• Title/Summary/Keyword: Histochemical analysis

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Development of a Screening System for Plant Defense-Inducing Agent using Transgenic Tobacco Plant with PR-1a Promoter and GUS Gene

  • Oh, Sang-Keun;Lee, Seon-Woo;Kwon, Suk-Yoon;Choi, Do-Il
    • The Plant Pathology Journal
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    • v.21 no.3
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    • pp.288-292
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    • 2005
  • Pathogenesis-related protein-1a (PR-1a) is strongly induced in tobacco plants by pathogen attack, exogenous salicylic acid (SA) application and by other developmental processes. In order to develop a rapid screening system for the selection of plant defense-inducing compounds originated from various sources, we have transformed tobacco Samsun NN plants with a chimeric construct consisting of GUS $(\beta-glucuronidase)$. In the $T_1$ generation, three transgenic lines having stable GUS expression were selected for further promoter analysis. Using GUS histochemical assay, we observed strong GUS induction driven by PR-1a promoter in PR1a-GUS transgenic tobacco leaves in response to the exogenous application of SA or benzol (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH), a SA­derivative compound. In addition, GUS expression was maintained locally or systemically in PR1a-GUS transgenic line $\#5\;T_2$ generation) until after 3 days when they were treated with same chemicals. Our results suggested that the PR1a-GUS reporter gene system in tobacco plants may be applicable for the large-scale screening of defense-inducing substances.

Effects of Wounding and Inoculation Time on Agrobacterium -mediated Transformation in Capsicum annuum L. (상처처리와 접종시간이 Agrobacterium에 의한 고추 형질전환에 미치는 영향)

  • Jeon, Young-Ju;Park, Young-Doo;Choi, Geun-Won
    • Horticultural Science & Technology
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    • v.18 no.6
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    • pp.797-801
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    • 2000
  • The present study was conducted to improve the efficiency of transformation mediated by Agrobacterium tumefaciens in hot pepper. Both regeneration ratio and transformation frequency after the cocultivation with A. tumefaciens were affected by inoculation time and artificial wounding. Transformation frequency was increased over 50% by combining artificial wounding with 120 s of inoculation treatment. Confirmation for the transformation of regenerated shoots was carried out by histochemical ${\beta}$-glucuronidase assay and polymerase chain reaction analysis using npt II primer.

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GUS Expression by CaMV 35S and Rice Act1 Promoters in Transgenic Rice

  • Kwang-Woong Lee
    • Journal of Plant Biology
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    • v.37 no.3
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    • pp.371-380
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    • 1994
  • To determine the patterns and the levels of expression of the cauliflower mosaic virus (CaMV 35S) promoter and the rice actin 1 (Act1) promoter in rice, transgenic rice plants containing CaMV 35S-$\beta$-glucuronidase (GUS) and Act1-GUS constructs were generated and examined by fluorometric and histochemical analyses. The fluorometric analysis of stably transformed calluses showed that the activity of the rice Act1 promoter was stronger than that of the CaMV 35S promoter in rice cells. In a histochemcial study of the transgenic rices, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in parenchymal cells of vascular tissues of leaves and roots and mesophyll cells of leaves. These results are similar to those of potato, a dicot plant. In contrast, rice plant transformed with Act1-GUS fusion construct revealed strong GUS activity in parenchymal cells of vascular tissue, mesophyll cells, epidermal cells, bulliform cells, guard subsidiary cells of leaves and most cells of the root, suggesting that the rice Act1 promoter is more constitutive than the CaMV 35S promoter. It was also confirmed that in both types of transgenic rice little or no staining was localized in metaxylen tracheary elements of vascular tissue from leaves or roots. These results indicate that the rice Act1 promoter can be utilized more successfully for expression of a variety of foreign gene in rice than the CaMV 35S promoter.

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Structural Characteristics of Two Wheat Histone H2A Genes Encoding Distinct Types of Variants and Functional Differences in Their Promoter Activity

  • Huh, Gyung-Hye;Iwabuchi, Masaki
    • Proceedings of the Botanical Society of Korea Conference
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    • 1996.07a
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    • pp.26-38
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    • 1996
  • To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode different types of variants. Both genes had an intron in the coding region. In the promoters, some characteristics sequences, such as Oct and Nona motifs, which are conserved among plant histone genes were also found, and they were located in a short region (about 120 bp) upstream from the putative TATA box. Analyses of promoter activity with H2A-GUS fusion genes in the transient system using tobacco protoplasts revealed novel types of positive cis-acting sequences in the TH254 promoter: a direct repeat of a 13-bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). A quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUG chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were both active in developing seedlings and flower organs but regulated in different manner.

