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STUDY ON MUTATION OF P53 AND EXPRESSION OF MDM-2 IN DMBA INDUCED CARCINOMA OF HAMSTER BUCCAL POUCH (DMBA로 유도된 햄스터 협낭암종에서 p53 유전자 변이와 mdm-2 단백의 발현에 관한 연구)

  • Park, Yong-Sun;Kim, Kyung-Wook;Lee, Jae-Hoon;Kim, Chang-Jin
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.27 no.5
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    • pp.373-384
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    • 2001
  • Cellular proliferation is an intricately regulated process mediated by the coordinated interactions of critical growth control genes. Two of these factors in mammalian cells are the p53 and mdm-2 genes. A protein product of the mem-2 oncogene has been recently shown to associate with the protein encoded by the tumor suppressor gene p53. The p53 tumor suppressor protein is stabilized in response to DNA damage and other stress signals and causes the cell to undergo growth arrest or apoptosis, thus preventing the establishment of mutations in future cellular generations. Mutation or loss of p53 is a very common event in tumor progression. It occurs in about 50% of all tumors analysed including of colon, lung, breast and liver. The cellular mdm-2 gene, which has potential transforming activity that can be activated by overexpression, is amplified in a significant percentage of human sarcoma and in other mammalian tumors. Proteins encoded by the mdm-2 gene are able to bind to the p53 protein and, when overexpressed, can inhibit p53's transcriptional activation function, thus mdm-2 can act as a negative regulator of p53 function. Experimental study was performed to observe the relationship between p53 gene mutation and mdm-2 protein expression and apply the results to the clinical activity. 36 golden syrian hamster each weighing $60{\sim}80g$ were used and painted with 0.5% DMBA by 3 times weekly on the right buccal cheek(experimental side) for 6, 8, 10, 12, 14 and 16 weeks. Left buccal cheek(control side) was treated with mineral oil as the same manner to the right side. The hamsters were sacrificed on the 6, 8, 10, 12, 14 & 16 weeks. Normal and tumor tissues from paraffin block were examined for histology and immunohistochemistry observation, and were completely dissected by microdissection and DNA from both tissue were isolated by proteins K/phenol/chloroform extraction. Segments of the hamster p53 exons 5, 6, 7 and 8 were amplified by PCR using the oligonucleotide primers, and then confirmational change was observed by SSCP respectively. The results were as follows : 1. Dysplasia at 6 weeks, carcinoma in situ at 8 weeks and invasive carcinoma from 10 weeks could be observed in experimental groups. 2. p53 mutations were detected in 10 of the 36(28%) and the exons 6(6 of the 10 : 60%) was the most hot spot area among the highy conserved region(exons 5, 6, 7 & 8). 3. Immunohistochemical study confirmed 22 of the 36(61%) of p53 expression involving 10 of p53 mutations. 4. mdm-2 expression of was showed in 3 of the 36(8%) involving 1 of the 22 of p53 expression and 2 of the 14 of p53 non-expression. From the above results, mutation of p53 gene or expression of p53 protein may have the influence of the DMBA induced carcinoma of hamster buccal pouch but the expression of mdm-2 protein may not have relationship with tumorigenesis.

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Anti-thrombotic Activities of Hot-water Extracts Prepared from Various Parts of Nelumbo nucifera Gaertner (다양한 부위의 연(Lotus) 열수 추출물의 항혈전 활성)

  • Ahn, Seon-Mi;Sung, Hwa-Jung;Kim, Jong-Sik;Park, Jong-Yi;Sohn, Ho-Yong
    • Journal of Life Science
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    • v.28 no.10
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    • pp.1156-1162
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    • 2018
  • To investigate the anti-thrombotic activities of the lotus (Nelumbo nucifera Gaertner), various hot-water extracts were prepared from the leaf, pod of seed (PS), seed, embryo of seed (ES), root, and the node of root (NR) of the lotus. The highest extraction ratio was found in the NR (20.3%), followed by the seed, root, leaf, ES, and PS. These extracts had pH and acidity levels ranging from 5.6~6.5 and 0.06~0.20%, respectively. The seed extract showed 70% brix, whereas the leaf and PS extracts showed less than 0.1% brix. The highest contents of total polyphenol (179.7 mg/g), total flavonoids (161.4 mg/g), and reducing sugar (161.4 mg/g) were observed in the leaf extract, and the highest total sugar content (873.0 mg/g) in the seed extract. Anti-coagulation assay of the extracts of NR, leaf, and PS showed strong activities. In particular, at a concentration of 5 mg/ml, the PS extract had 15-fold extended thrombin, prothrombin, and activated partial thromboplastin times. However, only the ES extract showed activities inhibitory to platelet aggregation, with treatment with 0.25 mg/ml of ES extract decreasing platelet aggregation to 25.1%, a reduction comparable to that effected by aspirin. The extracts other than the seed extract showed no hemolysis activities against human RBC at treatments of up to 1 mg/ml. These results suggest that the NR, PS, seed, and ES, all byproducts of the lotus agriculture industry, have high potential as novel sources of anti-thrombotic agent.

