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In ovo vaccination using Eimeria profilin and Clostridium perfringens NetB proteins in Montanide IMS adjuvant increases protective immunity against experimentally-induced necrotic enteritis

  • Lillehoj, Hyun Soon;Jang, Seung Ik;Panebra, Alfredo;Lillehoj, Erik Peter;Dupuis, Laurent;Arous, Juliette Ben;Lee, Seung Kyoo;Oh, Sung Taek
    • Asian-Australasian Journal of Animal Sciences
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    • v.30 no.10
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    • pp.1478-1485
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    • 2017
  • Objective: The effects of vaccinating 18-day-old chicken embryos with the combination of recombinant Eimeria profilin plus Clostridium perfringens (C. perfringens) NetB proteins mixed in the Montanide IMS adjuvant on the chicken immune response to necrotic enteritis (NE) were investigated using an Eimeria maxima (E. maxima)/C. perfringens co-infection NE disease model that we previously developed. Methods: Eighteen-day-old broiler embryos were injected with $100{\mu}L$ of phosphate-buffered saline, profilin, profilin plus necrotic enteritis B-like (NetB), profilin plus NetB/Montanide adjuvant (IMS 106), and profilin plus Net-B/Montanide adjuvant (IMS 101). After post-hatch birds were challenged with our NE experimental disease model, body weights, intestinal lesions, serum antibody levels to NetB, and proinflammatory cytokine and chemokine mRNA levels in intestinal intraepithelial lymphocytes were measured. Results: Chickens in ovo vaccinated with recombinant profilin plus NetB proteins/IMS106 and recombinant profilin plus NetB proteins/IMS101 showed significantly increased body weight gains and reduced gut damages compared with the profilin-only group, respectively. Greater antibody response to NetB toxin were observed in the profilin plus NetB/IMS 106, and profilin plus NetB/IMS 101 groups compared with the other three vaccine/adjuvant groups. Finally, diminished levels of transcripts encoding for proinflammatory cytokines such as lipopolysaccharide-induced tumor necrosis $factor-{\alpha}$ factor, tumor necrosis factor superfamily 15, and interleukin-8 were observed in the intestinal lymphocytes of chickens in ovo injected with profilin plus NetB toxin in combination with IMS 106, and profilin plus NetB toxin in combination with IMS 101 compared with profilin protein alone bird. Conclusion: These results suggest that the Montanide IMS adjuvants potentiate host immunity to experimentally-induced avian NE when administered in ovo in conjunction with the profilin and NetB proteins, and may reduce disease pathology by attenuating the expression of proinflammatory cytokines and chemokines implicated in disease pathogenesis.

TLR9 Expression in Uterine Cervical Lesions of Uyghur Women Correlate with Cervical Cancer Progression and Selective Silencing of Human Papillomavirus 16 E6 and E7 Oncoproteins in Vitro

  • Hao, Yi;Yuan, Jian-Ling;Abudula, Abulizi;Hasimu, Axiangu;Kadeer, Nafeisha;Guo, Xia
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.14
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    • pp.5867-5872
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    • 2014
  • Background: Cervical cancer is listed as one of high-incidence endemic diseases in Xinjiang. Our study aimed to evaluate the expression of TLR9 in uterine cervical tissues of Uyghur women and examine associations with clinicopathological variables. We further characterized the direct effects of TLR9 upon the selective silencing of human papillomavirus (HPV) E6 and E7 oncoprotein expression in HPV 16-positive human cervical carcinoma cells treated with siRNA in vitro. Materials and Methods: Immunohistochemistry was applied to evaluate TLR9 expression in 97 formalin-fixed paraffin-embedded cervical samples from Uyghur women; 32 diagnosed with cervical squamous cell carcinomas (CSCC), 14 with low-grade cervical intraepithelial neoplasias (CINI), 10 medium-grade (CINII), 24 high-grade (CINIII), and 17 chronic cervicitis. $BLOCK-iT^{TM}$ U6 RNAi Entry Vector $pENTR^{TM}$/U6-E6 and E7 was constructed and transfected the entry clone directly into the mammalian cell line 293FT. Then the HPV 16-positive SiHa human cervical carcinoma cell line was infected with RNAi recombinant lentivirus. RT-PCR and Western blotting were used to determine the expression of TLR9 in both SiHa and HPV 16 E6 and E7 silenced SiHa cells. Results: Immunohistochemical staining showed that TLR9 expression was undetectable (88.2%) or weak (11.8%) in chronic cervicitis tissues. However, variable staining was observed in the basal layer of all normal endocervical glands. TLR9 expression, which was mainly observed as cytoplasmic staining, gradually increased in accordance with the histopathological grade in the following order: chronic cervicitis (2/17, 11.8%)