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Influence of fat levels and types of dietary fiber on lipid metabolism of rats (식이중 지방 수준과 Fiber 종류가 흰쥐의 지방대사에 미치는 영향)

  • 윤홍재
    • Journal of the Korean Home Economics Association
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    • v.23 no.2
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    • pp.45-53
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    • 1985
  • The influence of fat level and dietary fiber on serum and liver lipid concentration of the rat was investigated. Groups of rats were fed respectively 3%, 5%, 20% fat containing and free fiber diet until 5th week. From 5th week to 9th week high fat diet groups of rates were devided high fat diet group, high fat pectin supplemented diet group and high fat celulose supplemented diet group. Pectin and cellulose at 10% was supplemented to high fat diets. After rats were fed for 9 weeks, all rats were sacrificed to collect the liver and blood samples by heart puncture. Serum and liver lipids were determined on all rats and compared the histochemical lipid staining method with the biochemical analysis of serum and liver lipids. A high fat diet caused increased level of the total lipids, total cholesterol and free cholesterol contents in the liver and the serum. Rats fed pectin reduced body weight, werum lipid and liver lipid but cellulose had no effect to reduce serum and liver lipid concentration. These results indicate that dietary fat level led to changes in the lipid metabolism of rats and that pectin was more effective in lowering serum and liver lipid than cellulose.

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Biochemical Properties of Acetylcholinesterase from the Larval Head of Bombyx mori

  • Lee, Hwa-Jun;Lee, Heui-Sam;Lee, Pyeong-Jae;Cho, Il-Je;Lee, Sang-Mong;Moon, Jae-Yu
    • International Journal of Industrial Entomology and Biomaterials
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    • v.1 no.1
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    • pp.73-78
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    • 2000
  • We investigated some biochemical properties of acetylcholinesterase (AChE) in the Bombyx mori larval head. 1% Triton X-100 (v/v) was suitable for extracting AChE from the silkworm larval head but 1 M NaCl was not suitable. PAGE analysis showed a single band of AChE that was detected by histochemical staining using acetylthiocholine as a substrate. AChE was also partially purified with Sepharose 6B and DEAE-cellulose column. Finally, the specific activity of partially purified enzyme solution was 7.6. The study on inhibitor specificity indicated that the enzyme under study was a true cholinesterase (ChE) or AChE. AChE activity was maximum at the substrate concentration of $5{\times}10^{-4}$ M and the excess substrate inhibited the AChE activity. The optimal pH and temperature were pH 7.0-9.0 and 30-35$^{\circ}C$.

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The Relationship between Muscle Fiber Composition and Pork Taste-traits Assessed by Electronic Tongue System

  • Hwang, Young-Hwa;Ismail, Ishamri;Joo, Seon-Tea
    • Food Science of Animal Resources
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    • v.38 no.6
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    • pp.1305-1314
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    • 2018
  • To investigate relationships of electronic taste-traits with muscle fiber type composition (FTC) and contents of nucleotides, porcine longissimus lumborum (LL), psoas major (PM), and infra spinam (IS) muscles were obtained from eight castrated LYD pigs. FTC and taste-traits in these three porcine muscles were measured by histochemical analysis and electronic tongue system, respectively. IS had significantly higher proportion of type I fibers while LL had significantly higher proportion of type IIB than other muscles (p<0.05). IS had the highest inosine monophosphate (IMP) content while LL had the lowest IMP content (p<0.05). In contrast, LL had significantly higher hypoxanthine content compared to PM and IS (both p<0.05). For taste-traits, IS had significantly higher umami and richness values but lower sourness value than LL and PM (p<0.05). Sourness and astringency values of LL were significantly higher than those of IS (p<0.05). The proportion of type IIB fiber was positively correlated with sourness and astringency but negatively correlated with saltiness. These results suggest that sourness and astringency tastes are increased with increasing proportions of type IIB fibers in porcine muscles due to increase of hypoxanthine content. These results also imply that umami and richness tastes are increased with increasing contents of type I and IIA fibers because of increased IMP content in porcine muscles.