Uptake of Heavy Metal Ions by Water Dropwort (Oenanthe stolonifera DC.) and Identification of Its Heavy Metal-Binding Protein (미나리의 중금속 흡수량 측정 및 중금속 결합단백질의 동정)

  • Park, Young-Il;Kim, Hee-Guen;Kim, Yoo-Young;Kim, In-Soo
    • Applied Biological Chemistry
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    • v.39 no.6
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    • pp.494-500
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    • 1996
  • Uptake of hen metal ions by water dropwort (Oenanthe stolonifera DC.) and its cadmium-binding protein were studied to probe for good method to remove heavy metal contaminants from environments. The plant was cultured in the culture medium (pH 7.0) containing the various concentrations of $Cd^{2+}$, $Cr^{3+}$ or $Pb^{2+}$, for 3 and 7 days. The residual heavy metals deposited in roots linearly increased as the metal ions concentration increased up to 17 ppm for $Cd^{2+}$, 20 ppm for $Cr^{3+}$ and 50 ppm for $Pb^{2+}$. Above these concentrations, the plant growth was inhibited and the uptake rates of the metal ions decreased. The heavy metals absorbed by the plant were mostly deposited in roots. In particular, the residual concentration of lead in roots was about four times higher than those of cadmium and chromium. When cultured in the medium containing 20 ppm of each metal ion, 80% of cadmium, 90% of cromium and 96% of lead were deposited in roots out of the total residual metal ions in the plant. These values correspond to 6.1 mg of cadmium, 5.2 mg of chromium and 23.6 mg of lead per one gram of roots tissue on a dry weight basis. A cadmium-binding protein was partially purified by extraction, gel filtration and DEAE-Cellulose chromatography from water dropworts that was grown in the medium containing 20 ppm $Cd^{2+}$. The purified protein was a single band on SDS- and non-denaturing- polyacrylamide gel electrophoresis. Its molecular mass was estimated to be ca. 5,000 dalton by gel filteration. Analysis of amino acid composition of the protein indicated that it had a typical amino acid composition of heavy metal-binding protein in that it contained 27% of acidic amino acids and 9.9% of cysteine. However, it is likely that the protein is a new plant metal-binding protein, since its amino acid composition is somewhat different from those of phytochelatins that have been known so far.

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EVALUATION OF PERIODONTAL LIGAMENT CELL VIABILITY IN RAT TEETH ACCORDING TO VARIOUS EXTRA-ORAL DRY STORAGE TIMES USING MTT ASSAY (구강 외 노출시간에 따른 흰쥐 치아 치주인대세포 활성도의 MTT 검색법을 이용한 평가)

  • Jeon, In-Soo;Kim, Eui-Seong;Kim, Jin;Lee, Seung-Jong
    • Restorative Dentistry and Endodontics
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    • v.31 no.5
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    • pp.398-408
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    • 2006
  • The purpose of this study was to verify the usefulness of MTT analysis as a tool of measurement of the periodontal ligament cell viability from the extracted rat molar. A total of 80 Sprague-Dawley white female rat of 4 week-old with a body weight of 100 grams were used. The maxillary left and right, first and second molars were extracted under Ketamine anesthesia. Twenty-four teeth of each group (divided as five groups depending upon the time-lapse after extraction such as Immediate, 10, 20, 40 and 60 minutes) were immersed in $200{\mu}l$ of MTT solution (0.5 mg/ml) and processed for optical density measurements. Another 10 teeth of each group were treated as same as above and sectioned at $10{\mu}m$ for microscopic examination. All measurements values were divided by the value of hematoxylin-eosin staining which represented the volume of each corresponding samples. Immediate and 10 minute groups showed highest MTT values followed by 20, 40, and 60 minutes consecutively. Statistical significance (p<0.05) existed between all groups except in immediate versus 10 minute groups and 40 versus 60 minutes. Histological findings also showed similar findings with MTT results in crystal shape and crystal numbers between the experimental groups. These data indicate that in vivo MTT analysis nay be of value for evaluation of the periodontal ligament cell viability without time- consuming cell culturing processes.