Processing of Ready-to-Cook Food Materials with Dark Fleshed Fish 2. Processing of Ready-to-Cook Low Salt Mackerel Fillet (일시다획성 적색육어류를 이용한 중간식품소재 개발에 관한 연구 2. 저염 고등어 Fillet의 가공)

  • LEE Byeong-Ho;LEE Kang-Ho;YOU Byeong-Jin;SUH Jae-Soo;JEONG In-Hak;CHOI Byeong-Dae;JI Young-Ae
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.18 no.5
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    • pp.409-416
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    • 1985
  • In previous paper (Lee et al., 1983) processing method of sardine meat "surimi" was described as a part of the wort to develop new types of ready-to-cook food materials with dark fleshed fishes. As the other part of the work, processing of low salt mackerel fillet was investigated, in this paper, in which fresh mackerel was filleted, salted in brine or with dry salt for an adequate time until the expected salt concentration reached, washed, air dried (3 m/sec, 15 to $20^{\circ}C$), and finally packed individually in K-flex film bag by vacuum or $N_2$ gas substitution. Salting time and salt concentration of brine was decided by the salt level penetrated into the fillet. As the final salt level was fixed to 4 to $5\%$, salting for 20 hours with $10\%$ dry salt or in $15\%$ brine at $5^{\circ}C$ was enough to get that level of salt. If the final salt level was set 5 to $6\%$, salting for 20-24 hours with $15\%$ dry salt or in $20\%$ brine was adequate. Salt penetration, however, was not much influenced by salting method and temperature. Changes in VBN and salt soluble protein occurred more rapidly in cases of salting with dry salt at $20^{\circ}C$ than salted in brine at $5^{\circ}C$, although it was not significant in the period of 20 to 24 hours. Oxidation of lipid and histamine formation during salting at $20^{\circ}C$ could not be neglected if it was delayed loger than 25 hours. Insolubilizing the salt soluble proteins during the storage of salted fillet occurred rapidly regardless of storage temperature. Browning and histamine formation, however, was depended on temperature and packing condition. In case of air pack, deterioration by browning and rancid was deeply developed but not the case for the packs by vacuum or $N_2$ gas substitution. The shelf-life of the salted mackerel fillet based on panel scores of brown color and rancidity, appeared 21 days for the air packed, and more than 30 days for vacunm or $N_2$ gas packed fillet at $20^{\circ}C$.

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Quantification of Karanjin, Tannin and Trypsin Inhibitors in Raw and Detoxified Expeller and Solvent Extracted Karanj (Pongamia glabra) Cake

  • Panda, A.K.;Sastry, V.R.B.;Kumar, A.;Saha, S.K.
    • Asian-Australasian Journal of Animal Sciences
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    • v.19 no.12
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    • pp.1776-1783
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    • 2006
  • Despite being a rich source of protein (28-34%), karanj (Pongamia glabra) cake is found to be bitter in taste and toxic in nature owing to the presence of flavonoid (karanjin), tannin and trypsin inhibitor, thereby restricting its safe inclusion in poultry rations. Feeding of karanj cake at higher levels (>10%) adversely affected the growth performance of poultry due to the presence of these toxic factors. Therefore, efforts were made to detoxify karanj cake by various physico-chemical methods such as dry heat, water washing, pressure cooking, alkali and acid treatments and microbiological treatment with Sacchraromyces cerevisiae (strain S-49). The level of residual karanjin in raw and variously processed cake was quantified by high performance liquid chromatography and tannin and trypsin inhibitor was quantified by titrametric and colorimetric methods, respectively. The karanjin, tannin and trypsin inhibitor levels in such solvent and expeller pressed karanj cake were 0.132, 3.766 and 6.550 and 0.324, 3.172 and 8.513%, respectively. Pressure-cooking of solvent extracted karanj cake (SKC) substantially reduced the karanjin content at a cake:water ratio of 1:0.5 with 30-minute cooking. Among chemical methods, 1.5% (w/w) NaOH was very effective in reducing the karanjin content. $Ca(OH)_2$ treatment was also equally effective in karanjin reduction, but at a higher concentration of 3.0% (w/w). A similar trend was noticed with respect to treatment of expeller pressed karanj cake (EKC). Pressure cooking of EKC was effective in reducing the karanjin level of the cake. Among chemical methods alkali treatment [2% (w/w) NaOH] substantially reduced the karanjin levels of the cake. Other methods such as water washing, dry heat, HCl, glacial acetic acid, urea-ammoniation, combined acid and alkali, and microbiological treatments marginally reduced the karanjin concentration of SKC and EKC. Treatment of both SKC and EKC with 1.5% and 2.0% NaOH (w/w) was the most effective method in reducing the tannin content. Among the various methods of detoxification, dry heat, pressure cooking and microbiological treatment with Saccharomyces cerevisiae were substantially effective in reducing the trypsin inhibitor activity in both SKC and EKC. Based on reduction in karanjin, in addition to tannin and trypsin inhibitor activity, detoxification of SKC with either 1.5% NaOH or 3% $Ca(OH)_2$, w/w) and with 2% NaOH were more effective. Despite the effectiveness of pressure cooking in reducing the karanjin content, it could not be recommended for detoxification because of the practical difficulties in adopting the technology as well as for economic considerations.

