-lactate and 0.5% chitosan+Na-citrate. During fermentation, hot water soluble pectin (HWSP) of control increased, whereas HCI soluble pectin (HCISP) decreased. By addition of chitosan, however, the results became reverse. Chitosan addition appeared to be effective in improving preservation quality of Kimchi during fermention. The edible period become extended by using chitosan plus organic acids instead of using chitosan only. Overall. addition of 0.5% chitosan+Na-lactate seemed most effective in prolonging the edible periods during Kimchi fermentation.

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Effect of Powder of Stevia rebaudiana Leaves against Quality Characteristics during Salting of Rice Bran Danmooji (쌀겨단무지의 절임 중 품질 특성의 변화에 대한 스테비아 잎 분말 첨가의 영향)

  • Kim, Yong-Suk;Lee, Sun-Kyu;Jeong, Do-Yeong;Yang, Eun-Jin;Shin, Dong-Hwa
    • Food Science and Preservation
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    • v.14 no.5
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    • pp.497-503
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    • 2007
  • In order to develop a new rice bran danmooji, changes in physicochemical characteristics and texture of danmooji treated with rice bran, Stevia rebaudiana Bertoni leaf powder, succinic acid, or yeast extract were investigated during salting for 90 days. The PH of rice bran danmooji decreased from PH6.41 initially to pH 4.09 (control group), pH 4.10 (S. rebaudiana treatment S1), pH 3.84 (S. rebaudiana + succinic acid treatment S2), and pH 3.90 (S. rebaudiana+succinic acid+yeast extract treatment S3) after 90 days of salting. At this time, the salinities of rice bran danmooji of the S1, S52, and S3 groups were 2.32%, 1.94% and 2.15% respectively. The hardness of all groups decreased rapidly in the first 30 days of salting, and thereafter showed no changes. After 90 days of salting, the hardness of all groups was $1,186-1,368\;g/cm^2$ with no significant differences between groups. Redness, the a value, of the S2 and S3 groups treated with succinic acid, was lower than that of S3, whereas yellowness, the b value, of S3 treated with succinic acid and yeast extract was the highest of the three groups. Sensory evaluation of rice bran danmooji after 90 days of salting resulted in S3 attaining the highest scores for flavor, taste, texture, and overall acceptability. These results indicate nut high-quality rice bran danmooji may be prepared by addition of S. rebaudiana leaf powder, succinic acid and yeast extract to rice bran.