EFFECT OF NERVE GROWTH FACTOR GENE INJECTION ON THE NERVE REGENERATION IN RAT LINGUAL NERVE CRUSH-INJURY MODEL (백서 설신경 압박손상모델에서 신경성장인자 유전자 주입이 신경재생에 미치는 영향)

  • Gao, En-Feng;Chung, Hun-Jong;Ahn, Kang-Min;Kim, Soung-Min;Kim, Yun-Hee;Jahng, Jeong-Won;Lee, Jong-Ho
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.28 no.5
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    • pp.375-395
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    • 2006
  • Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

Enhancement of Inter-Image Statistical Correlation for Accurate Multi-Sensor Image Registration (정밀한 다중센서 영상정합을 위한 통계적 상관성의 증대기법)

  • Kim, Kyoung-Soo;Lee, Jin-Hak;Ra, Jong-Beom
    • Journal of the Institute of Electronics Engineers of Korea SP
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    • v.42 no.4 s.304
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    • pp.1-12
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    • 2005
  • Image registration is a process to establish the spatial correspondence between images of the same scene, which are acquired at different view points, at different times, or by different sensors. This paper presents a new algorithm for robust registration of the images acquired by multiple sensors having different modalities; the EO (electro-optic) and IR(infrared) ones in the paper. The two feature-based and intensity-based approaches are usually possible for image registration. In the former selection of accurate common features is crucial for high performance, but features in the EO image are often not the same as those in the R image. Hence, this approach is inadequate to register the E0/IR images. In the latter normalized mutual Information (nHr) has been widely used as a similarity measure due to its high accuracy and robustness, and NMI-based image registration methods assume that statistical correlation between two images should be global. Unfortunately, since we find out that EO and IR images don't often satisfy this assumption, registration accuracy is not high enough to apply to some applications. In this paper, we propose a two-stage NMI-based registration method based on the analysis of statistical correlation between E0/1R images. In the first stage, for robust registration, we propose two preprocessing schemes: extraction of statistically correlated regions (ESCR) and enhancement of statistical correlation by filtering (ESCF). For each image, ESCR automatically extracts the regions that are highly correlated to the corresponding regions in the other image. And ESCF adaptively filters out each image to enhance statistical correlation between them. In the second stage, two output images are registered by using NMI-based algorithm. The proposed method provides prospective results for various E0/1R sensor image pairs in terms of accuracy, robustness, and speed.

A Comparison of the Components and Biological Activities in Raw and Boiled Red Beans (Phaseolus radiatus L.) (생팥과 증자팥의 성분 및 생리활성 비교)

  • Lee, Ryun Kyung;Kim, Mi-Sun;Lee, Ye-Seul;Lee, Man-Hyo;Lee, Jong Hwa;Sohn, Ho-Yong
    • Microbiology and Biotechnology Letters
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    • v.42 no.2
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    • pp.162-169
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    • 2014
  • In the course of study for the development of functional food using red beans (azuki beans, Phaseolus radiatus L.), the ethanol extracts from raw-red bean (RRB) and boiled-red bean (BRB) were prepared, and the components and various biological activities of both were compared. It was observed that the extraction yield, and the total polyphenol content, of the BRB were 1.2 times higher than that of the RRB. However, the contents of total flavonoid, total sugar and reducing sugar in the BRB were 30, 27.9 and 30.8% respectively when compared with those of RRB. In relation to antioxidative activity, both RRB and BRB exhibited moderate DPPH anion, ABTS cation, and nitrite scavenging activities and reducing power, though in all cases RRB demonstrated stronger activities than BRB. The extracts of RRB and BRB did not reveal any antimicrobial activities. In a ${\alpha}$-amylase inhibitory activity assay, RRB was higher than BRB, while BRB showed higher ${\alpha}$-glucosidase inhibitory activity than RRB. A strong and particular activity was observed in an anti-thrombosis activity assay of RRB. The extract of RRB demonstrated strong inhibitions against prothrombin and blood coagulation factors, with moderate thrombin inhibition. However, the extract of BRB did not exhibit any significant anti-thrombosis activity. Our results indicate that RRB has different, but useful biological activities, and loss or elimination of the biologically active substances in RRB occurs during the production of BRB. Therefore, to develop more functional foods from red beans, a study of suitable boiling, heating and drying processes is essential, and the efficient re-use of boiled waste water from the boiling process is necessary. These results could be applied to the further development of functional red bean beverages and sweat red bean pastes.

Effect of Containers on the stability of Malathion emulsion concentrates (E.C.) (Malathion 유제(乳劑)의 포장용기(包裝容器)에 따른 경시변화(經時變化))