Development of Herbal Chicken Porridge and the Establishment of Optimizing the Mixing Ratio (한방 닭죽 개발 및 최적 배합비율 확립)

  • Kim, Kyung-Yeon;Baik, Moo-Yeol;Park, Cheon-Seok;Choi, Sung-Won;Kim, Chang-Nam;Kim, Byung-Yong
    • The Korean Journal of Food And Nutrition
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    • v.26 no.1
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    • pp.22-28
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    • 2013
  • The objectives of this study were to develop porridge with medicinal herbs and chicken breast meat and to find out the optimum mixing ratio. Several herb extracts such as Acanthopanacis senticosus, Rehmannia glutinosa, Disocorea japonica, and Poria cocos Wolf were used in the ratio of 2, 1, 1, and 1, respectively. The optimum processing condition for herbal chicken porridge was determined by a design expert program. Seventeen experimental points were selected, and herb extracts (82~101 g), chicken breast meat (30~50 g), and glutinous rice (40~60 g) were chosen as the independent variables. The measured responses were preference of taste, DPPH radical scavenging, SOD-like activity, and cost. The optimum formulation of herbal chicken porridge using the numerical analysis was set at herbal extracts (101 g), chicken breast meat (38.66 g), and glutinous rice (41.34 g) with a 0.714 desirability value. DPPH radical scavenging effect, preference of taste and the cost showed a linear model, whereas SOD-like activity showed a quadratic model indicating a higher interaction among the mixture. As a result of proximate composition of optimized herbal chicken porridge, the contents of moisture, carbohydrate, crude protein, crude lipid, and ash contents were 76.4, 9.0, 1.4, 0.5, and 1.6%, the calories of the porridge was 46.1 kcal/100 g.

The Effect of Atorvastatin on the Development of Puromycin Aminonucleoside(PAN)-induced Nephrosis in Rats (Puromycin을 투여한 백서에서 지질 변화가 신증의 진행에 미치는 영향)

  • Choi Kwang-Hae;Chung Hyo-Seuk;Kim Yong-Jin;Ha Jeong-Hee;Kim Heung-Sik;Park Yong-Hoon
    • Childhood Kidney Diseases
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    • v.7 no.1
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    • pp.9-15
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    • 2003
  • Purpose : Several studies have suggested that hyperlipidemia might be a causative factor contributing to the progression of initial glomerular injury through the development of glomerulosclerosis. We examined the potential beneficial effect of atorvastatin - which blocks the rate limiting step of cholesterol synthesis by inhibiting HMG-CoA reductase - in PAN-induced nephrosis. Materials and Methods : Glomerulosclerosis was induced in Sprague-Dawley male rats by repeated administration of PAN. Sprague-Dawley male rats were divided into 3 groups : group I(control), group II(PAN 20 mg/kg, subcutaneous injection), group III(PAN 20 mg/kg subcutaneous injection and atorvastatin 50 mg/kg/day per oral). On the 11th week, upon sacrifice of the experimental animals, blood sampling, 24-hr urine collection and nephrectomy were performed. Results : Group III had significantly lower BUN and higher serum albumin($30.9{\pm}17.2\;vs.\;17.3{\pm}2.5\;mg/dL;\;2.3{\pm}0.1\;vs.\;2.5{\pm}0.2\;g/dL$, P<0.05) compared with group II. In the lipid profiles, group III was associated with a reduction in total cholesterol and LDL($291{\pm}173\;vs.\;167{\pm}72\;mg/dL:\;57{\pm}53\;vs.\;27{\pm}12\;mg/dL$, P>0.05) compared with group II. Atorvastatin administration lowered the glomerular sclerosing index significantly(26.2% vs. 13.3%, P<0.05). Conclusion : Puromycin-induced glomerulosclerosis could be ameliorated by the reduction of hyperlipidemia with atorvastatin. This suggests that hyperlipidemia contributes to the pathogenesis of glomerulosclerosis.