Muscular and collagenous cerebellar choristoma in a dog

  • Ripplinger, Angel;de Melo, Stella Maris Pereira;Ferrarin, Denis Antonio;Schwab, Marcelo Luis;Wrzesinski, Mathias Reginatto;Rauber, Julia da Silva;Flores, Mariana Martins;Kommers, Glaucia Denise;Mazzanti, Alexandre
    • Journal of Veterinary Science
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    • v.23 no.2
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    • pp.30.1-30.6
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    • 2022
  • This report aims to describe the first case of muscular and collagenous choristoma in a dog. A 10-yr-old female mixed-breed dog presented with lateral recumbence, vocalization, positional vertical nystagmus, divergent strabismus, anisocoria, and status epilepticus. The clinical condition evolved to stupor and ultimately, death. Necropsy revealed a white mass causing an irregular increase in the volume of the cerebellar vermis. In histological analysis, a well circumscribed, unencapsulated mass was observed in the cerebellum, consisting of fibers of striated skeletal muscle and collagen fibers, mostly mineralized. Based on the histopathological and histochemical findings, a diagnosis of muscular and collagenous cerebellar choristoma was made.

The Effects of Chelidonii Herba on the Proliferation of Eosinophils and Activation of Immuno-cells in Asthma-induced Mouse (백굴채(白屈菜)가 천식유발 생쥐의 폐조직에서 호산구 증식과 면역 세포 활성화에 미치는 영향)

  • Roh, Seong-Soo
    • The Korea Journal of Herbology
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    • v.23 no.2
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    • pp.99-109
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    • 2008
  • Objectives : The present study was to investigate the effect of extract of Chelidonii herba (ECH) on the proliferation and activation of eosinophils which were prepared from lung cells of asthma-induced mouse by ovalbumin (OVA) treatment. Methods : C57BL/6 mouse was exposed to OVA three times a week for 6 weeks. The mouse lung tissues were dissected out, chopped and dossiciated with collagenase (1 ${\mu}g$/ml). Eosinophils were activated by rIL-3/rmIL-5 co-treatments. The lung cells were treated with ECH, incubated for 48 hr at $37^{\circ}C$, and analyzed by flow cytometery, ELISA, RT-PCR, and immuno-histochemical analysis. Results : In FACS analysis, number of granulocyte/lymphocyte, $CD3e^-$/$CCR3^+$, $CD3e^+$/$CD69^+$, $CD4^+$ and $CD23^+$/$B220^+$ in asthma-induced lung cells were significantly decreased by ECH treatment compared to the control group. And mRNA expression for IL-4, IL-5, IL-13, CCR3 and eotaxin in asthma-induced lung cells, which was induced by rIL-3 plus rmIL-5 treatments, was significantly decreased by ECH treatment. In ELISA analysis, production levels of IL-3, IL-5, IL-13 and histamine in asthma-induced lung cells, which were induced by rIL-3 plus rmIL-5 co-treatment, were significantly decreased by ECH treatment. ECH treatments significantly inhibited the proliferation of eosinohils prepared from asthma-induced mouse lung tissues compared to the non-ECH treated control cells. Immunohistochemical analysis revealed that ECH treatment significantly decreased the levels of eosipnphil activation compared to non-treated cells. Conclusions : The present data suggested that Chelidonium majus L. may have an effect on the inhibition of parameters associated with asthma responses in eosinpophils, and thus implicate the possibility for the clinical application of Chelidonium majus L.

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Highly efficient production of transgenic Scoparia dulcis L. mediated by Agrobacterium tumefaciens: plant regeneration via shoot organogenesis

  • Aileni, Mahender;Abbagani, Sadanandam;Zhang, Peng
    • Plant Biotechnology Reports
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    • v.5 no.2
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    • pp.147-156
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    • 2011
  • Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with ${\beta}$-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with $1mg\;l^{-1}$ indole-3-butyric acid and $15mg\;l^{-1}$ hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for ${\beta}$-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of $3.9{\pm}0.39$ transgenic plantlets per explant was achieved in the present transformation system. It took only 2-3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.