  • Lee, D.S.;Lee, J.Y.;Lee, S.H.
    • Applied Biological Chemistry
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    • v.7
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    • pp.15-19
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    • 1966
  • In order to investigate the stability of the major component of malathion E.C. product, dimethyl S-(1, 2-dicarboxyethoxyethyl) dithiophosphate, toward the quality of glasswares as container, the amount of extractable inorganic components, change of pH and decomposition of the major component of the product were examined during the storage in brown-colored bottles of 100 ml. volume from 3 different companies in comparison with that in a Pyrex flask. 1. Malathion E.C. product was put in three containers A,B and C, and any changes occurring in storage were analyzed at three intervals of 60, 120 and 240 days. 2. It was shown that the amounts of Si, Mg, K, Ca, and Na extracted during these periods of storage differed markedly depending on the qualify of container. Container A revealed ten times higher extraction of Na and Ca than container B and C in a 8-month period. 3. Three commercial containers revealed the shift of pH from 6.5 to alkaline reaction in the storage whereas the Pyrex flask did not show any detectable change. In particular, the pH in container A changed to 9.2 in 60 days and 9.9 in 240 days. 4. The decomposition of malathion was the greatest in container A which showed the decomposition of 7.37% in 240 days. On the other hand, 0.5% was decomposed in the Pyrex flask. 5. The decemposition of malathion had a high correlation with the change of pH of water· in the same container, $r^2$ being 0.899. From the above results, it is concluded that about 10% of malathion E.C. product is decomposed in a year due to the alkaline metallic salts extracted from the container when it is stored in glassware bottles of lower quality.

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Effect of Maturity Scores and Number of Extractions on the Chemical Properties of Water Extract from Hanwoo Shank Bones (한우 성숙도와 추출횟수가 사골용출액의 화학적 특성에 미치는 영향)

  • Kim, Jin-Hyoung;Cho, Soo-Hyun;Seong, Pil-Nam;Hah, Kyung-Hee;Jeong, Jin-Hyung;Lim, Dong-Gyun;Park, Beom-Young;Lee, Jong-Moon;Kim, Dong-Hun;Ahn, Chong-Nam
    • Food Science of Animal Resources
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    • v.27 no.4
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    • pp.463-468
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    • 2007
  • This study was conducted to investigate the effect of maturity scores [2 (bull), 2 (steer), 3-9 (cow)] and the number of extractions (up to 4 times) on the chemical properties of water extract from Hanwoo shank bones (arm, fore shank, round and hind shank). The turbidity, meat color (CIE L value), collagen, protein, caloric and chondroitin sulfate contents of samples were observed. The turbidity and lightness were higher for water extract of Hanwoo shank bones with a maturity score of 2 (bull and steer) than maturity scores of 3-9 (cow) (p<0.05). The turbidity and lightness of water extract from shank bones of all Hanwoo maturity scores significantly increased with the 1st and 2nd extractions, but significantly decreased with 3rd and 4th extractions (p<0.05). The collagen and protein contents were highest for water extract from Hanwoo shank bones of maturity score 2 (bull and steer) (p<0.05). The caloric and chondroitin sulfate contents were higher for water extract from Hanwoo shank bones of maturity score 2 (bull and steer) than maturity scores of 3-9 (cow) (p<0.05). As the number of extractions increased, the chondroitin sulfate content significantly decreased (p<0.05). Based on these results, differences correlating with maturity scores were found only with collagen and protein contents. Therefore, further studies should be considered to address whether different maturity scores affect the price of shank bones in the meat industry.

Oxidative Desulfurization of Marine Diesel Using Keggin Type Heteropoly Acid Catalysts (Keggin형 헤테로폴리산 촉매를 이용한 선박용 경유의 산화 탈황)

  • Oh, Hyeonwoo;Woo, Hee Chul
    • Clean Technology
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    • v.25 no.1
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    • pp.91-97
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    • 2019
  • Oxidative desulfurization (ODS) has received much attention in recent years because refractory sulfur compounds such as dibenzothiophenes can be oxidized selectively to their corresponding sulfoxides and sulfones, and these products can be removed by extraction and adsorption. In this work, The oxidative desulfurization of marine diesel fuel was performed in a batch reactor with hydrogen peroxide ($H_2O_2$) in the presence of various supported heteropoly acid catalysts. The catalysts were characterized by XRD, XRF, XPS and nitrogen adsorption isotherm techniques. Based on the sulfur removal efficiency of promising silica supported heteropoly acid catalysts, the ranking of catalytic activity was: $30\;H_3PW_{12}/SiO_2$ > $30\;H_3PMo_{12}/SiO_2$ > $30\;H_4SiW_{12}/SiO_2$, which appears to be related with their intrinsic acid strength. The $30\;H_3PW_{12}/SiO_2$ catalyst showed the highest initial sulfur removal efficiency of about 66% under reaction conditions of $30^{\circ}C$, $0.025g\;mL^{-1}$ (cat./oil), 1 h reaction time. However, through the recycle test of the $H_3PW_{12}/SiO_2$ catalyst, significant deactivation was observed, which was attributed to the elution of the active component $H_3PW_{12}$. By introducing cesium cation ($Cs^+$) into the $H_3PW_{12}/SiO_2$ catalyst, the stability of the catalyst was improved with changing the solubility, and the $Cs^+$ ion exchanged catalyst could be recycled for at least five times without severe elution.