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Inhibitory Effect of Dioscorea Bulbifera MeOH Extract on Pro-inflammatory Mediator In Vitro and In Vivo (황약자(黃藥子) 메탄올 추출물의 염증억제 효과)

  • Jung, Ji-Yun;Lee, Jong-Rok;Byun, Sung-Hui;Jung, Ji-Wook;Kim, Yong-Han;Kim, Sang-Chan
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.24 no.2
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    • pp.310-318
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    • 2010
  • Dioscorea bulbifera is one of the traditional medicinal herb. It commonly used in the treatment of hematemesis, epistaxis, tuberculous cervical lymphadenitis, laryngitis, acute infectious disease in East Asia. In the present study, we have demonstrated the anti-inflammatory effects of Dioscorea bulbifera MeOH extract (DBME) in macrophage cell line. To investigate mechanism of the anti-inflammatory activity, we examined the effects of the lipopolysaccaride (LPS)-induced production of nitric oxide (NO), prostaglandin $E_2$ ($PGE_2$), pro-inflammatory cytokines and expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), p-inhibitory ${\kappa}B{\alpha}$ (p-$I{\kappa}B{\alpha}$), and nuclear factor-${\kappa}B$ (NF-${\kappa}B$) in a murine macrophage cell line RAW 264.7. The RAW 264.7 cells were cultured in DMEM + serum medium for 24 hrs. After serum starvation for 24 hrs, the cells were treated with DBME 0.03, 0.10, 0.30 mg/$m{\ell}$ for 1 h, followed by stimulation with LPS (1 ${\mu}g/m{\ell}$) for activation of immune response. After treatment, cell viability was measured by MTT assay, and NO production was monitored by measuring the nitrite content in culture medium. The protein band of iNOS, COX-2, p-$I{\kappa}B{\alpha}$, and NF-${\kappa}B$ was determined by immunoblot analysis and levels of cytokine were analyzed by sandwich immunoassays. There were three experimental groups: carrageenan, DBME 0.3, 1.0 g/kg. Rats were administrated either carrageenan (40% PEG) or carrageenan + DBME (0.3, 1.0 g/kg body weight) for 4 days (p.o.). To induce acute paw edema, rats were injected 1% carrageenan (100 ${\mu}{\ell}$/rat, dissolved in sterilized saline). The effect of DBME in the carrageenan-induced rat paw edema. As results, DBME has an inhibitory effect on the production of NO, PGE2, TNF-${\alpha}$, IL-$1{\beta}$ and IL-6 and on the expression of iNOS, COX-2, p-$I{\kappa}B{\alpha}$ and translocation of NF-${\kappa}B$ to nuclear from cytosol. In addition, DBME effectively inhibited the increases of paw edema induced by carrageenan treatment in vivo. These results suggest that DBME can inhibit production of pro-inflammatory mediators and might be a useful source for treatment of acute inflammatory disease.

Comparison of Yield and Quality of Direct-Seeded Whole Crop Rice (총체 벼의 직파시기에 따른 수량 및 사료가치 비교)

  • Kim, Jong-Geun;Chung, Eui-Soo;Lee, Joung-Kyeong;Lim, Young-Chul;Yoon, Sei-Hyung;Kim, Meing-Joong
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.29 no.1
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    • pp.25-30
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    • 2009
  • This experiment was conducted to evaluate the effect of direct seeding time on the yield and quality of whole crop rice at experimental field of Grassland and Forages Research Center, National Institute of Animal Science, RDA from 2005 to 2006. Whole crop rice variety, "Namil", was direct seeded at five different calender date (25 April, 10 May, 25 May, 4 June and transplanted at 25 May). The heading date was delayed at late seeding date. Crude protein (CP) was increased with delayed direct seeding date. The content of ADF (acid detergent fiber) and NDF(neutral detergent fiber) increased with delayed seeding date. The average TDN (total digestible nutrient) content was 61.5% and it also decreased with delayed direct seeding date. The highest DM (dry matter) yield was seeded at 25 April and transplanted at 25 May, 15,185 and 15,815 kg/ha, respectively. Although delayed direct seeding date decrease the DM yield, mid-May will be recommendable as proper direct seeding date.

Characterization of Transgenic Tall Fescue Plants Overexpressing NDP Kinase Gene in Response to Cold Stress (NDP Kinase 유전자를 과발현시킨 형질전환 톨 페스큐 식물체의 저온 스트레스에 대한 내성 특성)

  • Lee, Sang-Hoon;Lee, Ki-Won;Kim, Kyung-Hee;Yun, Dae-Jin;Kwak, Sang-Soo;Lee, Byung-Hyun
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.29 no.4
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    • pp.299-306
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    • 2009
  • Oxidative stress is the main limiting factor in crop productivity. To solve global environmental problems using the plant biotechnology, we have developed on the oxidative stress-tolerant transgenic tall fescue plants via Agrobacterium-mediated genetic transformation method. In order to develop transgenic tall fescue (Festuca arundinacea Schreb.) plants with enhanced tolerance to multiple environmental stresses, nucleotide diphosphate kinase gene under the control of CaMV35S promoter were introduced into genome of tall fescue plants. Proteomic analysis revealed that transgenic tall fescue not only accumulated NDP kinase 2 protein in their cells, but also induced several other antioxindative enzyme-related proteins. When leaf discs of transgenic plants were subjected to cold stress, they showed approximately 30% less damage than wild-type plants. In addition, transgenic tall fescue plants showed normal growth when transgenic plants were subjected to $4^{\circ}C$ for 3 days treatments. These results suggest that transgene is important in ROS scavenging by induction of antioxidative proteins, and could improve abiotic stress tolerance in transgenic tall fescue plants.

Development of a new lactic acid bacterial inoculant for fresh rice straw silage

  • Kim, Jong Geun;Ham, Jun Sang;Li, Yu Wei;Park, Hyung Soo;Huh, Chul-Sung;Park, Byung-Chul
    • Asian-Australasian Journal of Animal Sciences
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    • v.30 no.7
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    • pp.950-956
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    • 2017
  • Objective: Effects of newly isolated Lactobacillus plantarum on the fermentation and chemical composition of fresh rice straw silage was evaluated in this study. Methods: Lactic acid bacteria (LAB) from good crop silage were screened by growing them in MRS broth and a minimal medium with low carbohydrate content. Selected LAB (LAB 1821) were Gram-positive, rods, catalase negative, and were identified to be Lactobacillus plantarum based on their biochemical characteristics and a 16S rRNA analysis. Fresh rice straw was ensiled with two isolated LAB (1821 and 1841), two commercial inoculants (HM/F and P1132) and no additive as a control. Results: After 2 months of storage at ambient temperature, rice straw silages treated with additives were well-preserved, the pH values and butyric and acetic acid contents were lower, and the lactic acid content and lactic/acetic acid ratio were higher than those in the control (p<0.05). Acidity (pH) was lowest, and lactic acid highest, in 1821-treated silage (p<0.05). The $NH_3-N$ content decreased significantly in inoculant-treated silage (p<0.05) and the $NH_3-N$ content in 1821-treated silage was lowest among the treatments. The dry matter (DM) content of the control silage was lower than that of fresh rice straw (p<0.05), while that of the 1841- and p1174-inoculant-treated silages was significantly higher than that of HM/F-treated silage. Microbial additives did not have any significant (p>0.05) effect on acid detergent fiber or neutral detergent fiber contents. Crude protein (CP) content and in vitro DM digestibility (IVDMD) increased after inoculation of LAB 1821 (p<0.05). Conclusion: LAB 1821 increased the CP, IVDMD, lactic acid content and ratio of lactic acid to acetic acid in rice straw silage and decreased the pH, acetic acid, $NH_3-N$, and butyric acid contents. Therefore, adding LAB 1821 improved the fermentation quality and feed value of rice straw silage.